Analytica Chimica Acta 644 (2009) 95–103

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Analytica Chimica Acta

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New 3-D microarray platform based on macroporouspolymer monoliths

M. Robera, J. Walterb, E. Vlakha, F. Stahlb, C. Kasperb, T. Tennikovaa,∗

a Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoy pr. 31, 199004 St. Petersburg, Russiab Institut für Technische Chemie, Leibnitz Hannover Universität, Hannover, Germany

a r t i c l e i n f o

Article history:Received 16 December 2008Received in revised form 3 April 2009Accepted 6 April 2009Available online 17 April 2009

Keywords:Macroporous monolithic materialsThree-dimensional protein microarrayBioanalysis

a b s t r a c t

Polymer macroporous monoliths are widely used as efficient sorbents in different, mostly dynamic,interphase processes. In this paper, monolithic materials strongly bound to the inert glass surface aresuggested as operative matrices at the development of three-dimensional (3-D) microarrays. For thispurpose, several rigid macroporous copolymers differed by reactivity and hydrophobic–hydrophilicproperties were synthesized and tested: (1) glycidyl methacrylate-co-ethylene dimethacrylate(poly(GMA-co-EDMA)), (2) glycidyl methacrylate-co-glycerol dimethacrylate (poly(GMA-co-GDMA)),(3) N-hydroxyphthalimide ester of acrylic acid-co-glycidyl methacrylate-co-ethylene dimethacry-late (poly(HPIEAA-co-GMA-co-EDMA)), (4) 2-cyanoethyl methacrylate-co-ethylene dimethacrylate(poly(CEMA-co-EDMA)), and (5) 2-cyanoethyl methacrylate-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate (poly(CEMA-co-HEMA-co-EDMA)). The constructed devices were used as

platforms for protein microarrays construction and model mouse IgG—goat anti-mouse IgG affinity pairwas used to demonstrate the potential of developed test-systems, as well as to optimize microanalyticalconditions. The offered microarray platforms were applied to detect the bone tissue marker osteopontindirectly in cell culture medium.

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. Introduction

The microarrays represent independent and quite promisingnalytical tool with great potential at different practical fields, suchs molecular biology, medicine, analytical biotechnology, pharma-ology, ecology and bioinformatics. Ekins and coworkers explainedn ambient analyte theory that microspot assays have to be much

ore sensitive that conventional ligand-binding assays due to theighest fractional occupancy of the captured antibodies and highestignal per area achieved in microspots [1,2].

The application of biological test-systems for high sensitive

pot-analysis is based on a specific binding of target moleculeprotein, DNA, RNA, peptide, etc.) to its biocomplementary ligandmmobilized on a solid surface. The efficiency of processes basedn a principle of biorecognition (bioaffinity interactions) directly

Abbreviations: GMA, glycidyl methacrylate; EDMA, ethylene dimethacrylate;DMA, glycerol dimethacrylate; HPIEAA, N-hydroxyphthalimide ester of acryliccid; CEMA, 2-cyanoethyl methacrylate; HEMA, 2-hydroxyethyl methacrylate;yOH, cyclohexanol; DoOH, dodecanol; PEG, poly(ethylene glycol); 2-D and 3-, two- and three-dimensional, respectively; BSA, bovine serum albumin; IgG,

mmunoglobulin G; OPN, osteopontin.∗ Corresponding author. Tel.: +7 812 323 04 61; fax: +7 812 323 68 69.

E-mail address: tennikova@mail.ru (T. Tennikova).

003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2009.04.015

© 2009 Elsevier B.V. All rights reserved.

depends on ligand immobilization that, in turn, is dramaticallyinfluenced by properties of used solid support.

General approaches applied to the matrix functionalizationwith, for example, proteins are physical adsorption, covalent attach-ment via specific surface interaction and bioaffinity (oriented)immobilization [3,4]. It is a real problem to define the best wayof protein immobilization for all developed systems, because theresult will depend both on the properties of biomolecule to beimmobilized and used solid matrix, that is optimal experimentalconditions should be found for every protein affinity pair [5,6].

Bioanalytical microarrays can be constructed in two formats,namely, 2-D and 3-D devices. First is based on the use of so-calledplanar two-dimensional supports. In this case, the substance ofinterest forms a specific pair with a ligand located on a rigid mono-layer representing microarray’s surface. Such devices based on glassslides, non-porous synthetic polymers and metals provide the uni-form signal and satisfactory results reproducibility [7]. At the sametime, 3-D microarrays are expected to reduce the steric hindranceproblems, to offer the higher affinity capacity and, consequently,

to increase a sensitivity of analysis, as well as to provide similar tosolution conditions for immobilized biomolecules and, therefore,to prevent the loosing of its binding activity.

