planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

Analytica Chimica Acta 469 (2002) 49–61

Planar waveguides for ultra-high sensitivityof the analysis of nucleic acids

Gert L. Duveneck∗, Andreas P. Abel, Martin A. Bopp,Gerhard M. Kresbach, Markus Ehrat

Zeptosens AG, Benkenstrasse 254, CH-4108 Witterswil, Switzerland

Received 10 August 2001; received in revised form 20 November 2001; accepted 6 December 2001

Abstract

In the first part of this paper, the need for analytical techniques capable of highly parallel and sensitive nucleic acidanalysis, with the capability of achieving very low limits of detection (LODs) and of resolving small differences in concen-tration, is described. Whereas the requirement for performing simultaneously multi-analyte detection is solved by the ap-proach of nucleic acid microarrays, requirements on sensitivity can often not be satisfied by classical detection technologies.Inherent limitations of conventional fluorescence excitation and detection schemes are identified, and the implementationof planar waveguides as analytical platforms for nucleic acid microarrays, with fluorescence excitation in the evanescentfield associated with the guided excitation light, is proposed. The relevant parameters for an optimization of sensitivity arediscussed.

In the second part of this paper, the specific formats of our planar waveguide platforms, which are compatible withestablished industrial standard formats allowing for integration into industrial high throughput environments, are presented,as well as the dedicated optical system for fluorescence excitation and detection that we developed. In a direct comparisonwith a state-of-the-art scanner, it is demonstrated that the implementation of genomic microarrays on planar waveguideplatforms allows for unprecedented, direct detection of low-abundant genes in limited amounts of sample. Otherwise, whenusing conventional fluorescence excitation and detection configurations, the detection of such low amounts of nucleic acidsrequires massive sample preparation and signal or target amplification steps.© 2002 Elsevier Science B.V. All rights reserved.

Keywords:Planar waveguide; Fluorescence; Nucleic acid analysis; Microarray

1. Introduction

Nucleic acids form a very large, unique class ofbiomolecules: the whole variety of living organismsis based on only five different, simple nucleotidebuilding blocks, and it is only the base sequence,from which nature’s abundance is derived. Due to

∗ Corresponding author. Tel.:+41-61-726-8188;fax: +41-61-726-8171; URL:http://www.zeptosens.com.E-mail address:[email protected] (G.L. Duveneck).

the few common building blocks, nucleic acids showrelatively similar physical and chemical properties,allowing for the development of analysis methods thatcan be applied to this whole class of analytes, whichhowever, have to be very specific in order, e.g. to dis-tinguish reliably different nucleic acids with differentsequences.

One possible approach is directed to identifying nu-cleic acids with specific, known sequences in a samplecontaining many different known or unknown nucleic

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50 G.L. Duveneck et al. / Analytica Chimica Acta 469 (2002) 49–61

acids and possibly other compounds different fromnucleic acids. This approach is based on the ability ofspecific hybridization of single-stranded nucleic acidswith strands of complementary sequence, used for an-alytical purposes such as sequencing by hybridization[1,2], separation based on differences of affinity, etc.Following this approach, single-stranded nucleic acidsare, e.g. immobilized as capture probes on a solid sup-port in order to hybridize with complementary nucleicacid sequences to be identified in a supplied sample.

The typical tasks to be solved in nucleic acid anal-ysis, such as determination of sequence variationsbetween individuals, e.g. for the determination ofsingle-nucleotide polymorphisms (SNPs) or determi-nation of changes of cellular expression as responsesto different drugs in gene expression analysis, requirethe development of highly parallel analysis methods,for the simultaneous determination of hundreds tothousands of different nucleic acids in one sample orthe analysis of hundreds of samples in parallel.

In the early 1990s, the development of nucleic acidmicroarrays began, for addressing this need for highlyparallel analysis [3–6]. Nowadays, complete genomescan be deposited in arrays of tens of thousands ofspots on a single carrier (“chip”). However, especiallyif mRNA of low abundance, i.e. 1–10 copies of mRNAin a single cell, which typically represent about 95%of the total mRNA [7], have to be determined andchanges of their expression levels to be monitored, thesensitivity of the analytical procedure, with emphasison the detection step, is of key importance.

In many cases, the availability of genomic mate-rial is limited. Therefore, a simple increase of sampleamounts is not feasible. The most common approachto (biochemically) amplify the available nucleic acidmaterial by polymerase chain reaction (PCR) [8] is of-ten associated with the well-known high variability ofbiological processes and non-linearity of the amplifi-cation process, which can bias the results especially forlow-abundant molecules. An optimized detection tech-nology should allow low detection limits independentfrom chemical or biological amplification schemes.

