320A Volume 57 Number 11 2003

focal pointBY EMILY A SMITH AND ROBERT M CORN

DEPARTMENT OF CHEMISTRY

UNIVERSITY OF WISCONSIN

MADISON WISCONSIN 53706

Surface PlasmonResonance Imaging as

a Tool to MonitorBiomolecular

Interactions in anArray Based Format

INTRODUCTION

N early all processes within aliving organism are drivenby biomolecular interactions

In order to fully understand the roleof a newly discovered gene or pro-tein within a biological system it isnecessary to know what molecules itinteracts with and what the possibleoutcomes of these interactions areOne consequence of biological di-versity is that there are many poten-tial interacting partners in living sys-tems (eg DNA RNA peptidesproteins lipids and carbohydrates)

Current Address Department of Chemistryand Biochemistry University of DelawareNewark Delaware 19716

with a broad range of chemical prop-erties The mechanisms throughwhich these partners interact are asdiverse as the interacting partnersthemselves they may interact veryweakly fo rm a strong chemicalbond have cooperative or multiplebinding interactions create an entitywith enzymatic activity etc

Methods that are capable of rapidlyscreening diverse sets of biomolecu-lar interactions are necessary in orderto decipher the wealth of informationthat exists for the genomes and pro-teomes of many organisms One wayto accomplish this screening is to at-tach a set of biomolecules (arbitrarilyde ned as probes) to a surface in anarray format and then expose the ar-ray to another set of biomolecules

(arbitrarily de ned as targets) that arein solution The use of an array for-mat to study biomolecular interac-tions has several bene ts over solu-tion-based methods the use of smallanalyte volumes the ability to per-form parallel screening of multipleinteractions and the ability to providestraightforward and rapid responseread-outs Numerous detection meth-ods have been used to study biomo-lecular interactions on surfaces in-cluding total internal re ection uo-rescence1 wave guides23 ellipsome-try45 atomic force microscopy67 andsurface plasmon resonance (SPR)8ndash10

In this article we will describe the useof SPR imaging as a method forscreening biomolecular interactions

Surface plasmon resonance is a

APPLIED SPECTROSCOPY 321A

sensitive label-free technique thatcan provide real-time data on ad-sorp tion andor desorption eventsthat occur at a metaldielectric inter-face There are several instrumentalformats that can be used in SPR ex-periments these can be roughlycategorized into three types of mea-surements scanning angle SPR11ndash14

scanning wavelength SPR15 andSPR imaging916ndash22 For all SPR for-mats the re ectivity of light incidenton a metald ielectric in terface ismonitored and correlated to changesin the local index of refraction of thedielectric layer adjacent to the metal lm The most widely used formatfor an SPR experiment is the scan-ning angle technique in which there ectivity of monochromatic inci-dent light upon a metal lm is mon-itored as a function of the incidentangle The popularity of the scan-ning angle technique can be partiallyattributed to the existence of com-mercially available instrumentationfrom Biacore23 which has made itpossible to use SPR as a detectionmethod for many applications in-cluding basic life science researchdrug discovery environmental mon-itoring and process analysis Boththe scanning angle and scanningwavelength measurements typicallyprovide only one or a few data pointsat a time In contrast SPR imagingmeasurements sometimes calledSPR microscopy use the changes inre ectivity from a gold thin lm thatoccur upon adsorption to generatedifference images to simultaneouslymonitor tens hundreds or more in-teractions in a parallel manner

The high-throughput capabilitiesof SPR imaging have made it an at-tractive tool for screening biomolec-ular interactions For example SPRimaging has been used in an arrayformat to study the hybridization ofDNA and RNA to nucleic acid ar-rays fabricated on gold lms9202425

Shown in Fig 1A is an example ofan SPR difference image of a two-component DNA array The differ-ence image was obtained by sub-tracting the images taken before andafter exposing the array to a 16-merthat was complementary to the im-

mobilized sequence 1 which waspatterned on the surface as shown inFig 1B A change in the re ectedlight intensity was observed onlywhere sequence 1 was immobilizedon the array demonstrating the spe-ci c hybridization of the target to thecomplementary immobilized se-quence Shown in Fig 1C is a plotof a line pro le that was acquiredfrom the SPR image (indicated bythe black line in Fig 1A) The plotshows that the hybridization of the16-mer complementary sequencecorresponds to less than a one per-cent change in the re ected light in-tensity and that there is no change inthe re ected light intensity for thenon-complementary sequence DNAarrays have also been used in con-junction with SPR imaging to mon-itor RNA hybridization9 for DNAword design in computational algo-rithms26 for single base mismatchdetection (discussed below) and tomonitor hairpin formation in DNAmonolayers27

The next section of this article willprovide a brief background to theSPR imaging technique including anin troduction to surface plasmonsFollowing this background sectionthe remainder of the article willhighlight SPR imaging instrumenta-tion array fabrication techniquesand the ability to obtain quantitativedata with SPR imaging A few ex-amples of the use of SPR imagingfor the study of biomolecular inter-actions will also be presented

SURFACE PLASMONRESONANCE THEORY

Surface plasmons (SPs) are oscil-lations of free electrons that propa-gate along the surface of a metalwhen it is in contact with a dielectricinterface Surface plasmons can havea range of energies that depend onthe complex dielectric function ofthe metal (laquom) and the dielectricfunction of the adjacent medium(laquod) as shown by the followingequation

v laquo laquom dk 5 (1)sp c laquo 1 laquom d

where ksp is the wave vector of theSP vc is the wave vector in a vac-uum and the dielectric constant isthe square of the index of refrac-tion28 One condition for the gener-ation of SPs is that laquom and laquod are ofopposite sign and thus SPs will notbe generated for all systems In ad-dition laquom and laquod are wavelength de-pendent and certain regions of theelectromagnetic spectrum may be re-quired to generate SPs One commonsystem used in SPR experiments is agold lm in contact with a water in-terface Gold has a negative dielec-tric function in the IR and visible re-gions of the electromagnetic spec-trum whereas water has a positivedielectric function29

Surface plasmons can be directlyexcited by electrons however theycan not be excited directly by lightbecause they have a longer wavevector than light waves of the sameenergy (klight 5 vc) The wave vectorof a photon must be increased toconvert the photon into SPs Thiscan be accomplished with the use ofeither a prism or a grating coupler28

For example SPs can be excited un-der conditions of attenuated total re- ection (ATR) using a prismmetal lmdielectric layer (designated theKretschmann con guration)30 and p-polarized incident light that has itselectric component in the plane ofincidence Shown in Fig 2 is a sche-matic diagram of a Kretschmanncon guration sample setup that hasbeen used for the study of biomolec-ular interactions with SPR imagingThe setup consists of a high-index-of-refraction glass prism a 45 nmAu lm (with a 1 nm Cr underlayer)a 2 nm self-assembled monolayer a5 nm sensing layer where adsorptionof the target molecule occurs and abulk water layer

The equation for the surface com-ponent of a photonrsquos wave vector un-der conditions of ATR becomes

vk 5 sin uIumllaquo (2)ATR pc

where laquop is the dielectric constant ofthe prism and u is the angle of in-cidence of the light on the metal lm28 SPs will be generated in the

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focal point

Fig 1 (A) SPR difference image of a two-component DNA array showing the hybridization of a sequence complementary to im-mobilized sequence 1 (B) schematic diagram showing the pattern of the immobilized DNA sequences for the SPR image shown inA and (C) line prole plotted as the change in percent reectivity obtained from the area indicated by the black line in the SPRimage

metal lm at angles where the pho-tonrsquos wave vector equals the SPrsquoswave vector The excitation of SPscorresponds to an attenuation of there ected light intensity as the incom-ing light generates SPs The anglewhere there is a complete attenuationof the re ected light corresponds tothe complete conversion of the in-coming light and is referred to as thesurface plasmon angle

Fresnel equations can be used topredict the re ectivity and phaseshift when incident light impinges onan interface containing two or morephases31 These calculations assumethat each phase is homogenous andparallel and they require knowledgeof the complex index of refraction ofeach of the phases which are depen-dent on the wavelength of the inci-dent light The re ection coef cient

for p-polarized incident light is givenby the formula

rEr 5 (3)p tE

where E r is the electric eld com-ponent in the plane of incidence ofthe light re ected from the interfaceand E t is the electric eld componentin the plane of incidence of the lighttransmitted through the inter faceThe light re ected from the interfacecan be calculated using the formula

Rp 5 zrpz2 (4)

For multiphase systems these cal-culations require matrix operationsComputer programs have been de-veloped to calculate the re ectedlight intensity for multiphase sys-tems where one or more of the phas-es are a material with a complex in-

dex of refraction (ie a metal lm)23 Shown in Fig 3 are the re-sults of 5-phase Fresnel calculationsused to simulate scanning angle SPRre ectivity curves from the samplesetup depicted in Fig 2 The re ec-tivity of light is plotted versus theangle of incidence for two lms withsensing layers that have an index ofrefraction (nf) of 140 or 146 An in-crease in the index of refraction ofthe sensing layer simulates adsorp-tion onto the metal lm Conditionsof ATR are met at angles greaterthan the critical angle which occursnear 506 degrees As mentionedpreviously the surface plasmon an-gle corresponds to the angle wherethere is a near complete attenuationof the re ected light The position ofthe surface plasmon angle is depen-dent on the index of refraction of the

APPLIED SPECTROSCOPY 323A

Fig 2 Diagram showing a sample setup used to monitor biomolecular interactions with SPR imaging The 5 phases are a high-index-of-refraction glass prism (n1 5 1712) a 45-nm-thick gold lm (n2 5 01451 1 48725) a 2 nm self-assembled monolayer(n3 5 145) a 5 nm sensing layer where adsorption occurs (nf ) and a bulk water layer (n5) Adsorption of biomolecules to thesensing layer increases the value of nf

sensing layer An expanded view ofthe region encompassing the surfaceplasmon angles for both lms isshown in Fig 3 (right inset)

Surface plasmons have a maxi-mum intensity in the metal lm andthey decay exponentially in a per-pendicular direction from the surfacein both the metal and the dielectriclayer28 The decay length of the SPsis dependent on the wavelength ofthe incident light and on the dielec-tric constants of both layers For agold lm a typical decay length ison the order of a few hundred nano-meters into the dielectric layer for

excitation with visible light Thismeans that SPR is a surface sensitivetechnique and that measurements canbe made even when a large excess ofanalyte is present in solution Anyspecies that is farther from the metal lm than the SP decay length willnot effect the generation of SPs

The propagation length of SPs isthe distance where its electric eldintensity in the metal lm drops to avalue of 1e and is determined by laquomlaquod and the wavelength of the inci-dent light 28 The SP propagationlength determines the lateral resolu-tion in SPR imaging In order to re-

solve two features they must be sep-arated by a minimum distance cor-responding to the propagation lengthof the SP Longer SP propagationlengths correlate to a lower lateralresolution and higher lateral resolu-tion can be achieved using shorterwavelength light

The sensitivity in SPR imagingthe ability to detect small changes inn f is also affected by the wavelengthof the incident light Incident light oflonger wavelengths produces sharperSPR curves (with narrow widths at50 re ectivity) and incident lightof shorter wavelengths produces

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focal point

Fig 3 Graph showing the scanning angle SPR reectivity curves that were obtained from 5-phase Fresnel calculations for thesystem shown in Fig 2 The index of refraction of the sensing layer (nf ) was 140 (solid line) or 146 (dotted line) The gure insetat right shows an expanded view of the region near the plasmon angles The location of the plasmon angle shifts to higher anglesfor the nf 5 146 lm relative to the nf 5 140 lm The gure inset at left shows an expanded view around the optimal angle forperforming SPR imaging experiments with this system At the optimal angle the largest shift in R is observed for these two lms

broad SPR curves (with large widthsat 50 re ectivity)32 Sharper SPRcurves produced by longer wave-length incident light yield largerchanges in re ectivity fo r givenchanges in nf than do broad curvesproduced by incident light withshorter wavelengths This means thatthere is a trade-off between lateralresolution and sensitivity The use oflonger wavelength light provideshigher sensitivity but lower lateralresolution The use of shorter wave-length light provides a higher lateralresolution but lower sensitivity3334

SURFACE PLASMONRESONANCE IMAGING

Surface Plasmon Resonance Im-aging Instrumentation Surface

plasmon resonance imaging is a xed angle experiment where thespatial changes in re ected light aremeasured across a substrate The the-oretical curves generated for a scan-ning angle SPR experiment can beused to understand the basis of thecontrast observed in an imaging ex-periment The left inset in Fig 3shows an expanded region around anincident angle of 535 degrees Aslice through the X-axis simulates aconstant angle experiment At 535degrees less light is re ected fromthe n f 5 140 lm than the nf 5 146 lm If a surface were patterned tocontain regions w ith both lmsmore light would be re ected fromthe regions with n f 5 146 than re-

gions containing the n f 5 140 lmat an angle of 535 degrees and itwould be possible to distinguish be-tween the two lms in an SPR im-age

The basic components in a typicalSPR imaging instrument are shownin Fig 4 These are a collimatedwhite light source a polarizer thesample stage collection optics anda charge-coupled device (CCD) con-nected to a CPU for image collectionand processing The use of a colli-mated white light source is preferredover the use of laser excitation dueto interference fringes that result inthe SPR image when laser excitationis utilized The polarizer is used toselect p-polarized light and the col-

