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Talanta 77 (2008) 652–658

Contents lists available at ScienceDirect

Talanta

journa l homepage: www.e lsev ier .com/ locate / ta lanta

dsorption versus covalent, statistically oriented and covalent, site-specific IgGmmobilization on poly(vinyl alcohol)-based surfaces

atarzyna Derwinska, Ursula Sauer, Claudia Preininger ∗

ustrian Research Centers GmbH – ARC, Department of Bioresources, 2444 Seibersdorf, Austria

r t i c l e i n f o

rticle history:eceived 16 January 2008eceived in revised form 19 June 2008ccepted 3 July 2008vailable online 16 July 2008

eywords:oly(vinyl alcohol)

a b s t r a c t

Plain poly(vinyl alcohol) (PVA) surfaces, PVA surfaces tailored with additives (chitosan, chitosan-oligo)and PVA surfaces crosslinked with homo- or hetero-bifunctional amino-linkers (ethylenediamine, hexam-ethylenediamine, adipic acid dihydrazide, 3-aminophenylboronic acid) were evaluated for their ability toimmobilize IgG. Immobilization strategies tested were adsorption as well as covalent, statistically orientedand covalent, site-specific binding of antibodies. The PVA surfaces were optimized with respect to the typeof PVA, to PVA concentration and to glass substrate type. The resulting hydrogel surface of choice consistsof 4% PVA coated onto adhesive glass. Comparison of modified and unmodified PVA surfaces revealed six

gGdsorptionrosslinkersminophenylboronic acid

surfaces which showed significantly higher loading capacity than plain PVA:PVA surfaces tailored with2% chitosan resulted in twice greater fluorescence, whereas PVA surfaces oxidized using HIO4 with andwithout further crosslinking using adipic acid dihydrazide revealed 2.6–2.8 times greater fluorescence.Yet the greatest fluorescence compared with plain PVA (up to 3.5 times as much) was achieved on PVAsurfaces coupled with 3-aminophenylboronic acid activated by means of either 1% or 2.5% glutaraldehyde.Meanwhile, fluorescence signals were similar for statistically oriented IgG and IgG bound site-specifically

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. Introduction

The choice of proper surface chemistry in protein chips is criti-al due to the structural complexity of proteins. The biochip surfaceherefore holds a central role in the protein chip development as themmobilization strategy applied influences sensitivity and speci-city of the chip experiment. Strength of the binding, orientationnd accessibility of the probes, density of bound probe moleculesnd the proportion of non-specific adsorption depend on the reac-ion chemistry.

Various chip surfaces have been reported [1,2] ranging fromilane and gold monolayers to functional polymers and hydrogels.he latter are considered especially suitable for protein immo-ilization, since they provide a liquid microenviroment that caneep the proteins hydrated and stabilize the structure, which isesponsible for the protein’s activity [3]. The hydrogels that haveeen reportedly used as immobilization matrices on protein chips

re: agarose [4], poly(acrylamide) [3,5,6], polyurethane [7], dextran8] and polyethyleneglycol (PEG). For example, Dominguez et al.9] fabricated antibody-entrapped hydrogel chambers by arrayingolutions of both tetra- or octa-amine functionalized peptide-

∗ Corresponding author. Tel.: +43 50550 3527; fax: +43 50550 3666.E-mail address: claudia.preininger@arcs.ac.at (C. Preininger).

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039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2008.07.016

meta-periodate.© 2008 Elsevier B.V. All rights reserved.

ased branch macromolecules and IgG on aldehyde glass slides.n contrast to many covalent attachment methods this approachs single-step, facile and rapid, keeps the antibody hydrated andn its original conformation (no modification by crosslinkers or byurface reactive groups as a result of immobilization). A similarpproach was reported by Rubina et al. [5], who used so-calledydrogel drop microchips of polyacrylamide for a quantitative

mmunoassay of plant and bacterial toxins. Derwinska et al. [7]eveloped polyurethane surfaces that compared with commercialydrogel slides showed significantly improved loading capacity,specially at low IgG concentrations. This agrees well with Zubtsovt al. [10], who compared the chip performance of hydrogel andon-hydrogel surfaces in direct and sandwich immunoassays con-luding that at the same concentration of spotted antibody hydrogelurfaces provide stronger fluorescence signals than non-hydrogellides. This effect was attributed to the relatively large molec-lar size of the antibodies and the improved capacity of gelurfaces.

