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Colloids and Surfaces B: Biointerfaces 61 (2008) 113–117

Short communication

Single-step biocompatible coating for sulfhydryl coupling ofreceptors using 2-(pyridinyldithio)ethylcarbamoyl dextran

Xin Li a, Christopher Abell b, Matthew A. Cooper a,∗a Akubio Ltd., 181 Cambridge Science Park, Cambridge CB4 0GJ, United Kingdom

b University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

Received 18 April 2007; received in revised form 27 June 2007; accepted 28 June 2007Available online 3 July 2007

bstract

2-(Pyridinyldithio)ethylcarbamoyl dextran (PDEC dextran) is developed herein as a novel biosensor coating material aimed for direct and facile

abrication of a sulfhydryl-specific capture surface on gold; the site-directed immobilization of a single free cysteine residue presented proteinhuman serum albumin, HSA) on PDEC dextran-coated surfaces without prior activation and subsequent specific binding of mouse monoclonalnti-HSA antibody to the resultant surface were demonstrated using surface plasmon resonance (SPR) assays. 2007 Elsevier B.V. All rights reserved.

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eywords: Biosensor; SPR; Coating; Sulfhydryl-specific; Coupling

. Introduction

The immobilization of molecules at surfaces in a controlledanner is essential for binding and functional assays on solid

hases. For proximity-based biosensors, careful control of thenterfacial architecture between the transducer and the solutionhase are required in order to maximize the signal due to recep-or binding, and minimize non-specific binding (NSB). To thisnd, several strategies for immobilizing biological moleculesn biosensor surfaces have been developed and broadly used inhe literature. Substrates have been decorated with oligo- andoly-ethylene glycol n-alkanethiolates that form self-assembledonolayer (SAM) coatings on metals [1–3]. These oligo-

thylene glycol containing SAMs are prone to auto-oxidationnd, being planar, provide only a limited capacity scaffold tohich materials can be attached. The use of a biocompatibleydrogel matrix attached to a gold surface via a SAM for appli-ation in surface plasmon resonance (SPR) biosensors has foundreat utility in analytical biochemistry [4]. In this case an n-

lkanethiol SAM with an omega-functionalized hydroxyl groupas activated using an epoxy intermediate for attachment of

arboxymethylated dextran. However, this procedure requires

∗ Corresponding author. Tel.: +44 1223 225336; fax: +44 1223 225336.E-mail address: mcooper@akubio.com (M.A. Cooper).

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our steps, the use of organic solvents and aggressive and toxiceagents such as epibromohydrin, bromoacetic acid and stronglkali.

Attachment of biological molecules via the sulfhydrylroups of available or engineered cysteine residues on aeceptor to a suitably sulfhydryl-reactive surface has longeen employed in protein chromatography [5]. For biosensingpplications, sulfhydryl reactive surfaces are normally gen-rated by converting a carboxylic acid moiety surface to a-(pyridinyldithio)ethaneamine (PDEA) surface [6]. However,he residual carboxylic acid groups impart a significant neg-tive charge to the polymer, which in turn leads to increasedSB via electrostatic interactions [7]. Direct attachment ofolymer coatings to metal surfaces via a electrostatically neu-ral sulfur is unfortunately compromised by the tendency toxidation of free sulfhydryl groups [8]. This can occur dur-ng purification, storage or deposition, and can also affecthe reactivity and ultra-structure of a polymer as sulfhydrylroups are converted into intra-polymer disulfide groups.uch sulfur-mediated cross-linking of a polymer or hydro-el results in a more viscous, gel-like structure that is lessuitable for use in biosensors due to concomitant compro-

ised diffusion of receptors and analytes within the hydrogel

9].In response to these limitations we have developed a novel

olymeric material that does not possess free sulfhydryl groups

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nd that can be directly chemisorbed to gold in a single stepsing aqueous solvents.

. Materials and methods

.1. Materials

Aldrithiol-2, 2-cysteamine hydrochloride, 4-nitrophenylhloroformate, 4-(dimethylamino)pyridine (DMAP), N-ethylmorpholine, anhydrous DMSO, anhydrous pyridine,,l-dithiothreitol (DTT), 2-mercaptopyridine, phosphateuffered saline (PBS), human serum albumin (HSA) and bovineerum albumin (BSA) were from Sigma–Aldrich. Dextran T70as purchased from Amersham Biosciences (Little Chalfont,ritain). Mouse monoclonal to human serum albumin (mAbnti-HSA) and mouse monoclonal to bovine serum albuminmAb anti-BSA) antibodies were from Abcam plc (Cambridge,ritain). AffiniPure Rabbit anti-mouse IgG antibody (Fc

gamma) Fragment Specific) was purchased from JacksonmmunoResearch Laboratory.