2. Fluorescence detection

For achieving the highest possible detectionsensitivity, analyte detection based on the use of

fluorescence labels or of radioactive labels is currentlyconsidered as state-of-the-art. The application of ra-dioactive marker compounds was the only possibilityfor detecting very low copy numbers of nucleic acids,without using biochemical amplification steps, for along time, due to the method-inherent high signal-to-noise ratio. However, the precautions to be takenwhen working with radioactive compounds, lead tothe demand for the development of methods of similarsensitivity, but easier handling.

Despite a current lack of capability for detection oflow-abundant sequences without biochemical amplifi-cation steps, nowadays analyte detection is most oftenperformed using fluorescent labels, which can, e.g.,be introduced into the sequence of interest upon in-sertion of fluorescently labeled building blocks duringthe transformation of mRNA to cDNA.

Upon spectrally selective excitation and detectionof fluorescence from different fluorophores, fluores-cence detection facilitates the determination of multi-ple analytes in parallel: fluorescence labels of differentexcitation and emission wavelengths, classically inthe red and the green, can be applied simultaneouslyin one or sequentially in different detection steps, e.g.to distinguish between the hybridization with nucleicacids in an unknown sample and in a reference probe.

If different fluorescence labels are applied in oneand the same detection step, it has to be establishedcarefully that the analysis results are not masked byenergy transfer processes between the different la-bels or by different effects of a changing molecularenvironment on the fluorescence quantum yields orby different efficiencies of the enzymatic processfor integrating different labeled nucleotides into thecDNA chain.

2.1. Detection configurations

Classical configurations for the detection of flu-orescence from surfaces are often based on epi-illumination or trans-illumination configurations: Ina trans-illumination configuration, the excitation lightpasses through a transparent support carrying the fluo-rescent sample. The excitation light source, the objectand the detector can be arranged in a straight line,as in the scheme of Fig. 1A, or the optical excitationand emission paths can be tilted with respect toeach other, if the direct passing of excitation light in

G.L. Duveneck et al. / Analytica Chimica Acta 469 (2002) 49–61 51

Fig. 1. Classical configurations for the detection of fluorescence from surfaces: (A) trans-illumination configuration: light source anddetector are on opposite sides with respect to the surface from where fluorescence is collected; (B) epi-illumination configuration, as it isapplied in many fluorescence scanners. The optical paths of excitation and emission light are separated by means of a dichroic mirror.

direction towards the detector shall be avoided. Theexcitation light may be focused onto the object, andthe emission light is collected by a lens system andfocused onto the detector, which may be a photo-multiplier or photodiode without lateral resolution ora laterally resolving detector like a charge-coupleddevice (CCD) camera. Discrimination of excitationlight against the fluorescence light to be detectedis most often performed using spectral filters, suchas interference filters, at least in the emission lightpath.

Sources of background light, besides imperfect dis-crimination of excitation light, can be any fluorescentobjects in the long path of the excitation light fromthe light source towards the detector, e.g. caused byfluorescent compounds in the sample matrix, or fluo-rescent impurities in the substrate material of the chip,or any other component of the optical system.

In an epi-illumination configuration (Fig. 1B), thedirect propagation of excitation light in directiontowards the detector is avoided. The light sourceand the detector are arranged on the same side withrespect to the sample and its support. Typically, a

dichroic mirror is used as beam splitter, which reflectsexcitation light towards the sample to be excited andtransmits the longer-wavelength fluorescence light. Adisadvantage of this configuration, which is the basicoptical scheme of many scanning systems for fluores-cence detection, is the unavoidable further reductionof transmission at the fluorescence wavelength due tothe dichroic mirror (see Fig. 1B).

In both configurations (Fig. 1A and B), aperturescan be placed into the optical path in order to selec-tively illuminate only the actual area of interest andto collect emission selectively from the same locationand to discriminate any light signals laterally asidefrom the sample. This principle is applied in confocalmicroscopes.

2.2. Limitations of classical fluorescence detectionconfigurations

Besides the limited collection efficiency of anyimaging system, typically well below 10%, a majorsource of undesired background signals is the rela-tively long part of the excitation light path with high


Fig. 2. (A) In convention fluorescence excitation configurations, the part of the optical path with high excitation intensities is much largerthan the beam waist and deeply penetrates the bulk medium. (B) Surface-confined excitation associated with light-guiding in an opticalwaveguide: the penetration depth of the evanescent field into the adjacent media (transparent support and sample solution) is only somehundred nanometers.

excitation light intensity before and after the surfacewhere the sample is located and analyte fluorescenceis generated (Fig. 2A). Typically, this path is four-to six-times larger than the beam waist, where thesample is located. Even for a diffraction-limited focusof the excitation beam, this path will still be a fewmicrometers long. Whereas the lateral resolution ofa fluorescence microscope system is improved whena sharper focus is applied, undesired photobleachingeffects, due to the extremely high intensities in theexcitation beam focus, are generated as well.