APPLIED SPECTROSCOPY 325A

lection optics consist of a narrowband pass lter typically centered inthe near-infrared region that is usedto select the excitation wavelengthfor the experiment The sample is lo-cated on a rotation stage in order tocontrol the incident angle of lightand consists of a prism a substrateonto which a Au lm is depositedand a ow cell

Attachment Chemistry and Ar-ray Fabrication Surface plasmonresonance imaging is performed ona noble metal lm therefore strate-gies for attaching probe molecules tothese lms are critical to the successof an SPR imaging experiment Nu-merous immobilization strategies ex-ist These can generally be catego-rized into three routes (1) a thiol-modi ed probe molecule can be re-acted directly with a gold lm toform a goldndashthiolate bond35 (2) apolymer layer such as dextran orpolylysine can be rst formed on thegold surface and the probe moleculecan be immobilized onto the poly-mer layer36 and (3) a self-assembledalkanethiol monolayer (SAM) con-tain ing a v-terminated functionalgroup can be formed on the gold lm which is used to immobilize amolecule (a lsquolsquolinkerrsquorsquo) that is capableof reacting with the probe mole-cule37ndash41

There are drawbacks to the rstand second immobilization schemesMany biomolecules will non-specif-ically adsorb to a gold lm and alarge portion of the probes may notbe biologically active if they are di-rectly attached to the gold lm Sur-faces fabricated using the second im-mobilization strategy may not be ro-bust and polymer layers can be aproblem when studying kinetics ofadsorption

Several characteristics of the thirdimmobilization scheme make it suit-able for use with SPR imaging Gen-erally the surfaces that result fromthis immobilization strategy are sta-ble and can be used for several assaycycles Using this immobilizationstrategy provides a way to controlthe surface density of the probe mol-ecule This is important for two rea-sons (1) the surface density of the

probe can affect the amount of targetthat binds to the surface and there-fore the amount of signal that is de-tected and (2) varying the probedensity can be used as a tool to studythe interactions of targets that bindto the probes through multivalent in-teractions (an example of this is theuse of SPR imaging to study carbo-hydratendashprotein interactions) Final-ly this strategy provides a way tocontrol the resulting surface proper-ties (ie hydrophilic hydrophobiccharged)42ndash47 This is important sinceeverything that adsorbs to the goldsurface will produce an SPR signalit is necessary to control the surfaceproperties so that only the desiredtarget molecules interact with thesurface

In addition to immobilizing probemolecules to a gold lm the lmmust be patterned so that severalprobes are immobilized at discretelocations on the substrate There areseveral methods that have been usedto pattern gold lms UV photopat-terning 17 4 1 polydimethylsiloxane(PDMS) microchannels48 microcon-tact printing49 and robotic spotting50

have been used in conjunction withSPR imaging The array shown inFig 1 was fabricated using the UVphotopatterning method in which aquartz mask is used to selectively ex-pose an alkanethiol-modi ed goldsurface to UV light At locationswhere the UV light shines on thesurface the alkanethiol is removedgenerating bare gold patches thatserve as a platform for generatingthe array elements Both the UVphotopatterning and PDMS micro-channel array fabrication strategieswill be discussed later in this articlein conjunction with speci c exam-ples of the use of SPR imaging tostudy biomolecular interactions

Quantitation of Results Gener-ated with Surface Plasmon Reso-nance Imaging In addition to theability to detect interacting partnerswith SPR imaging it is desirable toobtain quantitative information aboutthese interactions Quantitative datacan be obtained from SPR measure-ments by assuming that moleculesadsorbing to or desorbing from the

metal lm correlate to changes in theindex of refraction of the dielectriclayer and that changes in the indexof refraction correlate to changes inthe re ectivity of the incident lightIn order to obtain quantitative datawith SPR imaging it is necessary toknow over what regions there is alinear relationship between thechange in the re ected light intensity(DR) and the change in the indexof refraction of the sensing layer(Dnf) A series of 5-phase Fresnelcalculations of the system shown inFig 2 can be used to determine this9

The rst calculation is performedwith an n f value of 14 Each succes-sive calculation increments the n f

value by 0002 index of refractionunits Figure 5 shows the resultsfrom these calculations plotted as theabsolute value of DR for the indi-cated Dn f These calculations wereperformed for an excitation wave-length of 794 nm and the results atthree angles along the SPR curve areshown (the location of the anglesalong the SPR curve are shown inthe gure inset) The dotted lines inFig 5 show a linear relationship be-tween DR and Dnf At an angle of5352 degrees the greatest contrastis predicted as determined by themagnitude of DR The smallest de-viation from linearity is also ob-served at 5352 degrees A smallchange in the incident angle of 004degrees does not signi cantly affectthe overall signal or the amount ofdeviation that occurs This is not thecase for angles greater than 01 de-grees from the optimal angle of5352 degrees (not shown in Fig 5)At angles further than 01 degreesfrom the optimal angle there is asmaller change in the percent of re- ected light and these signals devi-ate from a linear relationship at largen f values While the data is fairly lin-ear for an angle higher than the sur-face plasmon angle (5412) theoverall contrast that would be ob-served is much smaller than for an-gles to the left of the surface plas-mon angle This is a result of theSPR curve not being symmetricabout the surface plasmon angle (seeFig 3)

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focal point

Fig 4 (A) A schematic diagram of an SPR imager Collimated white light is passed through a polarizer and is incident on thesample assembly Reected light passes through focusing optics a narrow band pass lter typically centered at a wavelength in thenear-infrared and is captured by a CCD camera (B) A schematic diagram of the sample assembly consisting of a glass prism thatis optically coupled to a glass substrate containing a thin layer (45 nm) of gold The sample is contained within a ow cell for in situmeasurements

The deviations from linear behav-ior for DR and Dn f are small pro-vided that the experiment is per-formed at the optimal angle If de-viations are present they can be pre-dicted and accounted fo r in theexperimental results As mentionedpreviously the percent change in re- ected light intensity due to the hy-bridization of a monolayer of 16-meroligonucleotides is less than 1 per-cent which falls within the regionwhere linear data is obtained withSPR imaging9 An example of theuse of SPR imaging to quantitate theamount of material adsorbing to agold lm is described in the Exam-ples section In this example theamount of protein adsorbing to ametal lm is measured with SPR im-aging and is used to construct ad-sorption isotherms for the interactionof proteins with immobilized carbo-hydrates

EXAMPLESDetection of DNA Hybridiza-

tion Single-Base Mismatch Detec-

tion in the Presence of Small Mol-ecules A recent example of the useof SPR imaging demonstrated thatthis technique can be used to moni-tor the hybridization of short oligo-nucleotides in the presence of smallmolecules that alter the oligonucle-otidersquos binding properties27 A sche-matic diagram of a small moleculenaphthyridine dimer that has beenshown to stabilize the binding of GndashG mismatches in double strandedDNA is shown in Fig 651ndash53 To dem-onstrate the GndashG mismatch stabiliz-ing properties of this molecule aDNA array was fabricated using UVphotopattern ing This involves acombination of self-assembly andUV photopatterning steps A baregold lm is modi ed with a self-as-sembled monolayer of an amine-ter-minated alkanethiol The amine-ter-minated surface is then reacted witha hydrophobic protecting group thatcan be reversibly removed from thesurface A quartz mask containingpatterned features is placed over the

modi ed gold lm and the surface isexposed to UV light resulting in thephotooxidation of the goldndashthiolatebond and generation of bare gold re-gions where the UV light is exposedto the surface The substrate is thenreplaced in the amine-terminated al-kanethiol solution and a monolayeris formed at areas where there is baregold The resulting surface containshydrophilic amine-terminated mono-layer regions surrounded by a hydro-phobic monolayer The probe mole-cules can be immobilized through aseries of reactions that are carriedout within the hydrophilic wells bydelivering small solution volumes tothe surface (ie spotting solutionsonto the surface with a pulled cap-illary) The hydrophobic backgroundprevents cross-contamination be-tween the array elements during theimmobilization reaction Once theprobe compounds are immobilizedon the surface the hydrophobic pro-tecting group is removed from thebackground This generates an

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

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focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

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Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

APPLIED SPECTROSCOPY 321A

sensitive label-free technique thatcan provide real-time data on ad-sorp tion andor desorption eventsthat occur at a metaldielectric inter-face There are several instrumentalformats that can be used in SPR ex-periments these can be roughlycategorized into three types of mea-surements scanning angle SPR11ndash14

scanning wavelength SPR15 andSPR imaging916ndash22 For all SPR for-mats the re ectivity of light incidenton a metald ielectric in terface ismonitored and correlated to changesin the local index of refraction of thedielectric layer adjacent to the metal lm The most widely used formatfor an SPR experiment is the scan-ning angle technique in which there ectivity of monochromatic inci-dent light upon a metal lm is mon-itored as a function of the incidentangle The popularity of the scan-ning angle technique can be partiallyattributed to the existence of com-mercially available instrumentationfrom Biacore23 which has made itpossible to use SPR as a detectionmethod for many applications in-cluding basic life science researchdrug discovery environmental mon-itoring and process analysis Boththe scanning angle and scanningwavelength measurements typicallyprovide only one or a few data pointsat a time In contrast SPR imagingmeasurements sometimes calledSPR microscopy use the changes inre ectivity from a gold thin lm thatoccur upon adsorption to generatedifference images to simultaneouslymonitor tens hundreds or more in-teractions in a parallel manner

The high-throughput capabilitiesof SPR imaging have made it an at-tractive tool for screening biomolec-ular interactions For example SPRimaging has been used in an arrayformat to study the hybridization ofDNA and RNA to nucleic acid ar-rays fabricated on gold lms9202425

Shown in Fig 1A is an example ofan SPR difference image of a two-component DNA array The differ-ence image was obtained by sub-tracting the images taken before andafter exposing the array to a 16-merthat was complementary to the im-

mobilized sequence 1 which waspatterned on the surface as shown inFig 1B A change in the re ectedlight intensity was observed onlywhere sequence 1 was immobilizedon the array demonstrating the spe-ci c hybridization of the target to thecomplementary immobilized se-quence Shown in Fig 1C is a plotof a line pro le that was acquiredfrom the SPR image (indicated bythe black line in Fig 1A) The plotshows that the hybridization of the16-mer complementary sequencecorresponds to less than a one per-cent change in the re ected light in-tensity and that there is no change inthe re ected light intensity for thenon-complementary sequence DNAarrays have also been used in con-junction with SPR imaging to mon-itor RNA hybridization9 for DNAword design in computational algo-rithms26 for single base mismatchdetection (discussed below) and tomonitor hairpin formation in DNAmonolayers27

The next section of this article willprovide a brief background to theSPR imaging technique including anin troduction to surface plasmonsFollowing this background sectionthe remainder of the article willhighlight SPR imaging instrumenta-tion array fabrication techniquesand the ability to obtain quantitativedata with SPR imaging A few ex-amples of the use of SPR imagingfor the study of biomolecular inter-actions will also be presented

SURFACE PLASMONRESONANCE THEORY

Surface plasmons (SPs) are oscil-lations of free electrons that propa-gate along the surface of a metalwhen it is in contact with a dielectricinterface Surface plasmons can havea range of energies that depend onthe complex dielectric function ofthe metal (laquom) and the dielectricfunction of the adjacent medium(laquod) as shown by the followingequation

v laquo laquom dk 5 (1)sp c laquo 1 laquom d

where ksp is the wave vector of theSP vc is the wave vector in a vac-uum and the dielectric constant isthe square of the index of refrac-tion28 One condition for the gener-ation of SPs is that laquom and laquod are ofopposite sign and thus SPs will notbe generated for all systems In ad-dition laquom and laquod are wavelength de-pendent and certain regions of theelectromagnetic spectrum may be re-quired to generate SPs One commonsystem used in SPR experiments is agold lm in contact with a water in-terface Gold has a negative dielec-tric function in the IR and visible re-gions of the electromagnetic spec-trum whereas water has a positivedielectric function29

Surface plasmons can be directlyexcited by electrons however theycan not be excited directly by lightbecause they have a longer wavevector than light waves of the sameenergy (klight 5 vc) The wave vectorof a photon must be increased toconvert the photon into SPs Thiscan be accomplished with the use ofeither a prism or a grating coupler28

For example SPs can be excited un-der conditions of attenuated total re- ection (ATR) using a prismmetal lmdielectric layer (designated theKretschmann con guration)30 and p-polarized incident light that has itselectric component in the plane ofincidence Shown in Fig 2 is a sche-matic diagram of a Kretschmanncon guration sample setup that hasbeen used for the study of biomolec-ular interactions with SPR imagingThe setup consists of a high-index-of-refraction glass prism a 45 nmAu lm (with a 1 nm Cr underlayer)a 2 nm self-assembled monolayer a5 nm sensing layer where adsorptionof the target molecule occurs and abulk water layer

The equation for the surface com-ponent of a photonrsquos wave vector un-der conditions of ATR becomes

vk 5 sin uIumllaquo (2)ATR pc

where laquop is the dielectric constant ofthe prism and u is the angle of in-cidence of the light on the metal lm28 SPs will be generated in the

322A Volume 57 Number 11 2003

focal point

Fig 1 (A) SPR difference image of a two-component DNA array showing the hybridization of a sequence complementary to im-mobilized sequence 1 (B) schematic diagram showing the pattern of the immobilized DNA sequences for the SPR image shown inA and (C) line prole plotted as the change in percent reectivity obtained from the area indicated by the black line in the SPRimage

metal lm at angles where the pho-tonrsquos wave vector equals the SPrsquoswave vector The excitation of SPscorresponds to an attenuation of there ected light intensity as the incom-ing light generates SPs The anglewhere there is a complete attenuationof the re ected light corresponds tothe complete conversion of the in-coming light and is referred to as thesurface plasmon angle