.2. Instrumentation and sensors

Biacore 2000 optical biosensor and Biacore SIA bare goldensor chips were purchased from Biacore AB (Uppsala, Swe-en).

.3. Preparation of 2-(pyridinyldithio)ethaneaminePDEA)

Aldrithiol-2 (4.41 g, 20 mmol) was dissolved in 20 ml ofethanol and 0.8 ml of acetic acid. Into the solution was

dded dropwise 2-cysteamine hydrochloride (1.14 g, 10 mmol)n 50 ml of methanol over a period of 1 h. The mixture wastirred at room temperature overnight. The resulting yellow solu-ion was concentrated in vacuo. The resulting yellow oil residueas then thoroughly washed by vigorous stirring with 50 ml ofiethyl ether. The clear supernatant was decanted off, and theesidual yellow solid dissolved in methanol (10 ml). To the solu-ion was added 50 ml of diethyl ether. The resultant precipitatesere collected by filtration. This procedure was repeated three

imes, and the pale white solid was purified by recrystallisationmethanol/diethyl ether) to give a white solid 1.5 g.

.4. Typical procedure for the preparation of 4-nitrophenylarbonated dextran

To a solution of dextran T70 (Amersham Biosciences, 1.6 g,9.6 mmol hydroxyl) in anhydrous dimethylsulfoxide (18 ml)nd anhydrous pyridine (16 ml), were added 4-nitrophenyl chlo-oformate (Aldrich, 800 mg, 4 mmol) and catalytic amountf 4-(dimethylamino)pyridine with gentle stirring and exter-al ice-water cooling. The reaction mixture was stirred at the

emperature for 5 h, and then slowly added into a mixture ofethanol and diethyl ether (1:1) (150 ml) with vigorous stir-

ing. The precipitates formed were collected by filtration, andispersed again in 150 ml of the same solvent mixture. The pre-

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iointerfaces 61 (2008) 113–117

ipitates were collected by filtration, and the above procedureas repeated three times. The white solid collected was driednder high vacuum to give 1.57 g of a white powder.

4-Nitrophenol (11.6 mg) and 4-nitrophenyl carbonated dex-ran T70 (25.9 mg) were dissolved in d6-DMSO (1 ml). 1H-NMR400 MHz, d6-DMSO): selected δ 6.90 (2H of 4-nitrophenol,), 7.54 (2H, d), 8.08 (2H of 4-nitrophenol, d), 8.30 (2H, d).unctionality degree: 10.5% (mol glucose unit).

.5. Typical procedure for the preparation of-(pyridinyldithio)ethylcarbamoyl dextran (PDEC dextran)

To a solution of 4-nitrophenyl carbonated dextran T701.57 g) in anhydrous dimethylsulfoxide (20 ml) and anhy-rous pyridine (6 ml), were added N-methylmorpholine (400 �l)nd 2-(pyridinyldithio)ethaneamine (640 mg). The mixture wastirred at 25 ◦C for 18 h, and then it was slowly added into a mix-ure of methanol and ether (1:1) (150 ml) with vigorous stirring.he precipitates formed were collected by filtration, and washedith the same solvent mixture as described above three times

150 ml each time). The collected white solid was dried underigh vacuum to give 1.0 g of white powder that was stored in a20 ◦C freezer until use.

1H-NMR (400 MHz, d6-DMSO): selected δ 7.21 (1H ofyridinyl, t), 7.42 (1H of carbamoyl N–H, br), 7.75 (1H ofyridinyl, d), 7.81 (1H of pyridinyl, t), 8.43 (1H of pyridinyl,).

.6. Instrument preparation

Biacore 2000 instrument were cleaned using a ‘Desorb’ethod at 25 ◦C before the start of the experiment. After dockingmaintenance chip, the instrument was consecutively primedith desorb solution 1 (0.5% sodium dodecyl sulphate, SDS),esorb solution 2 (50 mM glycine, pH 9.5) and finally degassedilliQ water.