When apertures are used to discriminate light fromoutside of the plane where the sample is located, theefficiency of fluorescence light collection is also fur-ther reduced, and the requirements on the flatness ofthe support, e.g. a microscope slide with immobilizedcDNA, are strongly increased, to values well below10�m [9], which is not satisfied by the specificationson thickness variations of standard microscope slides(50�m, [9]): if the sample is located outside of thefocus of the optical excitation and emission light path,not only the efficiency of fluorescence light collec-tion, but also the excitation light intensity is stronglyreduced.

3. Waveguide technology for fluorescenceexcitation

The disadvantages of large propagation lengths ofexcitation light through regions remote from the sur-

face where fluorescence excitation is intended, i.e.over long distances before and after the plane wherethe sensing surface is located, can be avoided uponfluorescence excitation in the evanescent field of ex-citation light guided in an optical waveguide.

When a light wave is guided in a medium of highrefractive index medium by total internal reflection,the associated electromagnetic field does not abruptlyvanish at the interface to the adjacent media, but has afinite penetration depth into these lower-index media.The field strength of this so-called evanescent field de-creases exponentially with distance from the waveg-uide, with a penetration depth of only some hundrednanometers (Fig. 2B). This surface-confined excitationfield provides the opportunity for selectively study-ing interactions occurring at the interface between a(high-index) solid body, acting as a waveguide, anda lower-index medium, whereas deeper in the samplemedium no potentially interfering signals are gener-ated [10].

This excitation field is not only sharper confined tothe sensing surface by at least one order of magnitude,compared with the classical excitation schemes (seeFig. 2), but it is also simultaneously available alongthe whole propagation length of guided excitationlight in the waveguide. Thus, simultaneous fluores-cence excitation on macroscopic surfaces combinedwith very high spatial selectivity of the excitationstep (with respect to distance from the interactingsurface) is enabled by optical waveguides. Washingsteps, for removing unbound fluorescent molecules in


a supplied sample, can be avoided, thus reducing thenumber of work steps in a bioassay [11]. Furthermore,real-time studies of the kinetics of analyte binding tothe surface are enabled, which cannot be performedwith scanning systems due to sequential excitationand detection along the scanned areas. Due to thesurface-confined fluorescence excitation and detec-tion, even turbid or scattering media can be applied ona waveguide biosensor, thus avoiding many tedioussteps of typical sample preparation.

The light can be launched into the high-indexmedium acting as the waveguide by front-face cou-pling, or by using a prism or a diffractive grating.

Front-face coupling is most often used for light cou-pling into fibers using a focusing spherical lens or intomacroscopic, self-supporting planar waveguides, likeglass or plastic plates of hundreds of micrometers tomillimeter thickness, using focusing cylindrical lens[12,13]. If, however, the dimensions of the waveguide,such as the layer thickness of a thin-film waveguide,are reduced to sub-wavelength size, this method of“end face firing” is no longer of practical value, as therequirements on alignment accuracy become too high.

Prism coupling [14,15], mainly used only for pla-nar waveguides, can be applied to film waveguides(with films of the order of micrometers or less). How-ever, in general, the application of an index-matchingfluid between the waveguide and the prism is re-quired in order to avoid reflections due to air-gapsand to improve the coupling efficiency and repro-ducibility. Thus, prism coupling is a valuable methodfor fast demonstration of the feasibility of a waveg-uide configuration in a research laboratory, but notfavorable for automation, e.g. for repetitive use withhigh reproducibility and large numbers of studiesto be performed with individual waveguide sensingplatforms.

The most elegant way for light coupling intothin-film waveguides, with waveguiding layers witha thickness of less than micrometers, is based ondiffractive gratings [16], structured, e.g. as periodicmodulations in at least one of the surfaces of thewaveguiding layer. The essential parameters for effi-cient light coupling into a grating waveguide struc-ture are the match with the resonance angle for thelight coupling and the position of the excitation lightbeam on the in-coupling grating. Both parametersare defined with tolerances (typically 0.01–0.1◦ for

the coupling angle1 , dependent on the grating depth,and 10–100�m for the lateral position on the grat-ing), which can be satisfied with regular positioningequipment, and optimization of the alignment forhighest coupling efficiency can be automated easilyin a feed-back loop with a signal proportional tothe in-coupled light intensity as the parameter to bemaximized.