Widely used hydrogels in biochips are based on acrylamide andextran, whereas PVA, by contrast, is used mainly as an encapsula-

ion material for enzymes and cells, in drug delivery, or as blends inbio)sensors. Photosensitive poly(vinyl alcohol-styrylpyridinium)PVA-SbQ), for example, is cited as having been used for surface-atterning of a bio-MEMS-based cell chip using recombinantscherichia coli [11], and according to [12] PVA was part of a

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olymer blend in a glucose biosensor. In order to produce mechan-cally stable PVA layers, often polymer blends or co-polymers areeveloped and employed that combine the mechanical strength

f the additive polymer blend or monomer with the biocompati-ility and hydrophilicity of PVA, for example mechanically stableVA ormosils using organically modified silicates (ormosils) forOD biosensing in seawater [13] or polysiloxane–PVA discs fab-icated through sol–gel process for binding of anti-S100 protein

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ig. 1. Binding principle schematically shown for (A) antibody adsorption on the PVA surfnd APBA and glutaraldehyde (right), and (C) site-specific attachment of IgG–CHO on PVA

77 (2008) 652–658 653

ntibody [14]. In addition, improved hydrogel strength has alsoeen obtained by crosslinking PVA with glutaraldehyde [14–16]r chitosan. For example, Yu et al. [15] reported on PVA func-

ionalized poly(dimethylsiloxane) surfaces that were activated forntibody binding using glutaraldehyde, whereas Kumar et al. [17]repared PVA membranes of different swelling index for entrap-ent of glucoseoxidase combining PVAs of low and high degree of

olymerization together with a photolinker. Furthermore, boronic

ace, (B) covalent binding on PVA surfaces crosslinked with 2EI, HDA and ADH (left),/chitosan and PVA crosslinked with ADH.

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cids were reported to covalently bind to 1,2 or 1,3 diols, such asVA which was taken advantage of in biomolecule immobilization18] as well as sensing of glucose and polysaccharides [19].

Herein, PVA was chosen because it is one of the most hydrophilicolymers and thus can pre-eminently preserve proteins’ activityue to its high water content, and furthermore prevent non-specificdsorption. To fully exploit the potential of PVA as coating materialn biochips we aim to optimize layer fabrication as well as surface

odification and antibody binding.In the following, we describe first additive-functionalized and

hen crosslinked PVA, which we employed as an immobilizationatrix for IgG and IgG activated with sodium periodate in order

o evaluate: (1) the suitability of modified and unmodified PVAsn protein arrays; and (2) the effectiveness of simple, one-step IgGdsorption versus multi-step statistically oriented and site-specificovalent immobilization.

. Materials and methods

.1. Materials

Dodecyl sulfate sodium salt (SDS) was provided by Mercknd phosphate buffered saline (PBS) by Gibco. AminosulfobetainASB-14), sodium deoxycholate, cysteamine chloride, octyl-�--1-thioglucopyranoside, glutaraldehyde (25% in water) and-aminophenylboronic acid monohydrate (98%) (APBA) were fromigma whereas 3-(decyldimethylammonio) propanesulfonatenner salt (SB3-10), 3-[(3-cholamidopropyl) dimethylammonio]--propanesulfonate (CHAPS), sodium N-dodecanoyl-N-ethylglycinate (sarcosyl) and hexadecyltrimethylammonium

romide (CTAB) were from Fluka. Tween-20 was purchased fromluka. Monochlortriazinyl-�-cyclodextrin sodium salt (MCT) wascquired from Wacker. All other reagents were analytical grade.