.7. PDEC dextran deposition onto Biacore SIA bare goldurface

After docking the SIA bare gold chip and priming the instru-ent with degassed MilliQ water, the gold sensor chip was

leaned by flowing over an aqueous solution of 100 mM NaOH% (w/v) Triton X100 for 4 min. Then a solution of 2 mg/mlw/v) underivatized dextran T70 in MilliQ water was injectedhrough one of the sensor flowcells as a reference surface and aolution of 2 mg/ml (w/v) PDEC dextran through the rest threeowcells for 7 min at a flow rate of 10 �l/min. The signal dif-erences before and after contacting with the polymer solutionsere recorded to represent the material loading on the sensor

hip surfaces, 1000 RU = 1 ng/mm2 [10].

.8. PDEC dextran coating stability

A NaOH solution (100 mM) was injected to flow over theDEC dextran-coated sensor surface for 1 min at a flow rate of0 �l/min. The injection was repeated 20 times followed by 20

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socsurface to 2 mg/ml (w/v) PDEC dextran solutions resulted indeposition of between 3500 RU and 4000 RU, which corre-sponds [10] to 3.5–4 ng/mm2 of polymer. A reference channelexposed to a negative control: underivatized 2 mg/ml (w/v) dex-

X. Li et al. / Colloids and Surfac

imes injection of 100 mM NaOH 1% (w/v) Triton X100. Theignal differences before and after contacting with the alkalineolutions were recorded to represent the material loss from theensor chip surfaces.

.9. Static deposition of PDEC dextran onto Biacore SIAare gold surface

Two Biacore SIA bare gold sensor chips were placed in a0 mm glass Petri dish with the gold coating side uppermost. Aolution of 100 mM NaOH 1% Triton in MilliQ water (0.2 ml)as dropped on one chip, and another 0.2 ml solution on thether and the dish covered. The chips were then incubated atoom temperature for 5 min, then washed with MilliQ water 10imes. In the same manner, the chips’ gold-coated surfaces wereontacted with 2 mg/ml PDEC dextran in MilliQ water solutionor 16 h, then cleaned with MilliQ water 10 times, and left to airry.

.10. Release and quantification of 2-mercaptopyridinerom PDEC dextran-coated sensor chips

PDEC dextran-coated sensor chips were placed in a 40 mmlass Petri dish with the polymer coating side uppermost. A solu-ion of 1 mM d,l-dithiothreitol (DTT) in MilliQ water (0.2 ml)as dropped on one chip, and another 0.2 ml solution on thether. The chips were then incubated at room temperature forh, and then the solutions were collected with a pipette. Thebsorbances of the solutions were recorded on a CARY 1EV–vis spectrophotometer (Varian Inc., Palo Alto, USA) at70 nm. Standard solutions were prepared at 2 �M, 4 �M, 6 �M,�M and 10 �M of 2-mercaptopyridine in 1 mM DTT aqueous

olution, and their absorbances were recorded on the CARY 1EV–vis spectrophotometer at 270 nm.

.11. Human serum albumin (HSA) immobilization

The instrument was primed with PBS buffer before conduct-ng the following biological assays.

A control polymer surface was generated by injecting 50 mM-cysteine in 100 mM formate buffer pH 4.3 into one of theDEC dextran-coated flowcell at a flow rate of 10 �l/min. Theontact time was 5 min.

A HSA solution (1 mg/ml) in 100 mM borate buffer pH 8.5owed over both the above control and the active PDEC dextranowcells for 5 min at a flow rate of 10 �l/min. The signal differ-nces before and after contacting with the protein solution wereecorded to represent the immobilization level of the protein onhe sensor chip surfaces. The exposure of the protein solution tooth control and active surfaces was repeated five times.

.12. Anti-HSA antibody interaction with the surface boundSA

Solutions of 333 nM mAb anti-HSA antibody in PBS pH 7.4Fig. 3i), 10 mM NaCl–HCl solution pH 3.0 (Fig. 3ii), 10 mMaCl–HCl solution pH 2.0 (Fig. 3iii), 333 nM mAb anti-BSA

Biointerfaces 61 (2008) 113–117 115

ntibody in PBS pH 7.4 (Fig. 3iv), 333 nM rabbit anti-mousegG antibody in PBS pH 7.4 (Fig. 3v), 10 mM NaCl–HCl solu-ion pH 2.0 (Fig. 3vi), 333 nM mAb anti-HSA antibody in PBSH 7.4 (Fig. 3vii) and again 10 mM NaCl–HCl solution pH 2.0Fig. 3viii) were consecutively flowed over a surface bound withSA at a flow rate of 10 �l/min. The exposure time of each

bove solution to the surface was 5 min. The signal differencesefore and after each solution injection were recorded to rep-esent antibodies association to or dissociation from the sensorhip surface.