One of the first examples of 3-D microarrays was polyacrylamidegel pads attached to the glass surface which were developed by

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irzabekov and coworkers [8,9]. These devices provided increasedffinity capacity, reduced protein denaturation and limited non-pecific protein binding. However, it was reported by Mirzabekovnd coworkers [8], as well as by Kusnezow et al. who tested aydrogel surface from Perkin Elmer [10], that three-dimensionalel structure played a role of additional barrier for diffusion ofarge molecules and required very long incubation times or somedditional experimental tricks to accelerate diffusion of macro-olecules, such as, the use of electrophoresis or mixing the

olutions with a micropump within the microarray chamber [8].icroarrays based on nitrocellulose membranes are another exam-

le of non-planar microunits. Due to their enormous bindingapacity they provide much higher signal intensity in comparisonith 2-D microarrays [11]. Nevertheless, it has some disadvan-

ages, such as significant non-specific protein binding which makesitrocellulose surfaces not suitable for analysis of complex proteinixtures, as well as quite high autofluorescense. Colorizing of aembrane in black colour was suggested as an original solution of

he last problem [12].In the early 1990s a new class of continuous media based on a

igid macroporous copolymer of glycidyl methacrylate and ethy-ene dimethacrylate, or poly(GMA-co-EDMA), synthesized by aulk polymerization and commonly called later as monolith, waseveloped and introduced to the World market as fast and effi-ient chromatographic stationary phase [13,14]. Similarly to theacroporous polymer beads, monoliths are characterized by fixed

orous properties, maintained constant even in a dry state. Theirnternal structure represents a system of interconnected polymer

icroglobules separated by pores, and their structural rigidity isecured by extensive cross-linking. Today monolithic materials areuccessfully used in a wide range of fast interphase dynamic pro-esses, such as high-performance liquid chromatography [15–18],ioconversion processes (flow-through enzyme reactors) [19,20],apillary electrochromatography [21] and solid phase extraction22].

Recently a new type of 3-D microarrays intended for analyti-al detection of influenza virus and based on a rigid macroporousoly(GMA-co-EDMA) monolithic layers, have been developed byur scientific group [23]. In this paper for the first time a quantita-ive comparison of affinity pair formation at flow-through (affinityhromatography) and non-flowing (microarray) conditions wasarried out.

The matter of present study was the application of newacroporous monolithic materials for 3-D microarray construc-

ion, as well as a comparison of their bioanalytical potentials.or these purposes, five developed and thoroughly character-zed macroporous monolithic copolymers were used: glycidyl

ethacrylate-co-ethylene dimethacrylate (poly(GMA-co-EDMA)),lycidyl methacrylate-co-glycerol dimethacrylate (poly(GMA-co-DMA)), N-hydroxyphthalimide ester of acrylic acid-co-glycidylethacrylate-co-ethylene dimethacrylate (poly(HPIEAA-co-MA-co-EDMA)), 2-cyanoethyl methacrylate-co-ethyleneimethacrylate (poly(CEMA-co-EDMA)) and 2-cyanoethylethacrylate-co-2-hydroxyethyl methacrylate-co-ethylene

imethacrylate (poly(CEMA-co-HEMA-co-EDMA)). Original func-ional groups in the structure of synthesized copolymers madeossible realization of one-step surface modification with protein

igands. The optimization of analytical procedure was performedsing model protein affinity pair, namely, mouse IgG—goat anti-ouse IgG conjugated with Alexa Fluor 555. The detection limit

ound for developed devices was compared with that observed

or 2-D microarrays based on aldehyde-modified glass slides. Toemonstrate the efficiency of suggested microarrays for deter-ination of target proteins in complex biological liquids, the

rocedure of qualitative detection of well-known bone tissuearker osteopontin in cell culture medium has been developed.

a Acta 644 (2009) 95–103

2. Materials and methods

2.1. Reagents

Glycidyl methacrylate (GMA, 97% pure), ethylene dimethacry-late (EDMA, 98% pure), cyclohexanol (99% pure), glyceroldimethacrylate (GDMA, 85% pure), 2-hydroxyethyl methacrylate(HEMA, 98% pure), 2-hydroxy-2-methylpropiophenon (Darocur-1173, 97% pure), 3-(trimethoxysilyl) propyl methacrylate, PEG-600, 1,4-dioxan were purchased from Sigma–Aldrich GmbH(Taufkirchen, Germany). N-hydroxyphthalimide ester of acrylicacid (HPIEAA) was synthesized from acryloyl chloride (97%pure) produced by Sigma–Aldrich GmbH (Taufkirchen, Germany)and N-hydroxyphthalimide (97% pure, Sigma–Aldrich GmbH(Taufkirchen, Germany)) in a presence of triethylamine (99%pure, Vekton (St. Petersburg, Russia)) dissolved in tetrahydro-furan (99% pure, Sigma–Aldrich GmbH (Taufkirchen, Germany))according to the published method [24]. 2-Cyanoethyl methacry-late was purchased from Yarsinthez (Yaroslavl’, Russia). Purifiedmouse IgG was obtained from Zytomed (Berlin, Germany), goatanti-mouse IgG conjugated with Alexa Fluor 555 was pur-chased from Molecular Probes (Eugene, USA). TopBlock waspurchased from Fluka (Buchs, Switzerland). Human osteopon-tin, monoclonal mouse anti-osteopontin IgG and polyclonalmouse biotinylated anti-osteopontin IgG were purchased fromR&D Systems (Wiesbaden, Germany). Cy3-conjugated strep-tavidin was obtained from Jackson ImmunoResearch Labora-tories (Suffolk, UK). The reaction of silanization was car-ried out in toluene (Vecton Ltd, Russia). Following bufferswere used for microanalytical manipulations: sodium boratebuffer (1.25 × 10−2 M Na2B4O7 × 10H2O, 8.8 × 10−3 M NaOH),PBS (0.137 M NaCl, 2.7 × 10−3 M KCl, 4.3 × 10−3 M Na2HPO4,1.4 × 10−3 M KH2PO4), SSC (3 M NaCl, 0.3 M Na3C6H5O7) containing2% sodium dodecyl sulfate. All buffers were prepared by dissolvingthe analytical grade salts in distilled water and were additionallypurified by filtration through a 0.45 �m Milex Millipore microfilter(Wien, Austria).