3.1. Thin-film planar waveguides

For light-guiding over long distances, e.g. intelecommunication, any interactions with the waveg-uide surface, leading to losses of the guided power,are highly undesired, i.e. intensities at the interfacebetween the waveguide and the adjacent media haveto be minimized. In contrast, the optimization ofwaveguide transducers for biochemical sensing appli-cations leads into a quite opposite direction, namelyto a maximization of the strength of an excitationfield at a waveguide surface interacting with theanalyte.

Highest intensities of the evanescent field areachieved with thin-film waveguides, with a refractiveindex as high as possible, typically above 2.0. Addi-tionally, in principle, the strength of the evanescentfield does increase with decreasing thickness of thewaveguide, as long as the thickness is above a cer-tain cut-off value [17], below which light-guiding isnot further possible. However, with decreasing thick-ness of the waveguiding film, also attenuation lossesdue to scattering caused by surface roughness doincrease, which reduce the field intensity availablefor surface-confined excitation. As a consequence,there is an optimum of the waveguide film thick-ness, which is between 100 and 200 nm for guidingvisible excitation light in waveguides with a refrac-tive index around 2. It has been demonstrated thatwaveguide transducers consisting of a 150 nm thinTa2O5 film on a planar glass substrate are optimumin terms of high, surface-confined excitation fieldstrengths as well as in terms of surface properties[18–20].

1 For given waveguide and grating parameters, i.e. given refrac-tive indices, thickness of the waveguiding layer and grating pe-riod, there is a well-defined angle under which excitation light ofa given wavelength can be coupled into the waveguiding layer.


Fig. 3. Excitation light can be coupled into thin-film waveguides using diffractive relief gratings modulated in the high-index waveguidingfilm (typical grating depth: 5–20 nm). Note: The position of in-coupling of the excitation light is remote from the position where fluorescenceis generated and detected.

Excitation light is coupled into the waveguidingTa2O5 film using diffractive relief gratings (Fig. 3),which are etched into the glass substrate and trans-ferred into the surface of the waveguiding filmupon its deposition process [17]. Highest efficienciesfor the in-coupling of the excitation light into thewaveguiding film are obtained for first diffractionorder coupling, which is achieved with short periodgratings.

Therefore, typically, a parallel excitation light beamfrom a laser light source is launched under the cou-pling angle onto the in-coupling grating. Comparedwith the classical fluorescence excitation configura-tions described above, the principle of fluorescenceexcitation in the evanescent field of planar waveg-uides, upon light coupling into the waveguide bymeans of gratings, is associated with further significantadvantages.

(i) The location of launching the excitation lightonto the coupling grating is remote from thearea where analyte fluorescence is excited. Thus,

discrimination of the excitation light is facili-tated due to the lateral separation, in contrast tothe classical configurations where the areas oflaunching the excitation light and of fluorescencegeneration are identical.

(ii) The main critical parameter for light couplingusing a grating is the match with the couplingangle in order to satisfy the resonance condition.Therefore, when using a parallel excitation lightbeam, the distance between the planar waveguidesurface and the excitation light source is of noimportance.

(iii) Once the excitation light has been coupled intothe waveguide, the associated evanescent field isestablished at the waveguide surface along thetrace of the guided light. Deviations from a per-fect surface flatness over macroscopic distances,i.e. in the range of more than 10–50�m, haveno effect on the available excitation light inten-sities, as long as the thickness of the waveguidelayer remains constant and no light scattering isgenerated. Thus, only the step of fluorescence


collection is impaired by imperfections of thesurface flatness (due to variations of the distancebetween the surface and the detector), in contrastto the described classical fluorescence excitationconfigurations.

For the transducers used in the experiments de-scribed in the following sections, with 320 nm periodgratings, the resonance condition for red light cou-pling (635 nm, TE0) is met at a launching angle of−10◦, measured with respect to the normal of thewaveguide surface. The negative coupling angle hasthe advantage, that any reflections of undiffractedexcitation light will travel away from the direction oflight propagation in the waveguide, on the surface ofwhich analyte detection is intended. The coupling an-gle changes with the launched excitation wavelength,for our waveguides by about 0.2◦/nm. Consequently,under coupling angles between−20 and+30◦, exci-tation light covering the whole visible spectrum (fromabout 450–700 nm) can be coupled into these wave-guides, thus, allowing use of all current, commer-cially available fluorescence labels that can be excitedin the visible.