A BSA control surface was generated in the same way asenerating HSA active flowcell described above. The associa-ion of mAb anti-HSA antibody to both the HSA active surfacend the BSA control surface, and the surfaces regenerationith 10 mM NaCl–HCl solution pH 2.0 were repeated seven

imes.

. Results and discussion

.1. Preparation of PDEC dextran

To prepare PDEC dextran, dextran T70 (1, Scheme 1) wasrstly activated with 4-nitrophenyl chloroformate in the pres-nce of excessive anhydrous pyridine and a catalytic amountf 4-(dimethylamino)pyridine (DMAP) at 0 ◦C for 5 h. Theunctionality degree of the resultant 4-nitrophenoxycarbonylextran (2, Scheme 1), which was evaluated with 1H-NMRsing 4-nitrophenol as the internal reference, was 10.5% molarf glucose unit, i.e. there was one active 4-nitrophenyl carbonateroup within 10 glucose units in average. 2 was then allowedo react with 2-(pyridinyldithio)ethane amine (PDEA) in a mix-ure of anhydrous DMSO and pyridine to produce 3 (Scheme 1),DEC dextran. Good solubility, up to 4% (w/v) in MilliQ waterithout forming gel over several weeks at room temperature, is

onsidered to be crucial for surface fabrication.

.2. PDEC dextran deposition and its stability

In order to investigate application in biosensing, a gold sen-or chip was first cleaned by flowing over an aqueous solutionf 100 mM NaOH 1% (w/v) Triton X100 for 4 min in a Bia-ore 2000 SPR biosensor (Biacore AB). Exposure of bare gold

Scheme 1. Preparation of PDEC dextran.

116 X. Li et al. / Colloids and Surfaces B: Biointerfaces 61 (2008) 113–117

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ig. 1. Stability of PDEC dextran surface to alkaline solutions, 20 times 1 minnjections of 100 mM NaOH in MilliQ water followed with 20 times 1 minnjections of 100 mM NaOH 1% Triton X100 in MilliQ water.

ran T70, exhibited a signal shift of <20 RU or 20 pg/mm2 (dataot shown). This suggests that, at least in part, chemisorption ofDEC dextran was mediated by the coordination of the reactiveulfur moiety to gold.

The chemical stability of the resultant coating sensor surfacesas investigated via repeated exposure to alkaline solutions

Fig. 1, 20 times 100 mM NaOH injections followed by 20 times00 mM NaOH 1% Triton X100 injections). Initial exposure ofDEC dextran to NaOH resulted in the loss of ∼1200 RU or1.2 ng/mm2, most likely due to removal of physisorbed rather

han chemisorbed material. However, upon subsequent repeatedxposure as described above, only 5 RU, or ∼5 pg/mm2 of mate-ial was removed from the surface. PDEC dextran was alsohown to be resistant to injected pulses of 1 M NaCl, 10 mMlycine pH 2.0, and 100 mM HCl. The same material stored at20 ◦C for 1 year showed similar levels of immobilization to the

reshly prepared material and was subsequently demonstrated toetain activity for receptor capture (vide infra).

.3. Human serum albumin (HSA) immobilization

Having demonstrated facile deposition and subsequent chem-cal resistance of PDEC dextran we wished to probe thetility of the sulfhydryl reactivity inherent in the polymeror biosensing applications with a pharmacologically relevanteceptor. The remaining sulfhydryl reactive functionality, (2-yridinyl)disulfanyl, in the polymer coating matrix on singlePR chip, which was fabricated in a Petri dish in a staticode, is in the range of 1.0–1.5 nmol that was quantified,ith a UV–vis spectrophotometer at the wavelength of 270 nm,

rom the concentration of 2-mercaptopyridine released by 1 mMTT treatment for 2 h. Human serum albumin (HSA,) was

hosen as a model protein as coupling of HSA to aldehyder carboxyl surfaces is problematic due to the extremely lowI (4.7) of the protein. In addition, amine-coupling results inrandom orientation on the surface and the different drug

inding sites can be sterically occluded by the linkage to theolid support near that site. HSA is a 66 kDa globular pro-

ein that possesses a single sulfhydryl group at residue 34,hich should allow a site-specific immobilization. In order to

scertain whether immobilization of HSA to PDEC dextranas mediated via the residue 34 cysteine, and not via simple