Fresnel equations can be used topredict the re ectivity and phaseshift when incident light impinges onan interface containing two or morephases31 These calculations assumethat each phase is homogenous andparallel and they require knowledgeof the complex index of refraction ofeach of the phases which are depen-dent on the wavelength of the inci-dent light The re ection coef cient

for p-polarized incident light is givenby the formula

rEr 5 (3)p tE

where E r is the electric eld com-ponent in the plane of incidence ofthe light re ected from the interfaceand E t is the electric eld componentin the plane of incidence of the lighttransmitted through the inter faceThe light re ected from the interfacecan be calculated using the formula

Rp 5 zrpz2 (4)

For multiphase systems these cal-culations require matrix operationsComputer programs have been de-veloped to calculate the re ectedlight intensity for multiphase sys-tems where one or more of the phas-es are a material with a complex in-

dex of refraction (ie a metal lm)23 Shown in Fig 3 are the re-sults of 5-phase Fresnel calculationsused to simulate scanning angle SPRre ectivity curves from the samplesetup depicted in Fig 2 The re ec-tivity of light is plotted versus theangle of incidence for two lms withsensing layers that have an index ofrefraction (nf) of 140 or 146 An in-crease in the index of refraction ofthe sensing layer simulates adsorp-tion onto the metal lm Conditionsof ATR are met at angles greaterthan the critical angle which occursnear 506 degrees As mentionedpreviously the surface plasmon an-gle corresponds to the angle wherethere is a near complete attenuationof the re ected light The position ofthe surface plasmon angle is depen-dent on the index of refraction of the

APPLIED SPECTROSCOPY 323A

Fig 2 Diagram showing a sample setup used to monitor biomolecular interactions with SPR imaging The 5 phases are a high-index-of-refraction glass prism (n1 5 1712) a 45-nm-thick gold lm (n2 5 01451 1 48725) a 2 nm self-assembled monolayer(n3 5 145) a 5 nm sensing layer where adsorption occurs (nf ) and a bulk water layer (n5) Adsorption of biomolecules to thesensing layer increases the value of nf

sensing layer An expanded view ofthe region encompassing the surfaceplasmon angles for both lms isshown in Fig 3 (right inset)

Surface plasmons have a maxi-mum intensity in the metal lm andthey decay exponentially in a per-pendicular direction from the surfacein both the metal and the dielectriclayer28 The decay length of the SPsis dependent on the wavelength ofthe incident light and on the dielec-tric constants of both layers For agold lm a typical decay length ison the order of a few hundred nano-meters into the dielectric layer for

excitation with visible light Thismeans that SPR is a surface sensitivetechnique and that measurements canbe made even when a large excess ofanalyte is present in solution Anyspecies that is farther from the metal lm than the SP decay length willnot effect the generation of SPs

The propagation length of SPs isthe distance where its electric eldintensity in the metal lm drops to avalue of 1e and is determined by laquomlaquod and the wavelength of the inci-dent light 28 The SP propagationlength determines the lateral resolu-tion in SPR imaging In order to re-

solve two features they must be sep-arated by a minimum distance cor-responding to the propagation lengthof the SP Longer SP propagationlengths correlate to a lower lateralresolution and higher lateral resolu-tion can be achieved using shorterwavelength light

The sensitivity in SPR imagingthe ability to detect small changes inn f is also affected by the wavelengthof the incident light Incident light oflonger wavelengths produces sharperSPR curves (with narrow widths at50 re ectivity) and incident lightof shorter wavelengths produces

324A Volume 57 Number 11 2003

focal point

Fig 3 Graph showing the scanning angle SPR reectivity curves that were obtained from 5-phase Fresnel calculations for thesystem shown in Fig 2 The index of refraction of the sensing layer (nf ) was 140 (solid line) or 146 (dotted line) The gure insetat right shows an expanded view of the region near the plasmon angles The location of the plasmon angle shifts to higher anglesfor the nf 5 146 lm relative to the nf 5 140 lm The gure inset at left shows an expanded view around the optimal angle forperforming SPR imaging experiments with this system At the optimal angle the largest shift in R is observed for these two lms

broad SPR curves (with large widthsat 50 re ectivity)32 Sharper SPRcurves produced by longer wave-length incident light yield largerchanges in re ectivity fo r givenchanges in nf than do broad curvesproduced by incident light withshorter wavelengths This means thatthere is a trade-off between lateralresolution and sensitivity The use oflonger wavelength light provideshigher sensitivity but lower lateralresolution The use of shorter wave-length light provides a higher lateralresolution but lower sensitivity3334

SURFACE PLASMONRESONANCE IMAGING

Surface Plasmon Resonance Im-aging Instrumentation Surface

plasmon resonance imaging is a xed angle experiment where thespatial changes in re ected light aremeasured across a substrate The the-oretical curves generated for a scan-ning angle SPR experiment can beused to understand the basis of thecontrast observed in an imaging ex-periment The left inset in Fig 3shows an expanded region around anincident angle of 535 degrees Aslice through the X-axis simulates aconstant angle experiment At 535degrees less light is re ected fromthe n f 5 140 lm than the nf 5 146 lm If a surface were patterned tocontain regions w ith both lmsmore light would be re ected fromthe regions with n f 5 146 than re-

gions containing the n f 5 140 lmat an angle of 535 degrees and itwould be possible to distinguish be-tween the two lms in an SPR im-age

The basic components in a typicalSPR imaging instrument are shownin Fig 4 These are a collimatedwhite light source a polarizer thesample stage collection optics anda charge-coupled device (CCD) con-nected to a CPU for image collectionand processing The use of a colli-mated white light source is preferredover the use of laser excitation dueto interference fringes that result inthe SPR image when laser excitationis utilized The polarizer is used toselect p-polarized light and the col-

APPLIED SPECTROSCOPY 325A

lection optics consist of a narrowband pass lter typically centered inthe near-infrared region that is usedto select the excitation wavelengthfor the experiment The sample is lo-cated on a rotation stage in order tocontrol the incident angle of lightand consists of a prism a substrateonto which a Au lm is depositedand a ow cell

Attachment Chemistry and Ar-ray Fabrication Surface plasmonresonance imaging is performed ona noble metal lm therefore strate-gies for attaching probe molecules tothese lms are critical to the successof an SPR imaging experiment Nu-merous immobilization strategies ex-ist These can generally be catego-rized into three routes (1) a thiol-modi ed probe molecule can be re-acted directly with a gold lm toform a goldndashthiolate bond35 (2) apolymer layer such as dextran orpolylysine can be rst formed on thegold surface and the probe moleculecan be immobilized onto the poly-mer layer36 and (3) a self-assembledalkanethiol monolayer (SAM) con-tain ing a v-terminated functionalgroup can be formed on the gold lm which is used to immobilize amolecule (a lsquolsquolinkerrsquorsquo) that is capableof reacting with the probe mole-cule37ndash41

There are drawbacks to the rstand second immobilization schemesMany biomolecules will non-specif-ically adsorb to a gold lm and alarge portion of the probes may notbe biologically active if they are di-rectly attached to the gold lm Sur-faces fabricated using the second im-mobilization strategy may not be ro-bust and polymer layers can be aproblem when studying kinetics ofadsorption

Several characteristics of the thirdimmobilization scheme make it suit-able for use with SPR imaging Gen-erally the surfaces that result fromthis immobilization strategy are sta-ble and can be used for several assaycycles Using this immobilizationstrategy provides a way to controlthe surface density of the probe mol-ecule This is important for two rea-sons (1) the surface density of the

probe can affect the amount of targetthat binds to the surface and there-fore the amount of signal that is de-tected and (2) varying the probedensity can be used as a tool to studythe interactions of targets that bindto the probes through multivalent in-teractions (an example of this is theuse of SPR imaging to study carbo-hydratendashprotein interactions) Final-ly this strategy provides a way tocontrol the resulting surface proper-ties (ie hydrophilic hydrophobiccharged)42ndash47 This is important sinceeverything that adsorbs to the goldsurface will produce an SPR signalit is necessary to control the surfaceproperties so that only the desiredtarget molecules interact with thesurface

In addition to immobilizing probemolecules to a gold lm the lmmust be patterned so that severalprobes are immobilized at discretelocations on the substrate There areseveral methods that have been usedto pattern gold lms UV photopat-terning 17 4 1 polydimethylsiloxane(PDMS) microchannels48 microcon-tact printing49 and robotic spotting50

have been used in conjunction withSPR imaging The array shown inFig 1 was fabricated using the UVphotopatterning method in which aquartz mask is used to selectively ex-pose an alkanethiol-modi ed goldsurface to UV light At locationswhere the UV light shines on thesurface the alkanethiol is removedgenerating bare gold patches thatserve as a platform for generatingthe array elements Both the UVphotopatterning and PDMS micro-channel array fabrication strategieswill be discussed later in this articlein conjunction with speci c exam-ples of the use of SPR imaging tostudy biomolecular interactions

Quantitation of Results Gener-ated with Surface Plasmon Reso-nance Imaging In addition to theability to detect interacting partnerswith SPR imaging it is desirable toobtain quantitative information aboutthese interactions Quantitative datacan be obtained from SPR measure-ments by assuming that moleculesadsorbing to or desorbing from the

metal lm correlate to changes in theindex of refraction of the dielectriclayer and that changes in the indexof refraction correlate to changes inthe re ectivity of the incident lightIn order to obtain quantitative datawith SPR imaging it is necessary toknow over what regions there is alinear relationship between thechange in the re ected light intensity(DR) and the change in the indexof refraction of the sensing layer(Dnf) A series of 5-phase Fresnelcalculations of the system shown inFig 2 can be used to determine this9

The rst calculation is performedwith an n f value of 14 Each succes-sive calculation increments the n f

value by 0002 index of refractionunits Figure 5 shows the resultsfrom these calculations plotted as theabsolute value of DR for the indi-cated Dn f These calculations wereperformed for an excitation wave-length of 794 nm and the results atthree angles along the SPR curve areshown (the location of the anglesalong the SPR curve are shown inthe gure inset) The dotted lines inFig 5 show a linear relationship be-tween DR and Dnf At an angle of5352 degrees the greatest contrastis predicted as determined by themagnitude of DR The smallest de-viation from linearity is also ob-served at 5352 degrees A smallchange in the incident angle of 004degrees does not signi cantly affectthe overall signal or the amount ofdeviation that occurs This is not thecase for angles greater than 01 de-grees from the optimal angle of5352 degrees (not shown in Fig 5)At angles further than 01 degreesfrom the optimal angle there is asmaller change in the percent of re- ected light and these signals devi-ate from a linear relationship at largen f values While the data is fairly lin-ear for an angle higher than the sur-face plasmon angle (5412) theoverall contrast that would be ob-served is much smaller than for an-gles to the left of the surface plas-mon angle This is a result of theSPR curve not being symmetricabout the surface plasmon angle (seeFig 3)

326A Volume 57 Number 11 2003

focal point

Fig 4 (A) A schematic diagram of an SPR imager Collimated white light is passed through a polarizer and is incident on thesample assembly Reected light passes through focusing optics a narrow band pass lter typically centered at a wavelength in thenear-infrared and is captured by a CCD camera (B) A schematic diagram of the sample assembly consisting of a glass prism thatis optically coupled to a glass substrate containing a thin layer (45 nm) of gold The sample is contained within a ow cell for in situmeasurements

The deviations from linear behav-ior for DR and Dn f are small pro-vided that the experiment is per-formed at the optimal angle If de-viations are present they can be pre-dicted and accounted fo r in theexperimental results As mentionedpreviously the percent change in re- ected light intensity due to the hy-bridization of a monolayer of 16-meroligonucleotides is less than 1 per-cent which falls within the regionwhere linear data is obtained withSPR imaging9 An example of theuse of SPR imaging to quantitate theamount of material adsorbing to agold lm is described in the Exam-ples section In this example theamount of protein adsorbing to ametal lm is measured with SPR im-aging and is used to construct ad-sorption isotherms for the interactionof proteins with immobilized carbo-hydrates

EXAMPLESDetection of DNA Hybridiza-

tion Single-Base Mismatch Detec-

tion in the Presence of Small Mol-ecules A recent example of the useof SPR imaging demonstrated thatthis technique can be used to moni-tor the hybridization of short oligo-nucleotides in the presence of smallmolecules that alter the oligonucle-otidersquos binding properties27 A sche-matic diagram of a small moleculenaphthyridine dimer that has beenshown to stabilize the binding of GndashG mismatches in double strandedDNA is shown in Fig 651ndash53 To dem-onstrate the GndashG mismatch stabiliz-ing properties of this molecule aDNA array was fabricated using UVphotopattern ing This involves acombination of self-assembly andUV photopatterning steps A baregold lm is modi ed with a self-as-sembled monolayer of an amine-ter-minated alkanethiol The amine-ter-minated surface is then reacted witha hydrophobic protecting group thatcan be reversibly removed from thesurface A quartz mask containingpatterned features is placed over the

modi ed gold lm and the surface isexposed to UV light resulting in thephotooxidation of the goldndashthiolatebond and generation of bare gold re-gions where the UV light is exposedto the surface The substrate is thenreplaced in the amine-terminated al-kanethiol solution and a monolayeris formed at areas where there is baregold The resulting surface containshydrophilic amine-terminated mono-layer regions surrounded by a hydro-phobic monolayer The probe mole-cules can be immobilized through aseries of reactions that are carriedout within the hydrophilic wells bydelivering small solution volumes tothe surface (ie spotting solutionsonto the surface with a pulled cap-illary) The hydrophobic backgroundprevents cross-contamination be-tween the array elements during theimmobilization reaction Once theprobe compounds are immobilizedon the surface the hydrophobic pro-tecting group is removed from thebackground This generates an