The glass slides with dimensions 25 mm × 75 mm × 1.2 mmwere obtained from BioVitrum (St. Petersburg, Russia). The glassaldehyde slides were purchased from CEL Associates Inc. (Pearland,USA).

2.2. Apparatus

The 125 W mercury lamp (Philips, Netherlands) of wide radi-ation spectrum and constant intensity was used for free-radicalpolymerization. The monolith morphology was studied using scan-ning electron microscope JSM-35 CF produced JEOL (Tokyo, Japan).The mean pore size and pore size distribution were determinedby mercury intrusion porosimetry using PASCAL 440 ThermoquestInstrument (Rodano, Italy).

For spotting of proteins to be immobilized on a microarray sur-face sciFLEXARRAYER S3, Scienion (Berlin, Germany) was used. Thewashing procedure was carried out using a Thermomixer Comfort(Eppendorf, Germany). The special secure seal hybridization cham-bers (Grace Biolabs, Bend, USA) were used to perform the couplingof immobilized mouse IgG with goat anti-mouse IgG conjugatedwith Alexa Fluor 555. Microarrays were scanned using ScannerGenePix 4000 B (Axon Instruments, USA). GenePix 6.0 software wasused to analyze the scan data.

2.3. Methods

2.3.1. Microarray fabricationThe first step of microarray construction was glass surface etch-

ing with hydrofluoric acid (HF) using special mask. The process was

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M. Rober et al. / Analytica C

arried out at intensive interfusion (reaction time was varied from5 to 70 min) and resulted in a formation of special wells of desir-ble shape. After that the manufactured slides were washed threeimes with water, boiled in 0.1 M NaOH for 40 min, washed threeimes with distilled water and, finally, dried at 100 ◦C.

To introduce double bonds necessary for further tripleopolymerization, glass slides were held in 15% solution of 3-trimethoxysilyl) propyl methacrylate in toluene for 17–20 h atoom temperature [25]. To avoid a photochemical destruction ofsed silane, a reactor was preliminarily wrapped up in aluminumoil. The functionalized glasses were consequently washed threeimes with toluene, acetone and ethanol and stored in ethanol in aark. Directly before use, the glasses were dried for 1.5 h at 35 ◦C.

Preliminary optimization of polymers synthesis was car-ied out: poly(GMA-co-EDMA) [26], poly(GMA-co-GDMA) [27],oly(HPIEAA-co-GMA-co-EDMA) [28], poly(CEMA-co-EDMA) andoly(CEMA-co-HEMA-co-EDMA) [29]. The mixture containingonomers, porogens and 2-hydroxy-2-methylpropiophenon

Darocur-1173) as initiator was applied for polymer macroporousayers preparation. The ratio functional monomer(s): cross-inker was equal to 60:40 vol%. The ratio porogens: monomersor all copolymers, except HPIEAA-containing one, was alsohosen to be 60:40 vol%, whereas for mentioned terpolymer itas 75:25 vol%. The ratios HPIEAA:GMA = 13.3:53.3 mol% andEMA:HEMA = 22:46 mol% were used. Initiator concentration 1%

n related to the mass of monomers was used for all five polymersroduction. All reagents for polymerization were mixed and theolution was purged with nitrogen for 5 min. Preliminary preparednd functionalized wells on a glass surface were filled with reac-ion mixture and polymerization was allowed to proceed underV-lamp at room temperature in nitrogen medium for 30 min.

.3.2. Optimization of microanalytical conditions using modelioaffinity system.3.2.1. Immobilization of mouse IgG on monolith surface. To estab-ish optimal conditions for immobilization process, the followingarameters were varied: pH and concentration of mouse IgGolution, volume of sample spotted onto a monolith surface, immo-ilization temperature.