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extran surface (©) and the capped surface (�) which was treated with 5 minnjection of 50 mM l-cysteine in 100 mM formate buffer pH 4.3 beforehand atow rate 10 �l/min.

hysisorbtion, a control polymer surface was generated by pre-reatment with 50 mM l-cysteine in 100 mM formate buffer pH.3. This step effectively ‘caps’ the reactive 2-pyridinyldithioroups in PDEC dextran as demonstrated by the detection of-mercaptopyridine in the UV adsorbance assay. HSA solutions1 mg/ml) in 100 mM borate buffer pH 8.5 were then repeatedlyxposed for 5 min to both unreacted PDEC dextran (Fig. 2, ©)nd the control, capped polymer (Fig. 2, �). Whilst ∼2500 RUr ∼2.5 ng/mm2 of protein was coupled to the PDEC dextranurface, less than 150 RU or 0.15 ng/mm2 was bound to the con-rol surface. This result suggests that coupling of HSA to theurface was indeed mediated by the reactive 2-pyridinyldithioroups of PDEC dextran. The result further demonstrates thatysteine-capped PDEC dextran is relatively resistant to non-pecific physisorbtion of HSA, despite the introduction of awitterionic charge arising from the cysteine residues on theurface.

.4. Anti-HSA interaction with the surface bound HSA

The above result confirms facile deposition and specificoupling of a protein using the novel polymer. However, toemonstrate utility in biosensing assays it is important to fur-her establish that the immobilization conditions and interfacial

icro-environment do not destroy protein activity for analyteinding. To probe the remaining activity of PDEC dextran sur-ace bound HSA, 333 nM of mouse monoclonal anti-HSA IgGntibody (mAb; 150 kDa) in PBS pH 7.4 was passed over aurface that had been coated with 2230 RU of immobilizedSA. This resulted in an equilibrium response for antibodyinding of 1750 RU (Fig. 3A-i), and a calculated maximal satu-ating response (Rmax) for binding of the bivalent antibody to aonovalent receptor of 2500 RU. When compared to the exper-

mentally determined value for Rmax of 2230 RU, this gives arotein activity level of 89%. We note, however, that the bind-ng stoichiometry of antibody:HSA could lie between the range

f 1:2 to 1:1, as not all of the surface immobilized HSA mayossess the correct spatial separation and orientation to bind stoi-hiometrically to antibody. With this caveat, the effective proteinctivity on the surface is thus predicted to lie within the region of

X. Li et al. / Colloids and Surfaces B:

Fig. 3. The specific binding of anti-HSA antibody to HSA surface, regenerationand reproducibility. (A) Antibodies binding to HSA (2230 RU immobilized)surface and regenerations: (i) 5 min injection of 50 �g/ml anti-HSA in PBS bufferpH 7.4; (ii) 5 min injection of 10 mM NaCl–HCl solution pH 3.0; (iii) 5 mininjection of 10 mM NaCl–HCl solution pH 2.0; (iv) 5 min injection of 50 �g/mlanti-BSA in PBS buffer pH 7.4; (v) 5 min injection of 50 �g/ml rabbit anti-mouse antibody in PBS buffer pH 7.4; (vi) 5 min injection of 10 mM NaCl–HClsolution pH 2.0; (vii) 5 min injection of 50 �g/ml anti-HSA in PBS buffer pH7.4; (viii) 5 min injection of 10 mM NaCl–HCl solution pH 2.0. (B) Seven timesrsd

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epeats of anti-HSA binding to HSA (1240 RU immobilized, solid line) activeurface and BSA (2650 RU immobilized, dotted line) control surface on PDECextran-coated Biacore sensor chip.

5–89%. The binding complex was not dissociated by 10 mMaCl–HCl solution pH 3.0 (Fig. 3A-ii), but was disrupted atH 2.0, restoring the initial baseline level signal trace (Fig. 3A-ii). To probe the specificity and selectivity of analyte-bindingo the active surface, 333 nM mouse monoclonal anti-BSAgG (Fig. 3A-iv) and 333 nM rabbit anti-mouse antibodiesFig. 3A-v) in PBS pH 7.4 were applied sequentially across theSA immobilized surface for 5 min. Neither protein resulted

n association of more than 90 RU or 90 pg/mm2 of mate-ial, confirming that the surfaces were highly bio-specific fornalyte.