Fig. 4. Zeptosens’ planar waveguide chip formats and flow cell arrangements, with a geometry that is compatible with standard microtiterplates: 96 microfluidic cells can be arranged on one plate, with a thin-film planar waveguide as the base plate (“ZeptoPLATE”), or sixflow cells in a row are provided on a waveguide plate of 14 mm× 57 mm (“ZeptoCHIP”), which can be inserted into a carrier of againmicrotiter plate format. Each micro-flow cell accommodates a whole microarray.

3.2. Zeptosens’ planar waveguide chip formatand flow cell arrangement

We have designed our waveguide sensing platformsin a format which is compatible with the industrialstandard footprint of a 96-well microtiter plate. Thisallows the customer to use established laboratoryequipment and robotics when working with our mi-croarray system. The dedicated optical system forread-out of our planar waveguide-based microarrays(“ZeptoREADER”) is designed for automated opera-tion and capability for high throughput, without neces-sity of any adjustments to be performed by the user.

Two different formats of the waveguide plate geom-etries and of dedicated flow cell arrangements allowfor “medium” and “high throughput” applications(Fig. 4).

In the “high throughput” format, the waveguideplate (75 mm× 114 mm) forms the continuous baseplate of an arrangement of 96 flow cells (8 rows× 12columns of flow cells, “ZeptoPLATE”). On the waveg-uide base plate is mounted a plastic structure. Togetherwith the base plate, it forms an array of 96 flow cells,each capable of accommodating a sample or reagent


volume as low as 15�l within the cell. The cell inlets,located close to the bottom of the externally visiblestructure (from top view, not visible in Fig. 4), can beaddressed by regular pipette tips, thus, allowing fillingof the cells, e.g. by means of manual or robotic mul-tipipettors. The outlets of the flow cells (formed liketubes located in one of the corners of each flow cell,see Fig. 4) end in reservoirs capable to receive a mul-tiple of the cell volume of liquid. These reservoirs arethe externally visible recesses of the plastic structure.Thus, the cells can be filled sequentially with differentreagents, or washing steps can be performed withoutthe need of removal of waste solutions.

In parallel to the columns of the 96-cell structure,0.5 mm broad relief diffraction gratings extend overthe whole width of the waveguide plate, spaced at adistance of about 9 mm, corresponding to the pitch ofthe flow cells in adjacent columns. The in-couplinggratings are located within the flow cells, close to theleft inner cell wall (according to the geometry dis-played in Fig. 4, see inset).

In the “medium throughput” format, the waveguideplate has a size of 14 mm× 57 mm. Together with aplastic structure, it forms a column of six flow cells(“ZeptoCHIP”) with the same pitch, of 9 mm betweenadjacent flow cells within one column, as in the “highthroughput” format. All internal flow cell dimensionsare identical with the ones of the ZeptoPLATE. TheZeptoCHIP can be inserted into a carrier (“Zepto-CARRIER”) again having the footprint of a microtiterplate. The carrier can accommodate up to five Zepto-CHIPs. This “medium throughput” format, with itsmodular design, has the advantage of larger flexibil-ity, if the measurement capacity of a whole plate withthe dimensions of a microtiter plate is not needed.

Each flow cell, in both formats, is dedicated forthe interaction of an analyte-containing sample with awhole array of recognition elements (capture probes)accommodated within the cell (right from the couplinggrating, in direction of a guided mode of in-coupledexcitation light, see inset of Fig. 4 and example in thefollowing sections).

4. Optical system

We developed a completely new optical excitationand detection system (“ZeptoREADER”) optimized

for the specific excitation geometries required for pla-nar waveguides. The system is designed to collect thatpart of fluorescence, which is generated in the evanes-cent field of the guided excitation light, but isotropi-cally emitted.2 The system allows for use of at leasttwo laser light sources emitting at different wave-lengths, provided by a laser diode emitting at 635 nmand a frequency-doubled semiconductor laser emittingat 532 nm. Optionally, a further light source, emittingat 492 nm, is available. By means of lenses and aper-tures, the excitation beam from each laser is shaped tomatch the geometry of the in-coupling grating locatedwithin a flow cell. Thus, fluorescent molecules withinthe evanescent field of the guided excitation light canbe excited simultaneously on the area of a whole mi-croarray, without the necessity of scanning for signalcollection within one microarray. Typical excitationlight intensities launched onto the in-coupling grating,through the glass support (substrate) of the waveguid-ing layer, are of the order of 0.1–2 mW.

The optical components are mounted in such a way,that only minor, automated adjustments of the waveg-uide chips with respect to the excitation light beam arerequired for matching the coupling condition for theactual wavelength. The excitation light intensity canbe attenuated using neutral density filters mounted ona filter wheel in the excitation light path.