Mouse IgG always was spotted on a monolith surface in 10 repli-ates in one column. The sample volume was varied from 100 to00 pL. Two printing buffers were used for immobilization of mousegG, namely, 0.01 M sodium borate buffer, pH 9.5, and 0.01 M PBS,H 7.5. The slides containing spotted probes of IgG were incubatedt 4 ◦C and 37 ◦C. Following reaction times were used for differentlides: 17 h for poly(GMA-co-EDMA) and poly(GMA-co-GDMA), 2 hor poly(HPIEAA-co-GMA-co-EDMA), 4 h for poly(CEMA-co-EDMA)nd poly(CEMA-co-HEMA-co-EDMA).

.3.2.2. Washing procedures and surface blocking. After immobiliza-ion process was stopped, the slides were consequently washedith 0.01 M PBS, 2 M NaCl and 0.01 M PBS; the time of washingas varied from 5 to 60 min for each solution. Then the slide’s sur-

ace was blocked with 1% BSA in PBS for 45 min at slight stirringnd ready-to-use microarray was finally washed with PBS for 5 minnd then twice with water for 5 min each time. For comparison, theame experiment without blocking step was also carried out.

.3.2.3. Coupling with anti-mouse IgG and detection of pair formation.he solutions of goat anti-mouse IgG conjugated with Alexa Fluor55 with concentrations 20 ng mL−1 and 200 ng mL−1 were pre-

ared by dilution of commercial probe with concentration 2 mg/mL

n 0.2% TopBlock solution in PBS. Slides with immobilized mousegG were immersed into obtained protein solution and incubatedt 25 ◦C and 300 rpm for 2 h. After coupling the slides were washedsing following washing buffers: 2× SSC (3 M sodium chloride and

a Acta 644 (2009) 95–103 97

0.3 M sodium citrate) containing 2% sodium dodecyl sulfate (pH 7.0)for 5 min, 1× SSC (pH 7.0) for 5 min, 0.5× SSC (pH 7.0) for 5 min.

Then microarrays were dried with CO2 and scanned using thesame gain for all slides. Signal intensity (signal mean, SM), rela-tive signal intensity (signal mean–background mean, SM–BM) andsignal to noise ratio ((signal mean − background mean)/standarddeviation of background, SNR) were calculated and compared.

2.3.2.4. 2-D glass microarray. Experiment with functionalized glassslide was carried out according to the protocol optimized earlier.Immobilization of mouse IgG solutions in PBS on the surface ofaldehyde-bearing glass slides was performed for 1 h at 4 ◦C. Wash-ing procedure was carried out using following scheme: 0.01 M PBSfor 5 min; 2 M NaCl for 5 min; 0.01 M PBS for 5 min. 1% solutionof BSA in PBS was used for surface blocking that was performedfor 45 min at slight stirring. The concentration of goat anti-mouseIgG conjugated with Alexa Fluor 555 and used for coupling was200 ng mL−1. It was established that this concentration representsthe detection limit for this type of 2-D glass microarrays. Couplingand washing procedures were carried out as it was described for3-D monolith-based microarray.

2.3.3. Analysis of osteopontin (OPN) in cell culture mediumMonoclonal anti-osteopontin IgG solution of concentration of

1 mg mL−1 was spotted on monolith surface (sample volume wasequal to 500 pL) and the slide was incubated at pH 7.5 and 37 ◦C.Washing procedure was carried out according to following scheme:0.01 M PBS for 40 min, 2 M NaCl for 40 min and 0.01 M PBS for40 min. Surface blockage was performed as it was described abovefor model experiments. After blocking, the slides were washedwith PBS containing 0.05% Tween 20 (3 × 5 min). The microarraysobtained were treated with standard solution of osteopontin. Theconcentration of osteopontin in PBS containing 0.2% TopBlock wasvaried from 0.001 to 1 �g mL−1. The reactions were carried out for2 h at 25 ◦C and 300 rpm. After the reaction proceeded the slideswere washed 3 times with 0.01 M PBS containing 0.05% Tween 20for 5 min. The same washing scheme was used also after reactionswith biotinylated antibodies and labelled streptavidin. 1 �g mL−1

solutions of biotinylated polyclonal antibodies against OPN in PBScontaining 0.2% TopBlock was used for following coupling that wascarried out for 2 h at 25 ◦C. Afterwards, the slides were washed with2× SSC (3 M sodium chloride and 0.3 M sodium citrate) contain-ing 2% sodium dodecyl sulfate (pH 7.0) for 5 min, with 1× SSC (pH7.0) for 5 min, with 0.5× SSC (pH 7.0) for 5 min. To detect affin-ity complex formation, the microarray surface was covered with1 �g mL−1 solution of streptavidin–Cy3 conjugate with PBS con-taining 0.2% TopBlock and the reaction was left to run for 30 minat 25 ◦C. After the reaction was stopped, the slides were washedaccording to the procedure described above. Finally, the slides weredried and scanned. The described procedure was applied to allmonolith-based microarrays developed in this study.

After preliminary experiments with standard osteopontin wereperformed and the limit of sensitivity was established, two samplesof cell culture medium were analyzed. One of them was a super-natant of SAOS-2 cell line from human osteosarcoma obtained after5 days of cultivation. The second sample was obtained also after 5days of cultivation of primary mesenchymal stem cells derived fromhuman adipose tissue.