.2. Chip fabrication and surface modification

Aqueous solutions of 1%, 4% and 10% PVA1 (Sigma-36306, Mw46,000–186,000, hydrolysis degree 99+%), PVA2 (Sigma-341584,w 89,000–98,000, hydrolysis degree 99+%), PVA3 (Sigma-363103,w 146,000–186,000, hydrolysis degree 87–89+%), and PVA4

Aldrich-9002895, Mw 85,000–146,000, hydrolysis degree 99+%),% PVA4/2% chitosan (food grade, Dalwoo) and 4% PVA4/2% oligo-hitosan (food grade, Dalwoo) were prepared and dip-coated ontoither plain glass (Sigma, 8902), Silane PrepTM (Sigma, S4651)r adhesive Histobond slides (Marienfeld, no. 08 100 00) usinghe KSVD dip coater by KSV Instruments (velocity: 100 mm/min;etention time: 60 s; retention time between layers: 2 min). ForgG adsorption (schematically shown in Fig. 1A) the PVA slidesere used without further treatment, whereas for covalent IgG

mmobilization adhesive glass slides were coated with 4% PVA4nd modified with bifunctional crosslinkers, such as ethylene-iamine (2EI) (Fluka), hexamethylenediamine (HDA), adipic acidihydrazide (ADH) (Aldrich), chitosan and chitosan-oligo subse-uently to HIO4 oxidation (the slides were immersed in 1% aqueousIO4 for 1 h and then washed twice with MiliQ water before incu-ating the slides in 1% crosslinker solution (pH 8) for 60 min). PVA4lides modified with 3-aminophenylboronic acid were prepared byncubating the slides in 0.1N 3-aminophenylboronic acid monohy-rate (pH 8.6) for 1 h. The slides were washed intensively in distilledater and blown dry with compressed air. Schemes of the resultingodified surfaces are presented in Fig. 1B.

.3. Chemical modification of IgG

The carbohydrate groups of IgG were activated using sodiumeta-periodate as described in [20]. Briefly, 3 mg ml−1 IgG in 0.1 M

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77 (2008) 652–658

odium acetate buffer (NaOAc) (pH 5.5) was incubated with sodiumeta-periodate (25 mg ml−1 in NaOAc buffer, pH 5.5). After 1 h

lycerol was added to stop the reaction. The activated antibodyas then filled into microfilterfuge tubes (Microcon YM-30, Mil-

ipore) and centrifuged to separate the antibody from excess oferiodate. Antibody samples were then washed twice with coldaOAc. The activated IgG (IgG–CHO) was arrayed onto plain 4%VA4, 4% PVA/2% chitosan and 4% PVA4 surfaces crosslinked withDH. The binding principle is shown in Fig. 1C. The concentrationf IgG–CHO was determined spectrophotometrically with respecto non-activated IgG using the NanoDrop (ND-1000, protein a 280

ode) and the loss of material due to chemical modification wasalculated as percentage of the starting material.

.4. Microarray printing

Glutaraldehyde crosslinked surfaces were prepared by spotting% and 2.5% glutaraldehyde in 1× PBS (pH 7.2) respectively ontoVA4/APBA surfaces prior to antibody spotting. Three replicates of.005–1 mg ml−1 rabbit IgG (technical grade, Sigma) were arrayednto PVA-based surfaces using the OmniGrid contact spotter fromeneMachines (pin SMP3). Unless stated otherwise, 1× PBS (pH 7.2)as used as print buffer. The spot-to-spot distance was 500 �m,

pot volume was 0.6 nl.

.5. Postarraying and blocking

After arraying, the slides were incubated in a humid chambert 4 ◦C overnight to complete probe immobilization. Surface block-ng was performed in 1× PBS (pH 7.2)/0.1% Tween-20 to rinse offnbound protein and deactivate reactive surface groups. Finally, thelides were washed twice in 1× PBS (pH 7.2) and then blown drysing compressed air or spun dry in the centrifuge (900 rpm formin).

.6. Determination of immobilization capacity

Immobilization capacity in fmoles/mm2 was calculated by tak-ng the median fluorescence minus the local background of 27eplicate spots of 0.05 mg ml−1 dye-labelled anti-IgG before andfter blocking (30 min) multiplied by spotted protein concentra-ion and divided by molecular mass of labelled protein and squareadius of the spot. 27 replicates were obtained by using three slidesith nine replicates each. Thereby data FA and FB were determinedsing the same chip. The calculation was done according to formula1) (I, immobilization capacity; FB, fluorescence before blocking; FA,uorescence after blocking; MLP, molecular mass of labelled pro-ein; R, spot radius (typically 120 �m); CLP, concentration of spottedrotein (0.05 mg ml−1)). The factor 1.9 × 106 is calculated from theolume of the protein solution per spot (0.6 nl/spot) and the �.