With these excitation parameters, a large numberof fluorescence labels can be applied for analyte de-tection in assays using our system. The capability ofa control of the excitation light intensities allows forminimizing the light exposure of the labels, thus, re-ducing photobleaching effects to levels typically lowerthan in commercial scanner systems.

Fluorescence excited in the evanescent field of thewaveguide, but isotropically emitted is collected by acamera objective and focused onto the photosensitivechip of a Peltier-cooled CCD camera. The imaged areawithin a flow cell is about 5 mm× 7 mm. Betweenthe two halves of the objective, a filter wheel with

2 It has been demonstrated [18–20] that both the fluorescence,that is excited in the evanescent field of the waveguide, butisotropically emitted, and the fraction that is coupled back intothe waveguide can be detected, e.g. using a second grating forits out-coupling towards a detector. If, however, multiple differentanalytes shall be detected in discrete measurement areas along thepath of the guided light, only the first detection method can beapplied.


interference filters is mounted for selective transmis-sion of fluorescence excited by the different laser lightsources. Signal collection times can be varied between80 ms and minutes, in order to allow detection of bothstrong and weak fluorescence intensities.

All liquid handling steps are performed separately,remote from the ZeptoREADER. After termination ofthe different assay steps (incubation with sample andreagent solutions, washing steps), a ZeptoPLATE orZeptoCARRIER with one or more ZeptoCHIPs is in-serted into the receiving slot of the ZeptoREADER,from where it is transported either directly to the mea-surement position or deposited in a stacker.

The ZeptoREADER is equipped with a stacker intowhich up to 10 ZeptoPLATEs or ZeptoCARRIERs, tobe measured sequentially, can be loaded. The system isoperated completely under computer control. Differentsystem parameters and commands can be set for eachof the measurement positions in the two-dimensionalflow cell arrangement, each cell accommodating a mi-croarray comprising hundreds of spots with differentcapture probes as recognition elements. Taking intoaccount the capacity of the stacker of hosting up toten ZeptoPLATEs, it is apparent that tasks for analy-sis of up to 960 microarrays can be placed in a singlesession.

After initiation of an analysis series, the first Zepto-PLATE or ZeptoCARRIER is moved into the mea-surement position for the first flow cell. Before start ofa measurement, it is equilibrated for a user-definableperiod of time at the measurement temperature set bythe user. The excitation light beam on the in-couplinggrating within a flow cell is adjusted automaticallyfor optimum in-coupling efficiency. During this ad-justment step, the excitation light intensity is stronglyattenuated to avoid unnecessary light exposure of anyfluorescent tracer molecules bound to the microarray.

Then images of the whole array are taken at theselected fluorescence wavelength, with the system set-tings defined by the user. Thus, images can be takensequentially from up to 960 microarrays in an unat-tended way. The images are analyzed by proprietarysoftware (“ZeptoVIEW”).

5. Examples of applications

In the following results from two experiments willbe reported, in order to demonstrate the performance

of planar waveguide technology combined withnucleic acid microarrays, using our dedicated chipformats and optical system. The first experiment,showing the results of the hybridization of fluores-cently labeled oligonucleotides with the complemen-tary capture probes immobilized in a small array(4 × 4 spots), mainly reflects the physical systemperformance, in comparison with a state-of-the-artfluorescence scanner. In the second experiment, ex-cellent performance of our system is demonstrated ina typical assay application.

5.1. Chip preparation

For the examples of applications described in thefollowing, waveguide plates (150 nm Ta2O5 on AF 45glass, size 14 mm×57 mm) dedicated for ZeptoCHIPswere used. For comparison of the performance of theZeptoREADER with a state-of-the-art confocal scan-ner, always one and the same waveguide plate was an-alyzed with the ZeptoREADER and with the scanner.

Both oligonucleotide and cDNA microarrays wereproduced by using a GMS 417 Arrayer (Affymetrix,Santa Clara, CA, USA). For the first example,amino-functionalized 25-mer oligonucleotides (Mi-crosynth) were immobilized in a 4× 4 array onsilanized planar waveguide plates.

For the second experiment, 96 double-strandedmouse brain cDNAs (200–800 base pairs long, kindlyprovided by LION Bioscience, Heidelberg, Germany)were spotted as capture probes in duplicate, thus,creating an array of totally 192 measurement areason the waveguide plate (16× 12 spots), which was,in this case, coated with a positively charged graftcopolymer (poly(l-lysine)-g-poly(ethylene glycol)(PLL-g-PEG), for immobilization by electrostaticinteraction [21,22]).