3. Results and discussion

3.1. Preparation of polymer-inorganic platforms

The present study is devoted to the development of new typeof platforms for construction of microarray based on macroporousmonolithic materials.

98 M. Rober et al. / Analytica Chimica Acta 644 (2009) 95–103

Table 1Characteristics of polymer matrixes applied for protein microarray.

Copolymer Average pore size (�m) Specific surface area (m2 g−1) Quantity of functional groups (mmol g−1)

Poly(GMA-co-EDMA) 1.5 25 4.0Poly(GMA-co-GDMA) 1.2 29 4.0Poly(HPIEAA-co-GMA-co-EDMA) 1.9 14 0.8a

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a Only HPIE-groups were considered as functional ones.

Because of significant fragility of thin monolithic layers, theacroporous copolymers have to be attached to the inert solid sur-

ace. Solid support used for monolith attachment should be stable

t different organic solvents and inorganic buffers. Glass matri-es are very often used for such purposes and undoubtedly satisfyll above-mentioned requirements. Besides that, the possibility ofanufacturing of operative wells at glass surface using relatively

imple procedures of its treatment (with, for example, hydroflu-

ig. 1. Dependence of signal intensity on pH of immobilized protein solution: (a) slideso-GDMA), (C) poly(CEMA-co-HEMA-co-EDMA), (D) poly(CEMA-co-EDMA); (b) graphicalolution. Conditions: immobilization temperature 37 ◦C; PMT gain = 450 for poly(GMA-co-Eor poly(CEMA-co-HEMA-co-EDMA) and poly(CEMA-co-EDMA).

4.11.2

oric acid, or by careful processing with thin milling cutter) alsorepresents an important advantage of this inorganic support.

The process of polymer-inorganic microarray platforms con-

struction included two steps: (1) manufacturing of operative wellson a glass surface and (2) polymer layer preparation.

Thus, the first step of monolith-based microarray platform fab-rication was a preparation of operative wells with carefully definedgeometry, as well as a glass surface activation providing the cova-

images after microanalysis performance: (A) poly(GMA-co-EDMA), (B) poly(GMA-presentation of the dependence of signal intensity on pH of immobilized proteinDMA), poly(GMA-co-GDMA) and poly(HPIEAA-co-GMA-co-EDMA); PMT gain = 250

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Fig. 2. Dependence of mean signal intensity on temperature of immobilization pro-cess performance. Conditions: pH 7.5, poly(GMA-co-EDMA) material.

M. Rober et al. / Analytica C

ent binding of polymer monolith to inorganic matrix. The operativeells were made to be rectangular of 18 mm × 60 mm well per stan-ard microscopic glass slide. Varying etching reaction time from 15o 70 min it was managed to obtain the 0.08–0.35 mm depth wells.he depth 0.2 mm was finally found to be optimal from the pointf view of further analytical procedure development. The forminghickness of synthesized macroporous monolithic support seemso be enough to provide, on the one hand, the sufficient amountf immobilized ligands, and, consequently, the sufficient adsorp-ion capacity of tested antibody which can be easily detected inpite of non-transparency of polymer support. On the other hand,his thickness allows avoiding significant printed zone spreadinghat is observed for the case of thinner polymer layers. In order tobtain the wells with mentioned rectangular geometry, the glassas incubated with HF for 30 min.

Both after etching with hydrofluoric acid and mechanicalreatment, the glass slides were incubated with NaOH to con-ert Si–F– and Si–O–Si–, respectively, into Si–OH-groups thatas necessary for the next matrix activation step (silanation).-(Trimethoxysilyl)propyl methacrylate was used as a couplinggent. The last step of the microarray platform fabrication was thehoto-initiated synthesis of macroporous polymer layers directlyn activated surface of wells performed.

As it was mentioned above, macroporous flow-througholy(GMA-co-EDMA) devices (disks, rod columns, capillaries) areidely used in the processes based on dynamic interphase mass

ransition (chromatography, flowing enzyme reactors, etc.). Suchtationary phases are demonstrated the numerous advantagesomparing to the packed columns. Their unique features [30] areuite useful for all discussed applications, but especially for thoseased on a principle of biorecognition (affinity chromatography)31,32]. Obviously, all positive features of latter could be transferredo modern and impetuously developing bioanalytical field, namely,

icroarray technology. The main difference between affinity chro-atographic separations and microarray analysis is the distinctiveechanism of interphase mass exchange. While in chromatogra-

hy on monoliths this process is governed by fast convection, inhe case of microarray, the mass transfer mechanism is based onlow molecular diffusion. However, the optimized porous structuref polymer monolithic materials along with very small thickness ofperative layer allow obtaining much better results in comparison,or example, with gel supports.

As it was declared, four new polymethacrylate materials wereeveloped for microarray platform fabrication. The characteristicsf used monoliths are presented in Table 1. Despite all positive andery well described in numerous papers and reviews features ofoly(GMA-co-EDMA) (including easy one-step biofunctionalizationf polymer matrix), the reaction between epoxy groups of a sor-ent and amino-bearing ligands is characterized by slow kinetics.

t is known that immobilization capacity reached its maximum onlyfter 16–18 h [33]. Therefore, to avoid any undesirable changes inound biomolecule, we met a problem to introduce more reactiveunctional groups into polymer structure. Besides, the porous struc-ure of new materials must be similar to that of recently successfullyested poly(GMA-co-EDMA).