= 1.9 × 106 FA

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.7. Direct immunoassay

Protein slides were processed with 4 ng �l−1 Dy633-labellednti-Rabbit IgG (Dyomics) in 1× PBS (pH 7.2)/0.1% Tween-20 at 4 ◦Cor 3 h, then washed twice in 1× PBS (pH 7.2) and spun dry in theentrifuge (900 rpm for 3 min).

.8. Fluorescence detection and data analysis

Slides were stored in the dark and scanned at �ex = 635 nm andem = 670 nm on the same day the immunoassay was performed.

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luorescence measurements were taken using a GenepixTM 4000Bon-confocal scanner from Axon Instruments. For data comparison,he photomultiplier tube (PMT) was kept constant within singlexperiments. All fluorescence (a.u.) data are background-corrected.dditionally, data flagged as bad, according to parameters set in theenepix software (e.g. spot diameter 30–480 �m; signals >100 a.u.uorescence), were filtered.

.9. Profilometry

Layer thicknesses were measured over 2.5 mm × 2.5 mm sur-ace areas using the Wyko NT1100 optical profiling system (Veeco)nd Vision32 Veeco software. The values are mean values for twoeasurements.

. Results and discussion

.1. IgG adsorption

.1.1. Choice of PVA4% PVA1, −2, −3 and −4 coated onto aminosilane substrates

ere tested in a direct on-chip immunoassay using IgG adsorbedn the PVA-surface. The following criteria were taken into accountn evaluation: fluorescence signals (a.u.), background (a.u.) andpot morphology. Fig. 2 compares the fluorescence obtained athe maximum loading concentration of IgG on PVA surfaces 1–40.5 mg ml−1 IgG; spot volume 0.6 nl; 300 pg IgG/spot). The flu-rescence signals were mean values calculated from 27 spotsthree slides, nine replicates each). As is evident from Fig. 2,

olecular weight (Mw) and hydrolysis degree of PVA plays anmportant role in the assay performance. Up to 30% stronger sig-als were achieved using PVA1 as compared to PVA3, despite theame molecular weight distribution. This is most likely due tohe increased number of hydroxy groups available for IgG load-ng. The influence of molecular weight on fluorescence signals, at aydrolysis degree of 99+%, is not entirely clear. It can nonethelesse observed that PVA2, with the lowest Mw, and PVA4, con-aining low and high Mw parts, produce the strongest signals.ignals for the highest Mw PVA tested (PVA1) were 40–60% weaker.VA2, while having a narrower molecular weight distribution thanVA4, shows a similar IgG loading curve and equivalent assayerformance. This indicates that the optimum molecular weight

or the tested application is between 90.000 and a maximum of46.000. PVA4 was chosen for further measurements, as the % coef-cient of variation (CV) was 11%, i.e. four times lower than forVA2.

ig. 2. Fluorescence achieved with 0.5 mg ml−1 IgG spotted on PVA1, PVA2, PVA3nd PVA4 surfaces. Bars indicate the distribution of Mw for the tested PVAs.

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ig. 3. Loading of 0.005–1 mg ml−1 IgG on ( ) plain, ( ) aminosilane and ( )dhesive glass slides covered with 4% PVA4.

.1.2. Choice of substrateThe choice of optimal substrate is crucial for microarray surface

evelopment. The substrate should allow good polymer adherenceithout peeling off during the microarray experiment and display

ow autofluorescence at the wavelengths of interest. Three differentubstrates were coated with 4% PVA4 and evaluated: unmodifiedlass (Sa = 5.81 nm), aminosilane glass (Sa = 1.73 nm) and adhesivelass (Sa = 12.5 nm). Due to pretreatment, the latter two substratesere expected to foster stronger PVA binding, resulting in a more