5.2. Hybridization assays

The hybridization assays were performed in mi-crofluidic cells of a ZeptoCHIP as described above,using a hybridization buffer containing standardsaline citrate. For the first experiment, the immo-bilized 25-mer capture probes were incubated withcomplementary Cy5-labeled 25-mer oligonucleotides(10−10 M, Microsynth) for 2 h at room temperature,


for hybridization, followed by a wash with buffersolution.

The protocol of the second hybridization assaywith cDNA capture probes followed standard hy-bridization assay protocols [23–25]. Incubation withCy5-labeled target cDNA (kindly provided by Qiagen,Hilden, Germany), which had been obtained by reversetranscription of 100 and 25 ng mRNA [24,25], respec-tively, was performed overnight. After completionof the hybridization, also a final wash with buffersolution was performed.

5.3. Optical measurements

Identical waveguide plates with fluorescent tracerprobes bound to their capture probes immobilized inmicroarrays, after completion of the assays describedabove, were measured in a fluorescence scanner (withan epi-illumination configuration) and in the Zepto-READER. In order to avoid any photobleachingeffects (due to exposure to excitation light) that couldfalsify results in favor of the signals to be recordedwith the ZeptoREADER, experiments were first per-formed with the fluorescence scanner.

5.3.1. Confocal fluorescence scannerDue to the short focal length of the collecting lens

of the scanner system, the plastic body of the Zepto-CHIP had to be removed for measurement in the scan-ner. The waveguide plate carrying the microarrayswith the bound, Cy5-labeled tracer probes was blowndry with nitrogen. In order to keep conditions as sim-ilar as possible for the first comparative study, fordetection of fluorescently labeled oligonucleotides,the measurement with the scanner was performed ina liquid environment, as it is regularly done with theZeptoREADER. A drop of buffer solution was de-posited on the waveguide plate. Then a cover slidewas placed onto the liquid-covered microarray, andthe waveguide plate was inserted into the scanner.

Waveguide plates used for the second experiment,for determination of the hybridization with fluorescentcDNA transcribed from mRNA from a real, biologicalsample were measured dry, as it is typically preferredwhen using a fluorescence scanner.

Fluorescence excitation was performed using alaser diode emitting at 635 nm, with a maximumoutput of 8 mW. The interference filter mounted in

the emission light path of the fluorescence scannerhad maximum transmission at 666 nm (12 nm band-width). Excitation and detection parameters of thescanner were set for maximum sensitivity (highestlaser power and photomultiplier voltage). The totalexposure time of a scanned array of 5 mm×7 mm wasabout 5 s.

5.3.2. ZeptoREADERAfter termination of measurements performed with

the scanner, waveguide plates were again combinedwith the plastic body of the ZeptoCHIP, inserted intothe ZeptoCARRIER, and the flow cells were filledwith buffer solution, before insertion of the Zepto-CARRIER into the ZeptoREADER, as described inthe earlier sections.

In the ZeptoREADER, fluorescence from surface-bound tracer molecules was excited in the evanes-cent field of the guided excitation light, as describedin the earlier sections. The excitation light intensitylaunched onto the in-coupling grating was 0.8 mW(635 nm). For detection of Cy5 fluorescence, an inter-ference filter with maximum transmission at 675 andabout 50 nm bandwidth was used in the emission lightpath. The exposure time for fluorescence excitationwas 10 s in case of the first and 30 s for the secondexperiment.

5.4. Results: planar waveguide system comparedwith confocal scanner

5.4.1. Oligonucleotide hybridization assayThe images taken of the 4×4 array, after hybridiza-

tion of the Cy5-labeled target with the immobilizedcomplementary capture probe are depicted in Fig. 5Aand C. Whereas a high contrast between the fluo-rescent spots and the background, corresponding toa signal-to-noise ratio around 200 (see Fig. 5B), isobserved under conditions of surface-confined exci-tation in the evanescent field of the guided excitationlight, measured with the ZeptoREADER (10 s expo-sure time), the fluorescence signals measured with theconfocal scanner under conditions of highest systemsensitivity (maximum laser intensity and photomul-tiplier voltage) do hardly exceed the background(signal-to-noise ratio of about 2). Traces of the fluo-rescence signals measured with the two systems alongthe last column of spots are superimposed in Fig. 5B.


Fig. 5. Images showing the fluorescence signals from the same microarray, after hybridization of Cy5-labeled 25-mer target oligonucleotides(10−10 M, concentration of sample solution) with immobilized complementary capture probes in a 4× 4 array: (A) taken with theZeptoREADER; (B) superposition of the signal traces along the last column of spots (see arrows; small signals from fluorescence scanner,larger signals from ZeptoREADER); and (C) taken with a confocal fluorescence scanner.