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

322A Volume 57 Number 11 2003

focal point

Fig 1 (A) SPR difference image of a two-component DNA array showing the hybridization of a sequence complementary to im-mobilized sequence 1 (B) schematic diagram showing the pattern of the immobilized DNA sequences for the SPR image shown inA and (C) line prole plotted as the change in percent reectivity obtained from the area indicated by the black line in the SPRimage

metal lm at angles where the pho-tonrsquos wave vector equals the SPrsquoswave vector The excitation of SPscorresponds to an attenuation of there ected light intensity as the incom-ing light generates SPs The anglewhere there is a complete attenuationof the re ected light corresponds tothe complete conversion of the in-coming light and is referred to as thesurface plasmon angle

Fresnel equations can be used topredict the re ectivity and phaseshift when incident light impinges onan interface containing two or morephases31 These calculations assumethat each phase is homogenous andparallel and they require knowledgeof the complex index of refraction ofeach of the phases which are depen-dent on the wavelength of the inci-dent light The re ection coef cient

for p-polarized incident light is givenby the formula

rEr 5 (3)p tE

where E r is the electric eld com-ponent in the plane of incidence ofthe light re ected from the interfaceand E t is the electric eld componentin the plane of incidence of the lighttransmitted through the inter faceThe light re ected from the interfacecan be calculated using the formula

Rp 5 zrpz2 (4)

For multiphase systems these cal-culations require matrix operationsComputer programs have been de-veloped to calculate the re ectedlight intensity for multiphase sys-tems where one or more of the phas-es are a material with a complex in-

dex of refraction (ie a metal lm)23 Shown in Fig 3 are the re-sults of 5-phase Fresnel calculationsused to simulate scanning angle SPRre ectivity curves from the samplesetup depicted in Fig 2 The re ec-tivity of light is plotted versus theangle of incidence for two lms withsensing layers that have an index ofrefraction (nf) of 140 or 146 An in-crease in the index of refraction ofthe sensing layer simulates adsorp-tion onto the metal lm Conditionsof ATR are met at angles greaterthan the critical angle which occursnear 506 degrees As mentionedpreviously the surface plasmon an-gle corresponds to the angle wherethere is a near complete attenuationof the re ected light The position ofthe surface plasmon angle is depen-dent on the index of refraction of the

APPLIED SPECTROSCOPY 323A

Fig 2 Diagram showing a sample setup used to monitor biomolecular interactions with SPR imaging The 5 phases are a high-index-of-refraction glass prism (n1 5 1712) a 45-nm-thick gold lm (n2 5 01451 1 48725) a 2 nm self-assembled monolayer(n3 5 145) a 5 nm sensing layer where adsorption occurs (nf ) and a bulk water layer (n5) Adsorption of biomolecules to thesensing layer increases the value of nf

sensing layer An expanded view ofthe region encompassing the surfaceplasmon angles for both lms isshown in Fig 3 (right inset)

Surface plasmons have a maxi-mum intensity in the metal lm andthey decay exponentially in a per-pendicular direction from the surfacein both the metal and the dielectriclayer28 The decay length of the SPsis dependent on the wavelength ofthe incident light and on the dielec-tric constants of both layers For agold lm a typical decay length ison the order of a few hundred nano-meters into the dielectric layer for

excitation with visible light Thismeans that SPR is a surface sensitivetechnique and that measurements canbe made even when a large excess ofanalyte is present in solution Anyspecies that is farther from the metal lm than the SP decay length willnot effect the generation of SPs

The propagation length of SPs isthe distance where its electric eldintensity in the metal lm drops to avalue of 1e and is determined by laquomlaquod and the wavelength of the inci-dent light 28 The SP propagationlength determines the lateral resolu-tion in SPR imaging In order to re-

solve two features they must be sep-arated by a minimum distance cor-responding to the propagation lengthof the SP Longer SP propagationlengths correlate to a lower lateralresolution and higher lateral resolu-tion can be achieved using shorterwavelength light

The sensitivity in SPR imagingthe ability to detect small changes inn f is also affected by the wavelengthof the incident light Incident light oflonger wavelengths produces sharperSPR curves (with narrow widths at50 re ectivity) and incident lightof shorter wavelengths produces

324A Volume 57 Number 11 2003

focal point

Fig 3 Graph showing the scanning angle SPR reectivity curves that were obtained from 5-phase Fresnel calculations for thesystem shown in Fig 2 The index of refraction of the sensing layer (nf ) was 140 (solid line) or 146 (dotted line) The gure insetat right shows an expanded view of the region near the plasmon angles The location of the plasmon angle shifts to higher anglesfor the nf 5 146 lm relative to the nf 5 140 lm The gure inset at left shows an expanded view around the optimal angle forperforming SPR imaging experiments with this system At the optimal angle the largest shift in R is observed for these two lms

broad SPR curves (with large widthsat 50 re ectivity)32 Sharper SPRcurves produced by longer wave-length incident light yield largerchanges in re ectivity fo r givenchanges in nf than do broad curvesproduced by incident light withshorter wavelengths This means thatthere is a trade-off between lateralresolution and sensitivity The use oflonger wavelength light provideshigher sensitivity but lower lateralresolution The use of shorter wave-length light provides a higher lateralresolution but lower sensitivity3334

SURFACE PLASMONRESONANCE IMAGING

Surface Plasmon Resonance Im-aging Instrumentation Surface

plasmon resonance imaging is a xed angle experiment where thespatial changes in re ected light aremeasured across a substrate The the-oretical curves generated for a scan-ning angle SPR experiment can beused to understand the basis of thecontrast observed in an imaging ex-periment The left inset in Fig 3shows an expanded region around anincident angle of 535 degrees Aslice through the X-axis simulates aconstant angle experiment At 535degrees less light is re ected fromthe n f 5 140 lm than the nf 5 146 lm If a surface were patterned tocontain regions w ith both lmsmore light would be re ected fromthe regions with n f 5 146 than re-

gions containing the n f 5 140 lmat an angle of 535 degrees and itwould be possible to distinguish be-tween the two lms in an SPR im-age

The basic components in a typicalSPR imaging instrument are shownin Fig 4 These are a collimatedwhite light source a polarizer thesample stage collection optics anda charge-coupled device (CCD) con-nected to a CPU for image collectionand processing The use of a colli-mated white light source is preferredover the use of laser excitation dueto interference fringes that result inthe SPR image when laser excitationis utilized The polarizer is used toselect p-polarized light and the col-

APPLIED SPECTROSCOPY 325A

lection optics consist of a narrowband pass lter typically centered inthe near-infrared region that is usedto select the excitation wavelengthfor the experiment The sample is lo-cated on a rotation stage in order tocontrol the incident angle of lightand consists of a prism a substrateonto which a Au lm is depositedand a ow cell

Attachment Chemistry and Ar-ray Fabrication Surface plasmonresonance imaging is performed ona noble metal lm therefore strate-gies for attaching probe molecules tothese lms are critical to the successof an SPR imaging experiment Nu-merous immobilization strategies ex-ist These can generally be catego-rized into three routes (1) a thiol-modi ed probe molecule can be re-acted directly with a gold lm toform a goldndashthiolate bond35 (2) apolymer layer such as dextran orpolylysine can be rst formed on thegold surface and the probe moleculecan be immobilized onto the poly-mer layer36 and (3) a self-assembledalkanethiol monolayer (SAM) con-tain ing a v-terminated functionalgroup can be formed on the gold lm which is used to immobilize amolecule (a lsquolsquolinkerrsquorsquo) that is capableof reacting with the probe mole-cule37ndash41

There are drawbacks to the rstand second immobilization schemesMany biomolecules will non-specif-ically adsorb to a gold lm and alarge portion of the probes may notbe biologically active if they are di-rectly attached to the gold lm Sur-faces fabricated using the second im-mobilization strategy may not be ro-bust and polymer layers can be aproblem when studying kinetics ofadsorption

Several characteristics of the thirdimmobilization scheme make it suit-able for use with SPR imaging Gen-erally the surfaces that result fromthis immobilization strategy are sta-ble and can be used for several assaycycles Using this immobilizationstrategy provides a way to controlthe surface density of the probe mol-ecule This is important for two rea-sons (1) the surface density of the

probe can affect the amount of targetthat binds to the surface and there-fore the amount of signal that is de-tected and (2) varying the probedensity can be used as a tool to studythe interactions of targets that bindto the probes through multivalent in-teractions (an example of this is theuse of SPR imaging to study carbo-hydratendashprotein interactions) Final-ly this strategy provides a way tocontrol the resulting surface proper-ties (ie hydrophilic hydrophobiccharged)42ndash47 This is important sinceeverything that adsorbs to the goldsurface will produce an SPR signalit is necessary to control the surfaceproperties so that only the desiredtarget molecules interact with thesurface

In addition to immobilizing probemolecules to a gold lm the lmmust be patterned so that severalprobes are immobilized at discretelocations on the substrate There areseveral methods that have been usedto pattern gold lms UV photopat-terning 17 4 1 polydimethylsiloxane(PDMS) microchannels48 microcon-tact printing49 and robotic spotting50

have been used in conjunction withSPR imaging The array shown inFig 1 was fabricated using the UVphotopatterning method in which aquartz mask is used to selectively ex-pose an alkanethiol-modi ed goldsurface to UV light At locationswhere the UV light shines on thesurface the alkanethiol is removedgenerating bare gold patches thatserve as a platform for generatingthe array elements Both the UVphotopatterning and PDMS micro-channel array fabrication strategieswill be discussed later in this articlein conjunction with speci c exam-ples of the use of SPR imaging tostudy biomolecular interactions

Quantitation of Results Gener-ated with Surface Plasmon Reso-nance Imaging In addition to theability to detect interacting partnerswith SPR imaging it is desirable toobtain quantitative information aboutthese interactions Quantitative datacan be obtained from SPR measure-ments by assuming that moleculesadsorbing to or desorbing from the

metal lm correlate to changes in theindex of refraction of the dielectriclayer and that changes in the indexof refraction correlate to changes inthe re ectivity of the incident lightIn order to obtain quantitative datawith SPR imaging it is necessary toknow over what regions there is alinear relationship between thechange in the re ected light intensity(DR) and the change in the indexof refraction of the sensing layer(Dnf) A series of 5-phase Fresnelcalculations of the system shown inFig 2 can be used to determine this9

The rst calculation is performedwith an n f value of 14 Each succes-sive calculation increments the n f

value by 0002 index of refractionunits Figure 5 shows the resultsfrom these calculations plotted as theabsolute value of DR for the indi-cated Dn f These calculations wereperformed for an excitation wave-length of 794 nm and the results atthree angles along the SPR curve areshown (the location of the anglesalong the SPR curve are shown inthe gure inset) The dotted lines inFig 5 show a linear relationship be-tween DR and Dnf At an angle of5352 degrees the greatest contrastis predicted as determined by themagnitude of DR The smallest de-viation from linearity is also ob-served at 5352 degrees A smallchange in the incident angle of 004degrees does not signi cantly affectthe overall signal or the amount ofdeviation that occurs This is not thecase for angles greater than 01 de-grees from the optimal angle of5352 degrees (not shown in Fig 5)At angles further than 01 degreesfrom the optimal angle there is asmaller change in the percent of re- ected light and these signals devi-ate from a linear relationship at largen f values While the data is fairly lin-ear for an angle higher than the sur-face plasmon angle (5412) theoverall contrast that would be ob-served is much smaller than for an-gles to the left of the surface plas-mon angle This is a result of theSPR curve not being symmetricabout the surface plasmon angle (seeFig 3)

326A Volume 57 Number 11 2003

focal point

Fig 4 (A) A schematic diagram of an SPR imager Collimated white light is passed through a polarizer and is incident on thesample assembly Reected light passes through focusing optics a narrow band pass lter typically centered at a wavelength in thenear-infrared and is captured by a CCD camera (B) A schematic diagram of the sample assembly consisting of a glass prism thatis optically coupled to a glass substrate containing a thin layer (45 nm) of gold The sample is contained within a ow cell for in situmeasurements

The deviations from linear behav-ior for DR and Dn f are small pro-vided that the experiment is per-formed at the optimal angle If de-viations are present they can be pre-dicted and accounted fo r in theexperimental results As mentionedpreviously the percent change in re- ected light intensity due to the hy-bridization of a monolayer of 16-meroligonucleotides is less than 1 per-cent which falls within the regionwhere linear data is obtained withSPR imaging9 An example of theuse of SPR imaging to quantitate theamount of material adsorbing to agold lm is described in the Exam-ples section In this example theamount of protein adsorbing to ametal lm is measured with SPR im-aging and is used to construct ad-sorption isotherms for the interactionof proteins with immobilized carbo-hydrates