table PVA-layer, as hydrogel swelling along the substrate is therebynhibited. The best results in terms of fluorescence signal, as shownn Fig. 3, were obtained on the adhesive substrate, whereas the plainlass and the silanized glass resulted in signals that were at least0% weaker. However, concerning the loading capacity saturationccurs at 0.5 mg ml−1 IgG (300 pg/spot), regardless of the substratesed. Although both aminosilane and adhesive glass provide reac-ive groups that are expected to bind PVA to the substrate moreffectively than plain glass, substrate modification evidently hado significant effect with regard to producing stable PVA surfaces.his behaviour contrasts previous studies on poly(urethane) (PU)7] which report comparable performance for both aminosilane anddhesive glass. An explanation for this might be found in the elec-rostatic interaction between PU and aminosilane. This is strongerhan between aminosilane and PVA, with PVA displaying only alight negative surface charge that decreases linearly from −2 mVt pH 5 to −6 mV at pH 9, while PU is more negatively charged,ith a � potential that decreases linearly from −9 mV at pH 5 to24 mV at pH 9. In the present case, the improved loading capac-

ty on PVA-coated adhesive substrates is most likely the result ofncreased substrate roughness (the roughness of adhesive glass isix times greater than that of aminosilane glass). Increased rough-ess leads to better coverage of the substrate by PVA and to the

ormation of a rougher PVA layer onto which a higher amount ofgG can be adsorbed, which in turn results in stronger fluorescenceignals.

.1.3. Optimization of PVA concentration

Adhesive slides coated with aqueous solutions of 1%, 4% and

0% PVA4 were evaluated with respect to mechanical stability overncubation time (3 h) and to loading capacity, as determined in anmmunoassay and by spotting labelled IgG. Fluorescence of spot-ed IgG processed with 4 ng �l−1 Dy633-labelled anti-Rabbit IgG

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ncreases with increasing PVA concentration: when PVA concentra-ion was increased by a factor of 4, signals were 2.5 times stronger,hereas five times stronger signals were obtained by increasing

VA concentration by a factor of 10. This is most likely due to betterubstrate coverage with PVA4 at higher hydrogel concentrations.

The thickness of a dip-coated film is influenced by the dipcoat-ng velocity, especially the withdrawal speed, the viscosity of theoating solution, which is a function of the polymer concentrationnd the acceleration due to gravity. At constant withdrawal speedhe amount of coating solution moving upwards with the substrates larger for a more viscous solution, since the drag force is propor-ional to the solution viscosity. In fact, the thickness of the hydrogelayer increased significantly with increasing PVA concentration:he layer thickness (dry state) for 1% and 4% PVA as determined byrofilometry was 46.5 nm and 407.5 nm, respectively. Thus, increas-

ng the hydrogel concentration by a factor of 4, enhances the layerhickness by nine times. The fact that the increase in layer thicknesss more pronounced might be related to a comparatively strongerncrease in viscosity with increasing PVA concentration.

Furthermore, increasing the PVA concentration resulted inmproved mechanical stability: when using 1% PVA surfaces twices much hydrogel dissolves out in solution during incubation (3 h)han with 4% PVA surfaces. As a consequence, hydrogel layer thick-ess for 1% and 4% PVA is reduced by 67.7% and 38.2%, respectively.part from the improved mechanical stability of surfaces consistingf high PVA concentration, the immobilization capacity is drasti-ally improved on thicker gels. This is obvious from the loadingurve as well as from the immobilization capacity calculated forabelled IgG: 1% PVA4–16 fmoles mm−2, 4% PVA4–91 fmoles mm−2,nd 10% PVA4 126 fmoles mm−2. Moreover, the improved IgG load-ng on 4% and 10% PVA4 surfaces may be a result of increasedensity of OH-groups on the surface that due to their polarityromote the interactions between local dipoles existing on the

nteracting molecules [2]. In further experiments 4% PVA4 surfacesere employed because of the lower viscosity of solutions of 4%

VA and thus easier fabrication compared with 10% PVA.

.1.4. Effect of print buffer composition on IgG adsorptionAdsorption is a simple, one-step immobilization method.

ttachment occurs as a result of electrostatic and/or hydrophobicnteraction forces. Thus, the printing solution containing additivesf varying polarity and ionic charge can influence the strengthf IgG adsorption by affecting the wettability of the surface, theinding kinetics and the net charge both of the surface and ofhe protein. Several additives ranging from 0.01% to 0.001% in× PBS (pH 7.2) have been tested: ASB-14, Tween-20, sarcosyl,DS, MCT, CHAPS, CTAB, octyl-�-d-1-thioglucopyranoside, SB3-10,odium deoxycholate, cysteamine chloride and glycerol [1,21–23].