Poly(HPIEAA-co-GMA-co-EDMA) and poly(CEMA-co-EDMA)ontaining highly reactive activated ester groups which allowast covalent attachment of proteins, were developed and testeds a base for discussed microarrays. In the case of terpolymer,MA is not considered as a reactive comonomer since the inter-ction between HPIE-groups of material and ligand amino groups

roceeds much faster, namely, within 2 h, versus the reactionetween amino and epoxy groups. Poly(GMA-co-GDMA) monolithnd poly(CEMA-co-HEMA-co-EDMA) were used as the examplesf monoliths with enhanced hydrophilicity that can be verymportant in bioanalysis.

Fig. 3. Dependence of mean signal intensity on concentration of immobilized mouseIgG. Conditions: immobilization temperature 37 ◦C; pH 7.5; (a) gain 450; (b) gain 250.

100 M. Rober et al. / Analytica Chimica Acta 644 (2009) 95–103

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for affinity chromatography with poly(GMA-co-EDMA). In affinitychromatography, the use of pH 7.5 seemed to be non-optimal valuebecause of resulting in lower ligand surface concentration that fol-lowed by decreasing complement’s adsorption capacity. Probably,the tendency observed in microarray format can be explained by

ig. 4. Influence of surface blocking SNR: (a) SNRs obtained without and with surfnd with surface blocking. Conditions: mouse IgG concentration 1 mg/ml; pH 7.5; im

.2. Optimization of ligand covalent binding to monolith-basedicroarrays

As it was mentioned above, a model bioaffinity system con-isting of mouse IgG and goat anti-mouse IgG conjugated withuorescent label Alexa 555 was used to demonstrate the potentialf developed platforms to be applied for protein microarrays.

It is well known that efficiency of solid-phase analysis based onffinity interactions of complementary biological molecules signif-cantly depends on a quantity of immobilized ligand. Therefore, thenfluence of immobilization conditions (including immobilizationemperature, concentration of a protein ligand to be immobilizednd pH) on microanalytical results was investigated. In order toetermine the optimal ligand’s concentration and pH of reaction,odium borate buffer (pH 9.5) and PBS (pH 7.5) were compared.

Sodium borate buffer was chosen since the reaction betweenpoxy, activated ester and amino groups proceed with highestate at alkaline conditions. Besides, from numerous publicationst is well known that protein immobilization on the surface ofoly(GMA-co-EDMA) at pH ∼9.0 results in highest reaction yield,.g. the highest amount of immobilized protein [34]. Moreover, thea value of �-aminogroup of lysine equal to 10 allows supposing itsigh activity at covalent binding to any mentioned reactive groupf a solid support. PBS was tested since physiological mild condi-ions are preferable from the point of view of maintaining of proteintructure responsible for its further specific binding to the proteino be analyzed. It was established that, in general, for all tested

olymer materials both pHs could be used for protein immobi-

ization. However, the mean signal intensity (SM) determined inollowing bioanalytical experiments was 2–3 times higher whenBS was applied as a buffer for protein immobilization (Fig. 1).he main reason of higher signal intensity when PBS is used as

ocking; (b) images of monolith’s surface after microanalysis performance withoutlization temperature 37 ◦C; poly(GMA-co-EDMA) material.

printing buffer is a retention of active protein conformation atpH ∼7.5. Besides that, the ligand density also plays an importantrole. It is interesting, but this data are opposite to those obtained

Fig. 5. Comparison of the maximal SNRs obtained using investigated monolithsand aldehyde glass slide. Conditions: pH 7.5; immobilization temperature 37 ◦C; forpoly(GMA-co-EDMA), poly(GMA-co-GDMA) and poly(HPIEAA-co-GMA-co-EDMA)and glass slides was used PMT gain 450, for poly(CEMA-co-EDMA) and poly(CEMA-co-HEMA-co-EDMA) was used PMT gain 250, goat anti-mouse IgG-Alexa Fluor 555conjugate concentration 200 ng/ml.

M. Rober et al. / Analytica Chimic

Fig. 6. Comparison of maximal SNRs obtained for developed monolith surfacesabpa

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t analyzed protein concentration equal to 20 ng/ml. Conditions: pH 7.5; immo-ilization temperature 37 ◦C; for poly(GMA-co-EDMA), poly(GMA-co-GDMA) andoly(HPIEAA-co-GMA-co-EDMA) was used PMT gain 450, for poly(CEMA-co-EDMA)nd poly(CEMA-co-HEMA-co-EDMA) was used PMT gain 250.

ignificant increasing of immobilized ligand amount at higher pHalues provided by much more higher reactivity of ester groups.n turn, high ligand density might complicate the affinity complexormation and, consequently, lead to analytical efficiency reduction.esides that, pH 7.5 is friendlier for protein’s native conformationontrary to alkaline conditions. Thus, for the next experiments PBSas been chosen as a ligand printing buffer for all discussed macro-orous polymer supports.