EXAMPLESDetection of DNA Hybridiza-

tion Single-Base Mismatch Detec-

tion in the Presence of Small Mol-ecules A recent example of the useof SPR imaging demonstrated thatthis technique can be used to moni-tor the hybridization of short oligo-nucleotides in the presence of smallmolecules that alter the oligonucle-otidersquos binding properties27 A sche-matic diagram of a small moleculenaphthyridine dimer that has beenshown to stabilize the binding of GndashG mismatches in double strandedDNA is shown in Fig 651ndash53 To dem-onstrate the GndashG mismatch stabiliz-ing properties of this molecule aDNA array was fabricated using UVphotopattern ing This involves acombination of self-assembly andUV photopatterning steps A baregold lm is modi ed with a self-as-sembled monolayer of an amine-ter-minated alkanethiol The amine-ter-minated surface is then reacted witha hydrophobic protecting group thatcan be reversibly removed from thesurface A quartz mask containingpatterned features is placed over the

modi ed gold lm and the surface isexposed to UV light resulting in thephotooxidation of the goldndashthiolatebond and generation of bare gold re-gions where the UV light is exposedto the surface The substrate is thenreplaced in the amine-terminated al-kanethiol solution and a monolayeris formed at areas where there is baregold The resulting surface containshydrophilic amine-terminated mono-layer regions surrounded by a hydro-phobic monolayer The probe mole-cules can be immobilized through aseries of reactions that are carriedout within the hydrophilic wells bydelivering small solution volumes tothe surface (ie spotting solutionsonto the surface with a pulled cap-illary) The hydrophobic backgroundprevents cross-contamination be-tween the array elements during theimmobilization reaction Once theprobe compounds are immobilizedon the surface the hydrophobic pro-tecting group is removed from thebackground This generates an

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

APPLIED SPECTROSCOPY 323A

Fig 2 Diagram showing a sample setup used to monitor biomolecular interactions with SPR imaging The 5 phases are a high-index-of-refraction glass prism (n1 5 1712) a 45-nm-thick gold lm (n2 5 01451 1 48725) a 2 nm self-assembled monolayer(n3 5 145) a 5 nm sensing layer where adsorption occurs (nf ) and a bulk water layer (n5) Adsorption of biomolecules to thesensing layer increases the value of nf

sensing layer An expanded view ofthe region encompassing the surfaceplasmon angles for both lms isshown in Fig 3 (right inset)

Surface plasmons have a maxi-mum intensity in the metal lm andthey decay exponentially in a per-pendicular direction from the surfacein both the metal and the dielectriclayer28 The decay length of the SPsis dependent on the wavelength ofthe incident light and on the dielec-tric constants of both layers For agold lm a typical decay length ison the order of a few hundred nano-meters into the dielectric layer for

excitation with visible light Thismeans that SPR is a surface sensitivetechnique and that measurements canbe made even when a large excess ofanalyte is present in solution Anyspecies that is farther from the metal lm than the SP decay length willnot effect the generation of SPs

The propagation length of SPs isthe distance where its electric eldintensity in the metal lm drops to avalue of 1e and is determined by laquomlaquod and the wavelength of the inci-dent light 28 The SP propagationlength determines the lateral resolu-tion in SPR imaging In order to re-

solve two features they must be sep-arated by a minimum distance cor-responding to the propagation lengthof the SP Longer SP propagationlengths correlate to a lower lateralresolution and higher lateral resolu-tion can be achieved using shorterwavelength light

The sensitivity in SPR imagingthe ability to detect small changes inn f is also affected by the wavelengthof the incident light Incident light oflonger wavelengths produces sharperSPR curves (with narrow widths at50 re ectivity) and incident lightof shorter wavelengths produces

324A Volume 57 Number 11 2003

focal point

Fig 3 Graph showing the scanning angle SPR reectivity curves that were obtained from 5-phase Fresnel calculations for thesystem shown in Fig 2 The index of refraction of the sensing layer (nf ) was 140 (solid line) or 146 (dotted line) The gure insetat right shows an expanded view of the region near the plasmon angles The location of the plasmon angle shifts to higher anglesfor the nf 5 146 lm relative to the nf 5 140 lm The gure inset at left shows an expanded view around the optimal angle forperforming SPR imaging experiments with this system At the optimal angle the largest shift in R is observed for these two lms

broad SPR curves (with large widthsat 50 re ectivity)32 Sharper SPRcurves produced by longer wave-length incident light yield largerchanges in re ectivity fo r givenchanges in nf than do broad curvesproduced by incident light withshorter wavelengths This means thatthere is a trade-off between lateralresolution and sensitivity The use oflonger wavelength light provideshigher sensitivity but lower lateralresolution The use of shorter wave-length light provides a higher lateralresolution but lower sensitivity3334

SURFACE PLASMONRESONANCE IMAGING

Surface Plasmon Resonance Im-aging Instrumentation Surface

plasmon resonance imaging is a xed angle experiment where thespatial changes in re ected light aremeasured across a substrate The the-oretical curves generated for a scan-ning angle SPR experiment can beused to understand the basis of thecontrast observed in an imaging ex-periment The left inset in Fig 3shows an expanded region around anincident angle of 535 degrees Aslice through the X-axis simulates aconstant angle experiment At 535degrees less light is re ected fromthe n f 5 140 lm than the nf 5 146 lm If a surface were patterned tocontain regions w ith both lmsmore light would be re ected fromthe regions with n f 5 146 than re-

gions containing the n f 5 140 lmat an angle of 535 degrees and itwould be possible to distinguish be-tween the two lms in an SPR im-age

The basic components in a typicalSPR imaging instrument are shownin Fig 4 These are a collimatedwhite light source a polarizer thesample stage collection optics anda charge-coupled device (CCD) con-nected to a CPU for image collectionand processing The use of a colli-mated white light source is preferredover the use of laser excitation dueto interference fringes that result inthe SPR image when laser excitationis utilized The polarizer is used toselect p-polarized light and the col-

APPLIED SPECTROSCOPY 325A

lection optics consist of a narrowband pass lter typically centered inthe near-infrared region that is usedto select the excitation wavelengthfor the experiment The sample is lo-cated on a rotation stage in order tocontrol the incident angle of lightand consists of a prism a substrateonto which a Au lm is depositedand a ow cell

Attachment Chemistry and Ar-ray Fabrication Surface plasmonresonance imaging is performed ona noble metal lm therefore strate-gies for attaching probe molecules tothese lms are critical to the successof an SPR imaging experiment Nu-merous immobilization strategies ex-ist These can generally be catego-rized into three routes (1) a thiol-modi ed probe molecule can be re-acted directly with a gold lm toform a goldndashthiolate bond35 (2) apolymer layer such as dextran orpolylysine can be rst formed on thegold surface and the probe moleculecan be immobilized onto the poly-mer layer36 and (3) a self-assembledalkanethiol monolayer (SAM) con-tain ing a v-terminated functionalgroup can be formed on the gold lm which is used to immobilize amolecule (a lsquolsquolinkerrsquorsquo) that is capableof reacting with the probe mole-cule37ndash41

There are drawbacks to the rstand second immobilization schemesMany biomolecules will non-specif-ically adsorb to a gold lm and alarge portion of the probes may notbe biologically active if they are di-rectly attached to the gold lm Sur-faces fabricated using the second im-mobilization strategy may not be ro-bust and polymer layers can be aproblem when studying kinetics ofadsorption

Several characteristics of the thirdimmobilization scheme make it suit-able for use with SPR imaging Gen-erally the surfaces that result fromthis immobilization strategy are sta-ble and can be used for several assaycycles Using this immobilizationstrategy provides a way to controlthe surface density of the probe mol-ecule This is important for two rea-sons (1) the surface density of the

probe can affect the amount of targetthat binds to the surface and there-fore the amount of signal that is de-tected and (2) varying the probedensity can be used as a tool to studythe interactions of targets that bindto the probes through multivalent in-teractions (an example of this is theuse of SPR imaging to study carbo-hydratendashprotein interactions) Final-ly this strategy provides a way tocontrol the resulting surface proper-ties (ie hydrophilic hydrophobiccharged)42ndash47 This is important sinceeverything that adsorbs to the goldsurface will produce an SPR signalit is necessary to control the surfaceproperties so that only the desiredtarget molecules interact with thesurface

In addition to immobilizing probemolecules to a gold lm the lmmust be patterned so that severalprobes are immobilized at discretelocations on the substrate There areseveral methods that have been usedto pattern gold lms UV photopat-terning 17 4 1 polydimethylsiloxane(PDMS) microchannels48 microcon-tact printing49 and robotic spotting50

have been used in conjunction withSPR imaging The array shown inFig 1 was fabricated using the UVphotopatterning method in which aquartz mask is used to selectively ex-pose an alkanethiol-modi ed goldsurface to UV light At locationswhere the UV light shines on thesurface the alkanethiol is removedgenerating bare gold patches thatserve as a platform for generatingthe array elements Both the UVphotopatterning and PDMS micro-channel array fabrication strategieswill be discussed later in this articlein conjunction with speci c exam-ples of the use of SPR imaging tostudy biomolecular interactions

Quantitation of Results Gener-ated with Surface Plasmon Reso-nance Imaging In addition to theability to detect interacting partnerswith SPR imaging it is desirable toobtain quantitative information aboutthese interactions Quantitative datacan be obtained from SPR measure-ments by assuming that moleculesadsorbing to or desorbing from the

metal lm correlate to changes in theindex of refraction of the dielectriclayer and that changes in the indexof refraction correlate to changes inthe re ectivity of the incident lightIn order to obtain quantitative datawith SPR imaging it is necessary toknow over what regions there is alinear relationship between thechange in the re ected light intensity(DR) and the change in the indexof refraction of the sensing layer(Dnf) A series of 5-phase Fresnelcalculations of the system shown inFig 2 can be used to determine this9

The rst calculation is performedwith an n f value of 14 Each succes-sive calculation increments the n f

value by 0002 index of refractionunits Figure 5 shows the resultsfrom these calculations plotted as theabsolute value of DR for the indi-cated Dn f These calculations wereperformed for an excitation wave-length of 794 nm and the results atthree angles along the SPR curve areshown (the location of the anglesalong the SPR curve are shown inthe gure inset) The dotted lines inFig 5 show a linear relationship be-tween DR and Dnf At an angle of5352 degrees the greatest contrastis predicted as determined by themagnitude of DR The smallest de-viation from linearity is also ob-served at 5352 degrees A smallchange in the incident angle of 004degrees does not signi cantly affectthe overall signal or the amount ofdeviation that occurs This is not thecase for angles greater than 01 de-grees from the optimal angle of5352 degrees (not shown in Fig 5)At angles further than 01 degreesfrom the optimal angle there is asmaller change in the percent of re- ected light and these signals devi-ate from a linear relationship at largen f values While the data is fairly lin-ear for an angle higher than the sur-face plasmon angle (5412) theoverall contrast that would be ob-served is much smaller than for an-gles to the left of the surface plas-mon angle This is a result of theSPR curve not being symmetricabout the surface plasmon angle (seeFig 3)

326A Volume 57 Number 11 2003

focal point

Fig 4 (A) A schematic diagram of an SPR imager Collimated white light is passed through a polarizer and is incident on thesample assembly Reected light passes through focusing optics a narrow band pass lter typically centered at a wavelength in thenear-infrared and is captured by a CCD camera (B) A schematic diagram of the sample assembly consisting of a glass prism thatis optically coupled to a glass substrate containing a thin layer (45 nm) of gold The sample is contained within a ow cell for in situmeasurements

The deviations from linear behav-ior for DR and Dn f are small pro-vided that the experiment is per-formed at the optimal angle If de-viations are present they can be pre-dicted and accounted fo r in theexperimental results As mentionedpreviously the percent change in re- ected light intensity due to the hy-bridization of a monolayer of 16-meroligonucleotides is less than 1 per-cent which falls within the regionwhere linear data is obtained withSPR imaging9 An example of theuse of SPR imaging to quantitate theamount of material adsorbing to agold lm is described in the Exam-ples section In this example theamount of protein adsorbing to ametal lm is measured with SPR im-aging and is used to construct ad-sorption isotherms for the interactionof proteins with immobilized carbo-hydrates

EXAMPLESDetection of DNA Hybridiza-

tion Single-Base Mismatch Detec-

tion in the Presence of Small Mol-ecules A recent example of the useof SPR imaging demonstrated thatthis technique can be used to moni-tor the hybridization of short oligo-nucleotides in the presence of smallmolecules that alter the oligonucle-otidersquos binding properties27 A sche-matic diagram of a small moleculenaphthyridine dimer that has beenshown to stabilize the binding of GndashG mismatches in double strandedDNA is shown in Fig 651ndash53 To dem-onstrate the GndashG mismatch stabiliz-ing properties of this molecule aDNA array was fabricated using UVphotopattern ing This involves acombination of self-assembly andUV photopatterning steps A baregold lm is modi ed with a self-as-sembled monolayer of an amine-ter-minated alkanethiol The amine-ter-minated surface is then reacted witha hydrophobic protecting group thatcan be reversibly removed from thesurface A quartz mask containingpatterned features is placed over the

modi ed gold lm and the surface isexposed to UV light resulting in thephotooxidation of the goldndashthiolatebond and generation of bare gold re-gions where the UV light is exposedto the surface The substrate is thenreplaced in the amine-terminated al-kanethiol solution and a monolayeris formed at areas where there is baregold The resulting surface containshydrophilic amine-terminated mono-layer regions surrounded by a hydro-phobic monolayer The probe mole-cules can be immobilized through aseries of reactions that are carriedout within the hydrophilic wells bydelivering small solution volumes tothe surface (ie spotting solutionsonto the surface with a pulled cap-illary) The hydrophobic backgroundprevents cross-contamination be-tween the array elements during theimmobilization reaction Once theprobe compounds are immobilizedon the surface the hydrophobic pro-tecting group is removed from thebackground This generates an