he choice of additives was based on their widespread use in elec-rophoresis for the prevention of aggregation and scientific reportsn improved printing solutions. In Fig. 4a fluorescence image ofrocessed IgG spotted in various printing solutions is presented.

ig. 4. Spot images of IgG spotted in various buffers based on 1× PBS (pH 7.2) (Flu:280, CV 29.5%). Spots in PBS containing 0.005% Tween-20 (Flu: 7600 a.u., CV 9.5%),.01% thioglucopyranose (Flu: 6335 a.u., CV 7.7%) and 0.01% ASB14 (Flu: 8527 a.u.,V 39%), respectively are highlighted.

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ith (6) ADA, (7) 2 EA, (8) 1, 6 HMA, (9) chitosan and (10) chitosan-oligo; and PVA4rosslinked with (11) aminophenyl boronic acid and additionally with (12) 1%, and13) 2.5% glutaraldehyde in comparison with unmodified 4% PVA4.

he triplicate spots of the most suitable print buffers as comparedith plain PBS are highlighted. The corresponding fluorescence

ntensity values and coefficients of variation (%CV) are indicatedn the figure caption. The use of PBS containing 0.005% Tween-0 (non-ionic), 0.01% octyl-�-d-1-thioglucopyranoside (non-ionic)nd 0.01% ASB14 (anionic) resulted in 1.8, 1.5 and 2 times strongeruorescence signals respectively than those obtained using PBSithout additives. These additives, obviously provide the greatest

olubilizing power and do not denature the antibody as strongly asther agents tested. As reported also by Brogan et al. [24] adjust-ent of additive concentration is critical, i.e. addition of 0.005%

ween-20 led to signal enhancement by 30%, whereas addition of.01% Tween-20 resulted in 1.5 times weaker fluorescence signalss compared to signals obtained in plain 1× PBS (pH 7.2).

.2. Covalent IgG immobilization on modified PVA

1 mg ml−1 IgG in 1× PBS was spotted onto PVA surfaces, ontoVA surfaces activated with HIO4, onto surfaces activated androsslinked with amino-functional linkers of various lengths (2EA;AD; DAH), and with 3-aminophenylboronic acid; and finally ontolass slides covered with PVA/chitosan and PVA/oligo-chitosan.gG was bound to the surfaces in a statistically oriented manner,ince some orientation already exists due to preferential bind-ng between the reactive surface groups and the amino- and thiolroups of the antibody. Fig. 5 shows the percentage of signal changeor each modified surface as compared with the plain PVA4 surface.s is evident from the figure, only modification with ADH, acti-ation with HIO4 and modification using APBA with and withoutlutaraldehyde led to significantly enhanced signals. Modificationith ADH addresses the thiol-groups in the cystein units, whereas

ctivated PVA can bind both amino and thiol groups present inhe antibody. The greatest signal enhancement was achieved upon

odification with APBA. This modification procedure is based onhe well-known interaction of boronic acids with polyols, such asVA or sugars. APBA is crosslinked to the PVA surface forming ayclic ester between the diol group of the PVA and the boronic acidroup of the crosslinker. The amino function was used for couplinggG via its thiol groups, or its amino groups, in the latter case if

lutaraldehyde was employed as an additional crosslinker. Func-ionalization of the chip surface by the addition of 2% chitosanesults in higher loading capacity as indicated by the two timesnhanced fluorescence, whereas 4% chitosan and the addition ofligochitosan led to a deterioration of the on-chip assay.

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ig. 6. Comparison of 0.1 mg ml−1, 0.25 mg ml−1, 0.5 mg ml−1 and 1 mg ml−1spotted) IgG and ( ) IgG–CHO on plain PVA4 surfaces, PVA4 crosslinked with adipic

cid dihydrazide (ADH) and PVA4/chitosan surfaces.