To reveal the optimal temperature used for ligand immobiliza-ion, the slides were incubated at 4 ◦C, as it was often used iniological practice, and 37 ◦C, that should be preferable in termsf a rate of the reaction. The main reason of reaction performancet 4 ◦C is the apprehension to loose the binding activity of immo-ilized protein because of, the same as in the case of elevated pH,ome probable distortion of protein conformation. However, it isnown that any multiple-bond covalent immobilization of proteinolecule inside three-dimensional porous space leads to its sta-

ilization regarding the preservation of molecular conformation.herefore, in spite of a risk of partial protein denaturation, we reg-stered higher SM values for all tested polymer materials whereigand immobilization was performed at 37 ◦C. Fig. 2 illustrates onexample of a dependence SM on reaction temperature.

The quantity of immobilized mouse IgG necessary for obtain-ng of high signal intensity was also optimized by variation ofts concentration from 0.2 up to 1.0 mg mL−1 (Figs. 1a and 3). Itas shown that in the case of poly(GMA-co-EDMA) monolith, SM

alue reached its maximum at 1.0 mg mL−1 of mouse IgG concen-

ration used for printing, whereas for poly(GMA-co-GDMA) theptimal ligand concentration lied in interval 0.6–1.0 mg mL−1 thatorresponded to maximal level of signal intensity. In the casef poly(HPIEAA-co-GMA-co-EDMA), high SM value was alreadychieved at 0.2 mg mL−1 and only slightly raised at further IgG con-

able 2imes of immobilization procedures, durations of analysis, intrafield and interfiled coeffic

ype of microarray surface Time ofimmobilization (h)

Duration ofanalysis (h)

Inva

ldehyde-modified glass slide 1 3.5 17oly(GMA-co-EDMA) 16 5.25 8oly(GMA-co-GDMA) 16 5.25 8oly(HPIEAA-co-GMA-co-EDMA) 2 5.25 17oly(CEMA-co-EDMA) 4 5.25 10oly(CEMA-co-HEMA-co-EDMA) 4 5.25 10

a Acta 644 (2009) 95–103 101

centration increasing. When poly(CEMA-co-HEMA-co-EDMA) wasused, the mean signal intensity increased linearly with increasing ofprotein concentration. At the same time, for poly(CEMA-co-EDMA)it was found that SM increased with increasing of mouse IgG con-centration from 0.2 to 0.8 mg mL−1 and significantly decreasedat 1 mg mL−1 (Fig. 3b). Evidently, the reason of this phenomenonis abundant density of immobilized mouse IgG which leads tosteric hindrances for affinity complex formation. The reason ofdescribed effects of different IgG concentrations on registered SM,the most probably, can be explained by different content of reac-tive groups and their binding activity in synthesized monolithicpolymers. Thus, the conclusion is that such parameter as protein tobe immobilized concentration has to be selected for each supportindividually.

To optimize analytical conditions, it was necessary to establish asufficient sample volume used for spotting procedure. The variationof this parameter from 100 to 700 pL was tested to make a rightchoice. It was found that sample volume equal to 500 pL appeared tobe sufficient to reach the saturation of polymer surface with proteinligand.

It is common for microarray-based analytical protocols to carryout the procedure of surface blocking after ligand immobilization.BSA-containing buffer is widely used for this purpose. Since in ourstudy very big protein (IgG) with molecular mass ∼150 kDa wasused as immobilized ligand, the application of smaller BSA as ablocking agent looks acceptable. To confirm the necessity of surfaceblocking, two experiments carried out under the same conditionswere performed. At one of them, the slide with blocked by 1%BSA in PBS surface was incubate with fluorescently labelled goatanti-mouse IgG, while another one was carried out analogously butwithout blocking procedure. It is clear from Fig. 4 that the blockingof monolith’s surface leads to higher SNR that testifies a better spotquality. The same result was observed for all monolithic supportsunder discussion.

The last step of discussed investigation was devoted to veryimportant establishment of optimal time of slide washing afterligand immobilization step. Washing time was varied form 5 to60 min. It was shown that the longer time the slides were washed,the higher SM was registered. I was found that only after 40 minwashing the mean signal intensity became to be constant. Firstof all, this result can be explained by slow diffusional mobility ofunbound big antibodies that seriously limited their exit from mono-lith’s pores with sufficiently wide size distribution. A necessity ofstabilization of bound protein conformation can be considered as asecond reason of results obtained.

3.3. Comparison of efficiency of monolith-based 3-D microarraysand 2-D glass slides

The microanalytical results obtained using different macrop-

orous monolithic materials as operative supports and aldehyde-bearing glass slides were compared.