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

324A Volume 57 Number 11 2003

focal point

Fig 3 Graph showing the scanning angle SPR reectivity curves that were obtained from 5-phase Fresnel calculations for thesystem shown in Fig 2 The index of refraction of the sensing layer (nf ) was 140 (solid line) or 146 (dotted line) The gure insetat right shows an expanded view of the region near the plasmon angles The location of the plasmon angle shifts to higher anglesfor the nf 5 146 lm relative to the nf 5 140 lm The gure inset at left shows an expanded view around the optimal angle forperforming SPR imaging experiments with this system At the optimal angle the largest shift in R is observed for these two lms

broad SPR curves (with large widthsat 50 re ectivity)32 Sharper SPRcurves produced by longer wave-length incident light yield largerchanges in re ectivity fo r givenchanges in nf than do broad curvesproduced by incident light withshorter wavelengths This means thatthere is a trade-off between lateralresolution and sensitivity The use oflonger wavelength light provideshigher sensitivity but lower lateralresolution The use of shorter wave-length light provides a higher lateralresolution but lower sensitivity3334

SURFACE PLASMONRESONANCE IMAGING

Surface Plasmon Resonance Im-aging Instrumentation Surface

plasmon resonance imaging is a xed angle experiment where thespatial changes in re ected light aremeasured across a substrate The the-oretical curves generated for a scan-ning angle SPR experiment can beused to understand the basis of thecontrast observed in an imaging ex-periment The left inset in Fig 3shows an expanded region around anincident angle of 535 degrees Aslice through the X-axis simulates aconstant angle experiment At 535degrees less light is re ected fromthe n f 5 140 lm than the nf 5 146 lm If a surface were patterned tocontain regions w ith both lmsmore light would be re ected fromthe regions with n f 5 146 than re-

gions containing the n f 5 140 lmat an angle of 535 degrees and itwould be possible to distinguish be-tween the two lms in an SPR im-age

The basic components in a typicalSPR imaging instrument are shownin Fig 4 These are a collimatedwhite light source a polarizer thesample stage collection optics anda charge-coupled device (CCD) con-nected to a CPU for image collectionand processing The use of a colli-mated white light source is preferredover the use of laser excitation dueto interference fringes that result inthe SPR image when laser excitationis utilized The polarizer is used toselect p-polarized light and the col-

APPLIED SPECTROSCOPY 325A

lection optics consist of a narrowband pass lter typically centered inthe near-infrared region that is usedto select the excitation wavelengthfor the experiment The sample is lo-cated on a rotation stage in order tocontrol the incident angle of lightand consists of a prism a substrateonto which a Au lm is depositedand a ow cell

Attachment Chemistry and Ar-ray Fabrication Surface plasmonresonance imaging is performed ona noble metal lm therefore strate-gies for attaching probe molecules tothese lms are critical to the successof an SPR imaging experiment Nu-merous immobilization strategies ex-ist These can generally be catego-rized into three routes (1) a thiol-modi ed probe molecule can be re-acted directly with a gold lm toform a goldndashthiolate bond35 (2) apolymer layer such as dextran orpolylysine can be rst formed on thegold surface and the probe moleculecan be immobilized onto the poly-mer layer36 and (3) a self-assembledalkanethiol monolayer (SAM) con-tain ing a v-terminated functionalgroup can be formed on the gold lm which is used to immobilize amolecule (a lsquolsquolinkerrsquorsquo) that is capableof reacting with the probe mole-cule37ndash41

There are drawbacks to the rstand second immobilization schemesMany biomolecules will non-specif-ically adsorb to a gold lm and alarge portion of the probes may notbe biologically active if they are di-rectly attached to the gold lm Sur-faces fabricated using the second im-mobilization strategy may not be ro-bust and polymer layers can be aproblem when studying kinetics ofadsorption

Several characteristics of the thirdimmobilization scheme make it suit-able for use with SPR imaging Gen-erally the surfaces that result fromthis immobilization strategy are sta-ble and can be used for several assaycycles Using this immobilizationstrategy provides a way to controlthe surface density of the probe mol-ecule This is important for two rea-sons (1) the surface density of the

probe can affect the amount of targetthat binds to the surface and there-fore the amount of signal that is de-tected and (2) varying the probedensity can be used as a tool to studythe interactions of targets that bindto the probes through multivalent in-teractions (an example of this is theuse of SPR imaging to study carbo-hydratendashprotein interactions) Final-ly this strategy provides a way tocontrol the resulting surface proper-ties (ie hydrophilic hydrophobiccharged)42ndash47 This is important sinceeverything that adsorbs to the goldsurface will produce an SPR signalit is necessary to control the surfaceproperties so that only the desiredtarget molecules interact with thesurface

In addition to immobilizing probemolecules to a gold lm the lmmust be patterned so that severalprobes are immobilized at discretelocations on the substrate There areseveral methods that have been usedto pattern gold lms UV photopat-terning 17 4 1 polydimethylsiloxane(PDMS) microchannels48 microcon-tact printing49 and robotic spotting50

have been used in conjunction withSPR imaging The array shown inFig 1 was fabricated using the UVphotopatterning method in which aquartz mask is used to selectively ex-pose an alkanethiol-modi ed goldsurface to UV light At locationswhere the UV light shines on thesurface the alkanethiol is removedgenerating bare gold patches thatserve as a platform for generatingthe array elements Both the UVphotopatterning and PDMS micro-channel array fabrication strategieswill be discussed later in this articlein conjunction with speci c exam-ples of the use of SPR imaging tostudy biomolecular interactions

Quantitation of Results Gener-ated with Surface Plasmon Reso-nance Imaging In addition to theability to detect interacting partnerswith SPR imaging it is desirable toobtain quantitative information aboutthese interactions Quantitative datacan be obtained from SPR measure-ments by assuming that moleculesadsorbing to or desorbing from the

metal lm correlate to changes in theindex of refraction of the dielectriclayer and that changes in the indexof refraction correlate to changes inthe re ectivity of the incident lightIn order to obtain quantitative datawith SPR imaging it is necessary toknow over what regions there is alinear relationship between thechange in the re ected light intensity(DR) and the change in the indexof refraction of the sensing layer(Dnf) A series of 5-phase Fresnelcalculations of the system shown inFig 2 can be used to determine this9

The rst calculation is performedwith an n f value of 14 Each succes-sive calculation increments the n f

value by 0002 index of refractionunits Figure 5 shows the resultsfrom these calculations plotted as theabsolute value of DR for the indi-cated Dn f These calculations wereperformed for an excitation wave-length of 794 nm and the results atthree angles along the SPR curve areshown (the location of the anglesalong the SPR curve are shown inthe gure inset) The dotted lines inFig 5 show a linear relationship be-tween DR and Dnf At an angle of5352 degrees the greatest contrastis predicted as determined by themagnitude of DR The smallest de-viation from linearity is also ob-served at 5352 degrees A smallchange in the incident angle of 004degrees does not signi cantly affectthe overall signal or the amount ofdeviation that occurs This is not thecase for angles greater than 01 de-grees from the optimal angle of5352 degrees (not shown in Fig 5)At angles further than 01 degreesfrom the optimal angle there is asmaller change in the percent of re- ected light and these signals devi-ate from a linear relationship at largen f values While the data is fairly lin-ear for an angle higher than the sur-face plasmon angle (5412) theoverall contrast that would be ob-served is much smaller than for an-gles to the left of the surface plas-mon angle This is a result of theSPR curve not being symmetricabout the surface plasmon angle (seeFig 3)

326A Volume 57 Number 11 2003

focal point

Fig 4 (A) A schematic diagram of an SPR imager Collimated white light is passed through a polarizer and is incident on thesample assembly Reected light passes through focusing optics a narrow band pass lter typically centered at a wavelength in thenear-infrared and is captured by a CCD camera (B) A schematic diagram of the sample assembly consisting of a glass prism thatis optically coupled to a glass substrate containing a thin layer (45 nm) of gold The sample is contained within a ow cell for in situmeasurements

The deviations from linear behav-ior for DR and Dn f are small pro-vided that the experiment is per-formed at the optimal angle If de-viations are present they can be pre-dicted and accounted fo r in theexperimental results As mentionedpreviously the percent change in re- ected light intensity due to the hy-bridization of a monolayer of 16-meroligonucleotides is less than 1 per-cent which falls within the regionwhere linear data is obtained withSPR imaging9 An example of theuse of SPR imaging to quantitate theamount of material adsorbing to agold lm is described in the Exam-ples section In this example theamount of protein adsorbing to ametal lm is measured with SPR im-aging and is used to construct ad-sorption isotherms for the interactionof proteins with immobilized carbo-hydrates

EXAMPLESDetection of DNA Hybridiza-

tion Single-Base Mismatch Detec-

tion in the Presence of Small Mol-ecules A recent example of the useof SPR imaging demonstrated thatthis technique can be used to moni-tor the hybridization of short oligo-nucleotides in the presence of smallmolecules that alter the oligonucle-otidersquos binding properties27 A sche-matic diagram of a small moleculenaphthyridine dimer that has beenshown to stabilize the binding of GndashG mismatches in double strandedDNA is shown in Fig 651ndash53 To dem-onstrate the GndashG mismatch stabiliz-ing properties of this molecule aDNA array was fabricated using UVphotopattern ing This involves acombination of self-assembly andUV photopatterning steps A baregold lm is modi ed with a self-as-sembled monolayer of an amine-ter-minated alkanethiol The amine-ter-minated surface is then reacted witha hydrophobic protecting group thatcan be reversibly removed from thesurface A quartz mask containingpatterned features is placed over the

modi ed gold lm and the surface isexposed to UV light resulting in thephotooxidation of the goldndashthiolatebond and generation of bare gold re-gions where the UV light is exposedto the surface The substrate is thenreplaced in the amine-terminated al-kanethiol solution and a monolayeris formed at areas where there is baregold The resulting surface containshydrophilic amine-terminated mono-layer regions surrounded by a hydro-phobic monolayer The probe mole-cules can be immobilized through aseries of reactions that are carriedout within the hydrophilic wells bydelivering small solution volumes tothe surface (ie spotting solutionsonto the surface with a pulled cap-illary) The hydrophobic backgroundprevents cross-contamination be-tween the array elements during theimmobilization reaction Once theprobe compounds are immobilizedon the surface the hydrophobic pro-tecting group is removed from thebackground This generates an

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

APPLIED SPECTROSCOPY 325A

lection optics consist of a narrowband pass lter typically centered inthe near-infrared region that is usedto select the excitation wavelengthfor the experiment The sample is lo-cated on a rotation stage in order tocontrol the incident angle of lightand consists of a prism a substrateonto which a Au lm is depositedand a ow cell

Attachment Chemistry and Ar-ray Fabrication Surface plasmonresonance imaging is performed ona noble metal lm therefore strate-gies for attaching probe molecules tothese lms are critical to the successof an SPR imaging experiment Nu-merous immobilization strategies ex-ist These can generally be catego-rized into three routes (1) a thiol-modi ed probe molecule can be re-acted directly with a gold lm toform a goldndashthiolate bond35 (2) apolymer layer such as dextran orpolylysine can be rst formed on thegold surface and the probe moleculecan be immobilized onto the poly-mer layer36 and (3) a self-assembledalkanethiol monolayer (SAM) con-tain ing a v-terminated functionalgroup can be formed on the gold lm which is used to immobilize amolecule (a lsquolsquolinkerrsquorsquo) that is capableof reacting with the probe mole-cule37ndash41

There are drawbacks to the rstand second immobilization schemesMany biomolecules will non-specif-ically adsorb to a gold lm and alarge portion of the probes may notbe biologically active if they are di-rectly attached to the gold lm Sur-faces fabricated using the second im-mobilization strategy may not be ro-bust and polymer layers can be aproblem when studying kinetics ofadsorption

Several characteristics of the thirdimmobilization scheme make it suit-able for use with SPR imaging Gen-erally the surfaces that result fromthis immobilization strategy are sta-ble and can be used for several assaycycles Using this immobilizationstrategy provides a way to controlthe surface density of the probe mol-ecule This is important for two rea-sons (1) the surface density of the

probe can affect the amount of targetthat binds to the surface and there-fore the amount of signal that is de-tected and (2) varying the probedensity can be used as a tool to studythe interactions of targets that bindto the probes through multivalent in-teractions (an example of this is theuse of SPR imaging to study carbo-hydratendashprotein interactions) Final-ly this strategy provides a way tocontrol the resulting surface proper-ties (ie hydrophilic hydrophobiccharged)42ndash47 This is important sinceeverything that adsorbs to the goldsurface will produce an SPR signalit is necessary to control the surfaceproperties so that only the desiredtarget molecules interact with thesurface

In addition to immobilizing probemolecules to a gold lm the lmmust be patterned so that severalprobes are immobilized at discretelocations on the substrate There areseveral methods that have been usedto pattern gold lms UV photopat-terning 17 4 1 polydimethylsiloxane(PDMS) microchannels48 microcon-tact printing49 and robotic spotting50

have been used in conjunction withSPR imaging The array shown inFig 1 was fabricated using the UVphotopatterning method in which aquartz mask is used to selectively ex-pose an alkanethiol-modi ed goldsurface to UV light At locationswhere the UV light shines on thesurface the alkanethiol is removedgenerating bare gold patches thatserve as a platform for generatingthe array elements Both the UVphotopatterning and PDMS micro-channel array fabrication strategieswill be discussed later in this articlein conjunction with speci c exam-ples of the use of SPR imaging tostudy biomolecular interactions