.3. Covalent, site-specific immobilization of modified IgG

In order to attach IgG to the surface site-specifically, IgG wasctivated with NaIO4 by oxidation of the carbohydrate residues inhe Fc fragment of the antibody and formation of reactive aldehyderoups (refer to Section 2). Three types of surfaces were employedor activated IgG immobilization: plain 4% PVA4, activated 4% PVA4rosslinked with adipic acid dihydrazide and 4%PVA/4% Chitosan.ig. 6 compares the fluorescence signals obtained with 0.1 mg l−1,.25 mg l−1, 0.5 mg l−1 and 1 mg l−1 spotted IgG and IgG–CHOespectively after processing with 4 ng �l−1 Dy633-labelled anti-abbit IgG. As is obvious from the figure, no improvement in loadingas achieved with oriented immobilization using activated IgG.oreover, data reproducibility did not improve either. This agreesell with Kusnezow et al. [25] who reported similar signal-to-noise

atios when using activated and non-activated antibodies as wells a loss of about 40% of antibody due to activation and purifi-ation steps (herein the loss was 33%). Moreover, Wacker et al.26] demonstrated that direct spotting and site-specific immobi-ization of IgG via streptavidin–biotin attachment is similar withegard to signal intensity, assay sensitivity and assay reproducibil-ty. By contrast, Peluso et al. [27] reported that specific orientationf capture agents (oriented IgG (biotinylated on carbohydrate onc domain), oriented Fab’fragments (biotinylated in hinge region))onsistently increases the analyte-binding capacity of streptavidinurfaces, with up to 10-fold improvements over surfaces with ran-omly oriented capture agents (randomly biotinylated IgG andab’fragments). This concurs strongly with Luk et al. [28] whoeported measuring for ribonuclease inhibitor (RI) a four-timesreater binding capacity (compared with random immobilization)n the case of RNase A immobilized with a preferred orientation.owever, one has to keep in mind that there is no common opinions to the effect of orientation on the assay performance using pro-ein chips and that the preparation and purification of specificallyctivated antibodies and antibody fragments is tedious; in additionreat material losses have to be accepted when applying this pro-edure. Clearly, in striving to achieve oriented antibodies one needso accept the trade-off of increased costs for more starting materials well as increased effort in preparation and purification of thentibody over and against possible improvement of on-chip assayerformance.

.4. Reproducibility of fabrication and storage stability of PVA

lides

PVA slides were fabricated in batches of 50 slides using the KSVipcoater. The software controlled dipping speed was 100 mm/minor immersion and 100 mm/min for withdrawal (hold time 60 s).

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77 (2008) 652–658 657

he slide-to-slide variation was encountered in data analysis in thatot only replicate spots (nine per slide), but also replicate slidesthree) were employed in each chip experiment. The inter-slideariation as described by the CV was typically 46%; the intra-slideariation was 33%.

The PVA slides were stored in the fridge to prevent them fromrying out. The slides were stable for at least 2 months withouteterioration of assay performance. However, when stored at roomemperature for more than 3 weeks slides lost their binding abil-ty resulting in reduced fluorescence signals. This is in contrast to

elo-Junior et al. [14], who reported a 30% activity loss in 6 months,hereas there was almost no decrease in loading capacity dur-

ng the first 2 months. Furthermore, spot morphology significantlyeteriorated which became evident by the deformation of spots.pots “frayed out”, most likely due to surface degradation. As spotorphology has a critical impact on data analysis, reproducibility

f data declined (CV 63%).

. Conclusions

Several immobilization chemistries were evaluated using chipurfaces based on PVA. In this, one-step IgG adsorption was opti-ized with regard to the type of PVA (Mw 85,000–146,000), the

VA concentration (4%) and the glass support type (adhesive).odification of PVA surfaces improved the loading capacity sig-

ificantly: addition of 2% chitosan resulted in two times strongerignals, whereas activation of PVA surfaces using periodate withr without subsequent crosslinking with adipic acid dihydrazider slide modification using 3-phenylboronic acid led to signalmprovement by a factor of up to 2.8. A minimum of a threefoldncrease in fluorescence was achieved with PVA surfaces modifiedy applying a two-step process using 3-phenylboronic acid and glu-araldehyde. Especially in sandwich immunoassays high loading ofntibody is important as the assay sensitivity is directly related tohe immobilization capacity. From the results presented it is clearhat one- or two-step processes relying on either adsorption orovalent, statistically oriented immobilization of IgG are in any caseore efficient, less tedious and result in better assay performance

han covalent, site-specific immobilization using IgG with activatedarbohydrate residues.

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