From these data, a 60- to 100-fold higher sensiti-vity (derived from the signal-to-noise ratio) of theZeptoREADER, compared with the confocal scanner,is determined.

5.4.2. cDNA hybridization assayThe second experiment was directed to a compar-

ison of the performance of the two systems for thedetection of the results of a typical assay, showing thehybridization of different nucleic acids supplied in asample to a microarray with 96 different cDNAs ascapture probes.

At the higher sample concentration (cDNA derivedfrom 100 ng mRNA), the image taken with the Zepto-READER shows fluorescence signals, of differentintensity, from almost all 192 measurement areas(spots), representing the 96 different capture probesin duplicate (Fig. 6A). High signal reproducibilityis evident from almost equal intensity of the spotpairs representing same capture probes. Signals rep-resenting the highest nucleic acid concentrations inthe sample are in saturation. In contrast, in the imagetaken by the scanner, many spot pairs do not showdetectable fluorescence signals (Fig. 6B).

Traces of the fluorescence signals obtained alongthe sixth row of spots with the two systems are

superimposed in Fig. 6D. For three selected pairs ofspots, measured signal-to-noise ratios range from 5to 10 for the scanner data and from 300 to 750 forthe ZeptoREADER results. In perfect agreement withthe first experimental example, but now for an assaywith a nucleic acid sample that underwent the fullsampling, sample extraction, sample preparation andlabeling procedure, again, a 60-fold higher sensitiv-ity of the ZeptoREADER, in comparison with thescanner, is determined.

At the lower sample concentration, i.e. for a sampleamount of 25 ng mRNA, in case of the ZeptoREADERimage, most capture probes are still represented bycorresponding fluorescence signals from bound targetcDNA (Fig. 6C), whereas, using the confocal scanner,weak fluorescence signals are observed only for themost abundant nucleic acids occurring in the sample(Fig. 6E).

This second example demonstrates the capa-bility of our planar waveguide-based reader sys-tem for biomolecular arrays of detecting high andlow-abundant species simultaneously in one sample.The dynamic measurement range of the ZeptoRE-ADER can still be expanded by several decadesupon taking multiple images at different exposuretimes.


Fig. 6. Microarray images showing the fluorescence signals from the same microarray, after hybridization with cDNA samples obtained byreverse transcription from 100 ng mRNA (A and B) and 25 ng mRNA (C and E), respectively, taken with the ZeptoREADER (A and C)and taken with a confocal fluorescence scanner (B and E). (D) Superposition of the trace of the fluorescence signals along sixth row ofspots (small signals from fluorescence scanner, larger signals from ZeptoREADER). Signal-to-noise ratios measured with the two systemsare given for three selected spot pairs (duplicates with identical immobilized capture probes).

6. Summary and conclusions

It has been demonstrated that the optical detectionsensitivity can be improved by about two orders ofmagnitude, compared to conventional fluorescenceexcitation and detection systems, by utilizing surface-confined fluorescence excitation in the evanescentfield of planar waveguides. Numerous further advan-tages associated with planar waveguide technology,compared to the classical excitation configurations,have been identified. We showed different for-mats of sensing platforms in which these advan-tages are combined with microarrays in order toenable highly parallel, ultra-sensitive nucleic acidanalysis.

The adaptation of the planar waveguide sensingplatforms to a format compatible with the estab-lished 96-well microtiter plate format allows theapplication of standard laboratory equipment andautomation for all sample and reagent manipula-tion steps and promotes the integration of our sys-tem into industrial high throughput environments.

The complete system automation allows for easysystem operation and is designed for high systemrobustness.

In a direct comparison with a state-of-the art scan-ner, it has been demonstrated that the implementationof genomic microarrays on planar waveguide plat-forms allows for unprecedented, direct detection oflow-abundant genes in limited amounts of sample,which otherwise require massive sample preparationand signal or target amplification steps. As the in-crease in sensitivity has been achieved by introducinga novel optical detection approach, the informationintegrity, e.g. with respect to expression levels, canbe maintained. It can be anticipated that our planarwaveguide-based microarray approach can take fulladvantage of further improvements, e.g. in samplepreparation and fluorescence labeling techniques.Last, but not least, it has to be emphasized that mi-croarrays on planar waveguides are not limited tonucleic acid analysis, but that any biomolecular assaybased on surface-immobilized recognition elementscan be implemented on planar waveguide platforms,


thus allowing, e.g. also the creation of protein andreceptor microarrays.

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planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

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