Regeneration of free HSA was achieved by injection of0 mM NaCl–HCl solution pH 2.0 (Fig. 3A-vi). Repeated injec-ion of 333 nM of anti-HSA mAb in PBS pH 7.4 resulted in anquilibrium level of 1730 RU (Fig. 3A-vii), within 2% of the ini-ial response (Fig. 3A-i). To further probe assay reproducibility,ew assay surfaces were used with HSA (1240 RU) and BSA2650 RU) challenged by mAb anti-HSA antibody binding and

egeneration repeated seven times. For these assays antibodyinding generated 1080 ± 25 RU on the HSA surface (Fig. 3B,olid line), and only 58 ± 2 RU on the reference BSA channelFig. 3B, dotted line).

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Biointerfaces 61 (2008) 113–117 117

. Conclusion

In conclusion we have fabricated a novel biosensor coat-ng material that possesses “activated” reactive groups forirect and facile deposition on gold surfaces with subsequentapture of sulfhydryl presenting receptors. This procedure isar simpler than published methods that rely on a multi-tep conversion of carboxylated dextran using carbodiimidesnd cross-linking reagents such as succinimidyl 4-(N-aleimidomethyl)-cyclohexane-1-carboxylate (SMCC) [4,8].

n addition, the novel coating overcomes several of the disadvan-ages reported for disulfide-containing polymers [11] on gold,hich exploit short alkyl chain moieties for attachment. Finally,

arly (1 year) stability trials suggest that the activated materials compatible with storage and shipping that is a pre-requisite toommercialization.

cknowledgement

The authors would like to express their gratitude to theational Institute of Allergy and Infectious Diseases for thenancial support, NIH Grant number AI-061243-02.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.colsurfb.2007.06.026.

eferences

[1] S.V.A. Rao, K.W. Anderson, L.G. Bachas, Oriented immobilization ofproteins, Mikrochim. Acta (1998) 127–143.

[2] K.L. Prime, G.M. Whitesides, Adsorption of proteins onto surfaces con-taining end-attached oligo(ethylene oxide)—a model system using SAM,J. Am. Chem. Soc. 115 (1993) 10714–10721.

[3] E. Ostuni, R.G. Chapman, R.E. Holmlin, S. Takayama, G.M. Whitesides,A survey of structure–property relationships of surfaces that resist theadsorption of protein, Langmuir 17 (2001) 5605–5620.

[4] S. Lofas, B. Johnsson, A novel hydrogel matrix on gold surfaces in surfaceplasmon resonance sensors for fast and efficient covalent immobilizationof ligands, J. Chem. Soc., Chem. Commun. (1990) 1526–1528.

[5] K. Brocklehurst, J. Carlsson, M.P. Kierstan, E.M. Crook, Covalent chro-matography, Biochem. J. 133 (1973) 573–584.

[6] M.A. Cooper, Optical biosensors in drug discovery, Nat. Rev. Drug Discov.1 (2002) 515–528.

[7] K.E. Sapsford, F.S. Ligler, Real-time analysis of protein adsorption to avariety of thin films, Biosens. Bioelectron. 19 (2004) 1045–1055.

[8] M. Burgener, M. Sanger, U. Candrian, Synthesis of a stable and specificsurface plasmon resonance biosensor surface employing covalently immo-bilized peptide nucleic acids, Bioconjugate Chem. 11 (2000) 749–754.

[9] P. Schuck, A.P. Minton, Kinetic analysis of biosensor data: elementary testsfor self-consistency, Trends Biochem. Sci. 21 (1996) 458–460.

10] E. Stenberg, B. Persson, H. Roos, C. Urbaniczky, Quantitative determina-

tion of surface concentration of protein with surface-plasmon resonanceusing radiolabeled proteins, J. Colloid Interf. Sci. 143 (1991) 513–526.

11] R.A. Frazie, G. Matthijs, M.C. Davies, C.J. Roberts, E. Schacht, S.J.Tendler, Characterization of protein-resistant dextran monolayers, Bio-mater. 21 (2000) 957–966.