Quantitation of Results Gener-ated with Surface Plasmon Reso-nance Imaging In addition to theability to detect interacting partnerswith SPR imaging it is desirable toobtain quantitative information aboutthese interactions Quantitative datacan be obtained from SPR measure-ments by assuming that moleculesadsorbing to or desorbing from the

metal lm correlate to changes in theindex of refraction of the dielectriclayer and that changes in the indexof refraction correlate to changes inthe re ectivity of the incident lightIn order to obtain quantitative datawith SPR imaging it is necessary toknow over what regions there is alinear relationship between thechange in the re ected light intensity(DR) and the change in the indexof refraction of the sensing layer(Dnf) A series of 5-phase Fresnelcalculations of the system shown inFig 2 can be used to determine this9

The rst calculation is performedwith an n f value of 14 Each succes-sive calculation increments the n f

value by 0002 index of refractionunits Figure 5 shows the resultsfrom these calculations plotted as theabsolute value of DR for the indi-cated Dn f These calculations wereperformed for an excitation wave-length of 794 nm and the results atthree angles along the SPR curve areshown (the location of the anglesalong the SPR curve are shown inthe gure inset) The dotted lines inFig 5 show a linear relationship be-tween DR and Dnf At an angle of5352 degrees the greatest contrastis predicted as determined by themagnitude of DR The smallest de-viation from linearity is also ob-served at 5352 degrees A smallchange in the incident angle of 004degrees does not signi cantly affectthe overall signal or the amount ofdeviation that occurs This is not thecase for angles greater than 01 de-grees from the optimal angle of5352 degrees (not shown in Fig 5)At angles further than 01 degreesfrom the optimal angle there is asmaller change in the percent of re- ected light and these signals devi-ate from a linear relationship at largen f values While the data is fairly lin-ear for an angle higher than the sur-face plasmon angle (5412) theoverall contrast that would be ob-served is much smaller than for an-gles to the left of the surface plas-mon angle This is a result of theSPR curve not being symmetricabout the surface plasmon angle (seeFig 3)

326A Volume 57 Number 11 2003

focal point

Fig 4 (A) A schematic diagram of an SPR imager Collimated white light is passed through a polarizer and is incident on thesample assembly Reected light passes through focusing optics a narrow band pass lter typically centered at a wavelength in thenear-infrared and is captured by a CCD camera (B) A schematic diagram of the sample assembly consisting of a glass prism thatis optically coupled to a glass substrate containing a thin layer (45 nm) of gold The sample is contained within a ow cell for in situmeasurements

The deviations from linear behav-ior for DR and Dn f are small pro-vided that the experiment is per-formed at the optimal angle If de-viations are present they can be pre-dicted and accounted fo r in theexperimental results As mentionedpreviously the percent change in re- ected light intensity due to the hy-bridization of a monolayer of 16-meroligonucleotides is less than 1 per-cent which falls within the regionwhere linear data is obtained withSPR imaging9 An example of theuse of SPR imaging to quantitate theamount of material adsorbing to agold lm is described in the Exam-ples section In this example theamount of protein adsorbing to ametal lm is measured with SPR im-aging and is used to construct ad-sorption isotherms for the interactionof proteins with immobilized carbo-hydrates

EXAMPLESDetection of DNA Hybridiza-

tion Single-Base Mismatch Detec-

tion in the Presence of Small Mol-ecules A recent example of the useof SPR imaging demonstrated thatthis technique can be used to moni-tor the hybridization of short oligo-nucleotides in the presence of smallmolecules that alter the oligonucle-otidersquos binding properties27 A sche-matic diagram of a small moleculenaphthyridine dimer that has beenshown to stabilize the binding of GndashG mismatches in double strandedDNA is shown in Fig 651ndash53 To dem-onstrate the GndashG mismatch stabiliz-ing properties of this molecule aDNA array was fabricated using UVphotopattern ing This involves acombination of self-assembly andUV photopatterning steps A baregold lm is modi ed with a self-as-sembled monolayer of an amine-ter-minated alkanethiol The amine-ter-minated surface is then reacted witha hydrophobic protecting group thatcan be reversibly removed from thesurface A quartz mask containingpatterned features is placed over the

modi ed gold lm and the surface isexposed to UV light resulting in thephotooxidation of the goldndashthiolatebond and generation of bare gold re-gions where the UV light is exposedto the surface The substrate is thenreplaced in the amine-terminated al-kanethiol solution and a monolayeris formed at areas where there is baregold The resulting surface containshydrophilic amine-terminated mono-layer regions surrounded by a hydro-phobic monolayer The probe mole-cules can be immobilized through aseries of reactions that are carriedout within the hydrophilic wells bydelivering small solution volumes tothe surface (ie spotting solutionsonto the surface with a pulled cap-illary) The hydrophobic backgroundprevents cross-contamination be-tween the array elements during theimmobilization reaction Once theprobe compounds are immobilizedon the surface the hydrophobic pro-tecting group is removed from thebackground This generates an

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

326A Volume 57 Number 11 2003

focal point

Fig 4 (A) A schematic diagram of an SPR imager Collimated white light is passed through a polarizer and is incident on thesample assembly Reected light passes through focusing optics a narrow band pass lter typically centered at a wavelength in thenear-infrared and is captured by a CCD camera (B) A schematic diagram of the sample assembly consisting of a glass prism thatis optically coupled to a glass substrate containing a thin layer (45 nm) of gold The sample is contained within a ow cell for in situmeasurements

The deviations from linear behav-ior for DR and Dn f are small pro-vided that the experiment is per-formed at the optimal angle If de-viations are present they can be pre-dicted and accounted fo r in theexperimental results As mentionedpreviously the percent change in re- ected light intensity due to the hy-bridization of a monolayer of 16-meroligonucleotides is less than 1 per-cent which falls within the regionwhere linear data is obtained withSPR imaging9 An example of theuse of SPR imaging to quantitate theamount of material adsorbing to agold lm is described in the Exam-ples section In this example theamount of protein adsorbing to ametal lm is measured with SPR im-aging and is used to construct ad-sorption isotherms for the interactionof proteins with immobilized carbo-hydrates

EXAMPLESDetection of DNA Hybridiza-

tion Single-Base Mismatch Detec-

tion in the Presence of Small Mol-ecules A recent example of the useof SPR imaging demonstrated thatthis technique can be used to moni-tor the hybridization of short oligo-nucleotides in the presence of smallmolecules that alter the oligonucle-otidersquos binding properties27 A sche-matic diagram of a small moleculenaphthyridine dimer that has beenshown to stabilize the binding of GndashG mismatches in double strandedDNA is shown in Fig 651ndash53 To dem-onstrate the GndashG mismatch stabiliz-ing properties of this molecule aDNA array was fabricated using UVphotopattern ing This involves acombination of self-assembly andUV photopatterning steps A baregold lm is modi ed with a self-as-sembled monolayer of an amine-ter-minated alkanethiol The amine-ter-minated surface is then reacted witha hydrophobic protecting group thatcan be reversibly removed from thesurface A quartz mask containingpatterned features is placed over the

modi ed gold lm and the surface isexposed to UV light resulting in thephotooxidation of the goldndashthiolatebond and generation of bare gold re-gions where the UV light is exposedto the surface The substrate is thenreplaced in the amine-terminated al-kanethiol solution and a monolayeris formed at areas where there is baregold The resulting surface containshydrophilic amine-terminated mono-layer regions surrounded by a hydro-phobic monolayer The probe mole-cules can be immobilized through aseries of reactions that are carriedout within the hydrophilic wells bydelivering small solution volumes tothe surface (ie spotting solutionsonto the surface with a pulled cap-illary) The hydrophobic backgroundprevents cross-contamination be-tween the array elements during theimmobilization reaction Once theprobe compounds are immobilizedon the surface the hydrophobic pro-tecting group is removed from thebackground This generates an

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

APPLIED SPECTROSCOPY 327A

Fig 5 Graph showing the absolute value of the change in percent reectivity (R) for the indicated change in the index of refrac-tion (nf ) of the sensing layer The data is plotted for three angles The gure inset indicates the location of these points along the SPRcurve The data was obtained from 5-phase Fresnel calculations of the system shown in Fig 2 The dotted lines correspond to linearrelationships between DR and Dnf Greater contrast is observed for larger values of DR (ie at angles to the left of the plasmonangle)

amine-terminated background that issubsequently reacted with a mole-cule known to inhibit the adsorptionof target compounds to the surfacesuch as the succinimide ester ofpolyethylene glycol

To test the GndashG mismatch stabi-lizing properties of the naphthyridinedimer shown in Fig 6 a four-com-ponent DNA array was fabricatedEach of the four immobilized se-quences in the array differed by onebase The position of this base is in-dicated by an X in sequence 1 (Fig7) The SPR difference image cor-responding to the introduction of se-quence 2 to the array shows that anSPR signal is only observed for thesequence containing the base cyto-sine (C) at the X position in se-quence 1 the complementary se-quence to sequence 2 (Fig 7A)However the SPR difference image

corresponding to the addition of se-quence 2 in the presence of the naph-thyridine dimer shows that in addi-tion to its complement sequence 2also hybridizes to the sequence thatforms a GndashG mismatch These re-sults demonstrate that SPR imagingis a promising tool for monitoringsingle base mismatches in short oli-gonucleotides and also demonstratesthe possibility of using SPR imagingto screen molecules that alter the ol-igonucleotidersquos hybridization prop-erties

Protein Binding to Carbohy-drate Arrays Surface plasmon res-onance imaging is an attractive toolfor the study of proteins becausethere is no need to uorescently ra-dioactively or enzymatically labelthe analyte in order for it to be de-tected with SPR This opens the pos-sibility of directly studying an iso-

lated protein with less sample pro-cessing and with less expense (iethe expense of the labeling reagents)It has been demonstrated that SPRimaging can be used to monitor pro-tein adsorption onto DNA22 pep-tide54 and carbohydrate arrays55

A recent example of the use ofSPR imaging to study proteins is thestudy of proteinndashcarbohydrate inter-actions55 It was shown that carbo-hydrate arrays could be fabricatedusing PDMS microchannels A sche-matic diagram of this procedure isshown in Fig 8 In this technique48

a three-dimensional s ilicon maskwas used as a template to fabricatechannels in the PDMS These micro-channels were composed of a seriesof parallel lines that had entranceand exit reservoirs at their ends forsample introduction When thePDMS was placed over a modi ed

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

328A Volume 57 Number 11 2003

focal point

Fig 6 Structure of the GndashG mismatch stabilizing naphthyridine dimer that was used to generate the data shown in Fig 7B Thenaphthyridine dimer (blue) is shown hydrogen bonding to two guanine bases (black)

gold lm a different probe could beintroduced and immobilized withineach channel (Fig 8 step A) Sub-sequent removal of the PDMS fromthe gold lm yielded an array ofprobe molecules immobilized in aset of discrete lines (Fig 8 step B)

A two-component carbohydratearray was used to monitor the ad-sorption of two carbohydrate bindingproteins (lectins) A schematic dia-gram of the carbohydrate ligands isshown in Fig 8 Compound 1 is amodi ed andashmannose ligand andcompound 2 is a modi ed andashgalac-tose ligand The two lectins studiedwere concanavalin A and jacalinConcanavalin A has a known af nityfor andashmannose and jacalin has ahigh af nity for andashgalactose Ad-sorption isotherms were constructedfor the interactions of these lectinswith the surface immobilized carbo-hydrates by monitoring the SPR im-aging signal while increasing theconcentration of the protein in solu-tion Shown in Fig 9 are the adsorp-tion isotherm for (squares) jacalin in-teracting with compound 2 and (cir-cles) concanavalin A interacting withcompound 1 Each data point wasobtained by measuring the SPR im-aging signal for the indicated proteinconcentration Two examples of theSPR images used to construct theisotherms are shown in Fig 9 Theimage on the left corresponds to theintroduction of the lectin jacalin to

the array and the image on the rightcorresponds to the introduction ofthe lectin concanavalin A to the ar-ray

The adsorption isotherms provideinfo rmation about the interactionstrength of the proteins with the car-bohydrate surfaces For example theisotherms shown in Fig 9 indicatethat jacalin has a higher af nity forthe immobilized andashgalactose ligandthan concanavalin A does for the andashmannose ligand A number of pos-sible applications for the use of SPRimaging to quantitate the interactionstrength of proteins with immobi-lized arrays could be envisionedThese include the screening of com-pounds that might be of therapeuticsigni cance such as molecules thatdisrupt or enhance the interactions ofproteins with DNA proteins or car-bohydrates

Antibody Binding to Protein Ar-rays A recent example from the labsof Professors McDermott and Har-rison at the University of Albertademonstrates the use of SPR imag-ing to study the binding of antibod-ies to protein arrays56 The fabrica-tion of the protein array utilizedPDMS microchannels as in the pre-vious example to pattern the surfaceof a gold lm that had been modi edwith a carboxylic acid monolayerImmobilization of the protein on thesurface was carried out by owingprotein solutions through the PDMS

microchannels To image the arraysthe PDMS was removed from thesurface and solutions of antibodywere owed over the array Shownin Fig 10 are the SPR images ob-tained by Kariuki and co-workers ofa three-component protein array con-taining the proteins human brino-gen (line 1) ovalbumin (line 2) andbovine IgG (line 3) Figure 10Ashows the SPR difference image thatwas obtained after the array was ex-posed to the antibody for human -brinogen and Figs 10B and 10Cshow the SPR difference images ob-tained after exposing the array to an-tibodies for anti-ovalbumin and anti-bovine IgG respectively These im-ages show that there is a high degreeof antibody binding speci city and asmall degree of non-speci c adsorp-tion of the antibody to the arraybackground which the authors statecould be improved upon further ef-forts to modify the array back-ground These results successfullydemonstrate the suitability of usingSPR imaging to study antibody bind-ing to protein arrays and opens thepossibility of using SPR imaging asa diagnostic tool for the study of an-tibodies

CONCLUSION AND FUTUREDIRECTIONS

The label-free detection high-throughput capabilities and simple

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

APPLIED SPECTROSCOPY 329A

Fig 7 SPR difference images of a four-component DNA array Each immobilized oligonucleotide differs by one base indicated byan X in sequence 1 The images were taken in the presence of (A) 1 mM sequence 2 or (B) 250 mM naphthyridine dimer with 1mM sequence 1 The image shown in A indicates that sequence 2 only hybridizes to the perfect match The image shown in Bindicates that sequence 2 hybridizes to both the perfect match and the GndashG mismatch oligonucleotide when naphthyridine dimer ispresent

instrumental format make SPR im-aging a useful tool for the study ofa variety of biomolecular interac-tions Examples of the use of SPRimaging to study biomolecular inter-actions have thus far been limitedto arrays composed of 2ndash10 com-ponents however SPR imaging hasthe potential to screen arrays com-posed of at least 30 000 species on a18 cm 3 18 cm substrate

It is expected that the high-throughput capabilities of SPR im-aging will aid in the study of proteinin teractions including proteinndashDNA proteinndashpeptide proteinndashpro-tein and proteinndashcell surface inter-actions in addition to those exam-ples discussed in this article Thereis however work that remains to beaccomplished to make SPR imaginga routine detection method for the

study of proteins This includes thedevelopment of new array attach-ment methods new array fabricationtechniques and improved analyteprocessing capabilities that more ef- ciently deliver solutions of targetproteins to the array surface Prelim-inary work has been done on the de-velopment of oriented arrays of fu-sion proteins for the study of pro-teinndashprotein interactions with SPR

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

330A Volume 57 Number 11 2003

focal point

Fig 8 Simplied schematic of the array fabrication process using polydimethylsiloxane microchannels The microfabricated PDMSchannels are placed on top of a modied gold lm Immobilization of the probe molecules occurs within the channels upon removalof the PDMS from the surface the probe ligands are immobilized in discrete lines on the gold lm A two-component array wasfabricated to generate the data shown in Fig 9 The two components are 1 (a modied andashmannose ligand) and 2 (a modied andashgalactose ligand)

Fig 9 Isotherms for (squares) the binding of jacalin to a surface containing compound 2 and (circles) the binding of concanavalinA to a surface containing compound 1 The relative protein surface coverage (fraction of occupied surface sites u) was determinedusing SPR imaging as the concentration of protein in solution was increased The data have been t to Frumkin isotherms (solidlines) which provide information on the strength of the interaction between the surface immobilized species and the adsorbing spe-cies The two SPR difference images are two-component carbohydrate arrays that were fabricated using the method shown in Fig8 The SPR image on the right shows the binding of the lectin concanavalin A to the mannose array elements and the SPR imageon the left shows the binding of the lectin jacalin to the galactose array elements The images were used to generate two of thedata points on the isotherms and to demonstrate the specicity of lectin binding to the immobilized carbohydrate ligands

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

APPLIED SPECTROSCOPY 331A

Fig 10 SPR difference images of athree-component protein array contain-ing the proteins human brinogen (line1) ovalbumin (line 2) and bovine IgG(line 3) SPR difference images obtainedafter exposing the protein array to (A)the antibody to human brinogen (B)the antibody to ovalbumin and (C) theantibody to bovine IgG Reproducedfrom Ref 56 with kind permission fromKluwer Academic Publishing

imaging This strategy uses a sur-face-based array of a capture agentto immobilize a set of fusion proteinsthat contain two domains one in-variant domain that binds to the cap-ture agent and a second variable do-main containing the probe proteinwhich is in direct contact with thetarget solution

Finally detection methods that in-crease the sensitivity of the SPR im-aging technique will be extremelyuseful in a number of applicationsthat require very low analyte con-centrations including environmentalmonitoring and DNA diagnosticsIncreased sensitivity can currently beachieved through the use of labeledtarget molecules (ie latex polysty-rene or gold nanoparticles conjugat-ed to the target molecule) or throughthe use of a sandwich assay inwhich the secondary binding eventof a large molecule provides the de-tection signal20255758 These methodsincrease the complexity of the SPRimaging measurement An alternatepromising method for achieving in-creased sensitivity in SPR imagingexperiments is through improved in-strumental design One recent reportdemonstrated improved sensitivitywith the technique of SPR interfer-ometry in which both the amplitudeand phase of the re ected light aremeasured59ndash61 Near the surface plas-mon angle there is a large shift in thephase of the re ected light Measur-ing both the re ectivity and thephase of the light in an SPR imagingexperiment may provide both an en-hanced sensitivity and an increaseddynamic range

Other improvements in SPR im-aging instrumentation are also inprogress For example a portable eld-ready SPR imager is being de-veloped for environmental monitor-ing and the demonstration of Fou-rier transform SPR (FT-SPR) spec-troscopy has expanded the use ofSPR to near-infrared wavelengths(1000ndash2500 nm)15 As a nal note itshould be mentioned that SPR indexof refraction measurements are justthe simplest of many possible sur-face plasmon spectroscopies SPRhas also been used for SPR uores-

cence6263 SPR Raman scattering64

SPR CARS65 SPR electro-opticalmeasurements66ndash68 and SPR second-harmonic generation at surfaces69

ACKNOWLEDGMENTS

This research is funded by the National Sci-ence Foundation (Grant CHE-0133151) Theauthors wish to thank Dr Hye Jin Lee DrAlastair Wark Greta Wegner and Berta Os-trander for their assistance in the preparationof the manuscript

1 J H Watterson P A E Piunno C CWust U J Krull Sens Actuators B 7427 (2001)

2 N Sloper and M T Flanagan BiosensBioelectron 11 537 (1993)

3 F S Ligler M Breimer J P Golden D

A Nivens J P Dodson T M Green DP Haders and O A Sadik Anal Chem74 713 (2002)

4 E Garcia-Caurel B Drevillon and A LS De Martino Appl Opt 44 7339(2002)

5 T Mutschler B Kiesser R Frank and GGauglitz Anal Bioanal Chem 374 658(2002)

6 L Y Li S F Chen S J Oh and S YJiang Anal Chem 74 6017 (2002)

7 J Wang and A J Bard Anal Chem 732229 (2001)

8 V Silin and A Plant Trends Biotechnol15 353 (1997)

9 B P Nelson T E Grimsrud M R LilesR M Goodman and R M Corn AnalChem 73 1 (2001)

10 I Gokce E M Raggett Q Hong R Vir-den A Cooper and J H Lakey J MolBiol 304 621 (2000)

11 L A Lyon M D Musick and M J Na-tan Anal Chem 70 5177 (1998)

12 R Advincula E Aust W Meyer and WKnoll Langmuir 12 3536 (1996)

13 D G Hanken C E Jordan B L Freyand R M Corn Surface Plasmon Reso-nance Measurements of Ultrathin Organ-ic Films at Electrode Surfaces (MarcelDekker New York 1996) vol 20

14 C E Jordan B L Frey F R Kornguthand R M Corn Langmuir 10 3642(1994)

15 A G Frutos S C Weibel and R MCorn Anal Chem 71 3935 (1999)

16 W Hickel D Kamp and W Knoll Na-ture (London) 339 186 (1989)

17 D Piscevic W Knoll and M J TarlovSupramol Sci 2 99 (1995)

18 B Rothenhausler and W Knoll Nature(London) 332 615 (1988)

19 L A Lyon W D Holliway and M JNatan Rev Sci Instrum 70 2076(1999)

20 C E Jordan A G Frutos A J Thieland R M Corn Anal Chem 69 4939(1997)

21 J M Brockman B P Nelson and R MCorn Annu Rev Phys Chem 51 41(2000)

22 E A Smith M G Erickson A T Uli-jasz B Weisblum and R M Corn Lang-muir 19 1486 (2003)

23 wwwbiacorecom24 D Piscevic R Lawall M Veith M Lil-

ey Y Okahata and W Knoll Appl SurfSci 90 425 (1995)

25 L He M D Musick S R NicewarnerF G Salinas S J Benkovic M J Natanand C D Keating J Am Chem Soc122 9071 (2000)

26 M Li H J Lee A E Condon and RM Corn Langmuir 18 805 (2002)

27 E A Smith M Kyo H Kumasawa KNakatani I Saito and R M Corn J AmChem Soc 124 6810 (2002)

28 H Raether Surface Plasmons on Smoothand Rough Surfaces and on Gratings(Springer-Verlag Berlin 1988)

29 P B Johnson and R W Christy PhysRev B 6 4370 (1972)

332A Volume 57 Number 11 2003

focal point

30 E Kretschmann and H Raether Z Na-turforsch A Phys Sci 23 2135 (1968)

31 W N Hansen J Opt Soc Am 58 380(1968)

32 B P Nelson A G Frutos J M Brock-man and R M Corn Anal Chem 713928 (1999)

33 H E de Bruijin R P H Kooyman andJ Greve Appl Opt 32 2426 (1993)

34 C E H Berger R P H Kooyman andJ Greve Rev Sci Instrum 65 2829(1994)

35 T M Herne and M J Tarlov J AmChem Soc 119 8916 (1997)

36 B L Frey and R M Corn Anal Chem68 3187 (1996)

37 K L Prime and G M Whitesides J AmChem Soc 115 10714 (1993)

38 J Lahiri L Isaacs B Grzybowski J DCarbeck and G M Whitesides Lang-muir 15 7186 (1999)

39 B T Houseman and M Mrksich AngewChem Int Ed Engl 38 782 (1999)

40 A G Frutos J M Brockman and R MCorn Langmuir 16 2192 (2000)

41 J M Brockman A G Frutos and R MCorn J Am Chem Soc 121 8044(1999)

42 C D Bain and G M Whitesides J AmChem Soc 110 3665 (1988)

43 C D Bain and G M Whitesides Science(Washington DC) 240 62 (1988)

44 R G Chapman E Ostuni L Yan andG M Whitesides Langmuir 16 6927(2000)

45 R G Chapman E Ostuni S TakayamaR E Holmlin L Yan and G M White-sides J Am Chem Soc 122 8303(2000)

46 E Ostuni R G Chapman R E HolmlinS Takayama and G M WhitesidesLangmuir 17 5605 (2001)

47 C D Bain E B Troughton Y T Tao JEverall G M Whitesides and R GNuzzo J Am Chem Soc 111 321(1989)

48 H Lee T T Goodrich and R M CornAnal Chem 73 5525 (2001)

49 A T A Jenkins T Neumann and A Of-fenhausser Langmuir 17 265 (2001)

50 M Zizlsperger and W Knoll Prog Col-loid Polym Sci 109 244 (1998)

51 K Nakatani S Sando and I SaitoBioorg Med Chem 9 2381 (2001)

52 K Nakatani S Sando H Kumasawa JKikucji and I Saito J Am Chem Soc123 12650 (2001)

53 K Nakatani S Sando and I Saito NatBiotech 19 51 (2001)

54 G J Wegner H J Lee and R M CornAnal Chem 74 5161 (2002)

55 E A Smith W D Thomas L L Kies-sling and R M Corn J Am Chem Soc125 6140 (2003)

56 J K Kariuki V Kanda M T Mc-Dermott and D J Harrison in Micro To-tal Analysis Systems 2002 Y Baba SShoji and A van der Berg Eds (KluwerAcademic Publisher Nara Japan 2002)vol 1 pp 230ndash232

57 T Wink S J van Zuiken A Bult andW P van Bennekom Anal Chem 70827 (1998)

58 J H Gu H Lu Y W Chen L Y LiuP Wang J M Ma and Z H Lu Supra-mol Sci 5 695 (1998)

59 P I Nikitin A A Beloglazov V E Ko-chergin M V Valeiko and T I Ksen-evich Sens Actuators B 54 43 (1999)

60 A N Grigorenko P I Nikitin and A VKabashin Appl Phys Lett 75 3917(1999)

61 A V Kabashin and P I Nikitin OptCommun 150 5 (1998)

62 S Roy J-H Kim J T Kellis A J Pou-lose C R Robertson and A P GastLangmuir 18 6319 (2002)

63 T Liebermann and W Knoll Langmuir19 1567 (2003)

64 R M Corn and M R Philpott J PhysChem 80 5245 (1984)

65 C K Chen A R D De Castro Y RShen and F DeMartini Phys Rev Lett43 946 (1979)

66 D G Hanken R R Naujok J M Grayand R M Corn Anal Chem 69 240(1997)

67 D G Hanken and R M Corn AnalChem 69 3665 (1997)

68 C Xia R Advincula A Baba and WKnoll Langmuir 18 3555 (2002)

69 R M Corn M Romagnoli M D Le-venson and M R Philpott J PhysChem 81 4127 (1984)