It was impossible to compare registered signal values atthe same instrumental gain for all slides, because the scan-ning of poly(CEMA-co-EDMA) and poly(CEMA-co-HEMA-co-EDMA)

ients of variation, limit of detection for all compared slides.

trafield coefficient ofriation (%)

Interfield coefficient ofvariation (%)

Limit of detection(pg mL−1)

14 0.2008 0.020

10 0.02019 0.02011 0.00211 0.020

102 M. Rober et al. / Analytica Chimica Acta 644 (2009) 95–103

F ed polc EMA-3

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ppo2ttsswmo5

ig. 7. Results of OPN detection in cell culture medium obtained for some of developo-EDMA) was used PMT gain 450, for poly(GMA-co-GDMA) and poly(CEMA-co-H00.

icroarrays demonstrated better resolution at lower gain. There-ore, the values of maximum SNR obtained at different gainsptimal for each test-system were used for their comparison. Ast seen from Fig. 5, the highest SNR was obtained for poly(CEMA-o-EDMA). The second nice result was detected for hydrophilicoly(GMA-co-GDMA). The sensitivity of test-system based onydrophilic poly(GMA-co-GDMA) was two times higher relating toonventional poly(GMA-co-EDMA) that probably can be explainedy better penetration of water-surrounding protein moleculesnto a three-dimensional porous space of hydrophilic material.he microarrays based on terpolymers, namely, poly(CEMA-co-EMA-co-EDMA) and poly(HPIEAA-co-GMA-co-EDMA), showed

he comparable results and, moreover, very close to that obtainedith poly(GMA-co-EDMA) material. Such result is explained by

ow content of functional groups in terpolymers (Table 1) that,n its turn, leads to the low ligand’s surface density. It should beoted that the content of functional monomer in the poly(CEMA-o-HEMA-co-EDMA) is three times lower in comparison witholy(CEMA-co-EDMA). For the case of poly(HPIEAA-co-GMA-co-DMA), the amount of activated ester groups is one and a halfime lower comparatively to CEMA-containing terpolymer. Finally,t was found that the sensitivity of analysis on microarray based onoly(CEMA-co-HEMA-co-EDMA), poly(HPIEAA-co-GMA-co-EDMA)nd poly(GMA-co-EDMA) was practically the same, whereas theeaction of ligand immobilization taking place for two terpolymersas much faster than for GMA-bearing copolymer. This fact can be

ounted as a great practical advantage of these devices in spite ofemonstrated modest sensitivity.

A comparison of developed microarray based on macroporousolymer monoliths with widely used glass biochips was alsoerformed. A detection limit for test-system based on macrop-rous supports, except poly(CEMA-co-EDMA), was found to be0 ng mL−1 (0.133 pmol mL−1), while for glass slide this value cameo 200 ng mL−1 (1.333 pmol mL−1) for used affinity system, e.g.he difference of one order of magnitude was observed. The sen-itivity limit was established according to a concentration of a

olution of complementary labelled antibody to which the slidesith immobilized ligand were immersed to reach affinity pair for-ation. Fig. 6 demonstrates SNR for monolith-based microarrays

btained using concentration of goat anti-mouse IgG-Alexa Fluor55 20 ng mL−1. As it seen from the figure, SNR of poly(CEMA-co-

ymer surfaces. Conditions: pH 7.5; immobilization temperature 37 ◦C; for poly(GMA-co-EDMA) was used PMT gain 350, for poly(CEMA-co-EDMA) was used PMT gain

EDMA) is quite higher and left the possibility to detect much lowerprotein amounts.

The reproducibility of results obtained was estimated byintrafield and interfield coefficient of variation [29]:

Coefficient of variation = standard deviationmean of signal intensity

The intrafield and interfiled coefficients of variation for com-pared surfaces are presented in Table 2. The means of intrafield andinterfiled coefficients of variation for glass slides are given accord-ing to data published elsewhere [35]. As it seen from Table 2, allmonoliths under investigation show a better reproducibility com-pared to 2-D glass slides, except poly(HPIEAA-co-GMA-co-EDMA)demonstrating the highest values of intrafield and interfiled coef-ficients of variation. The reason of this fact is probably connectedwith low reproducibility of this kind of monoliths production.

3.4. Microarray-based detection of bone tissue markerosteopontin

Osteopontin represents a phosphorilated bone matrix glyco-protein which has a wide area of tissue localization [36]. In vivoosteopontin is excreted to the blood plasma and its concentrationreflects the increased turnover associated with bone destruc-tion of aging, menopause and different conditions affecting bonemetabolism.

Preliminary experiments on determination of osteopontin wereperformed on poly(GMA-co-EDMA) microarray platform usingstandard system including monoclonal antiosteopontin, stan-dard osteopontin solutions, biotinilated captures antiosteopontinand streptavidin conjugated with Cy3. To determine a detec-tion limit, the concentration of osteopontin was varied from0.001 to 1 �g mL−1. The value was found to be 0.01 �g mL−1

(0.306 pmol mL−1).To examine the possibility of detection of bone tissue marker

osteopontin (bone tissue engineering) by means of suggested

microanalytical platform, two kinds of cell supernatants were used,namely, the culture medium of SAOS-2 cells derived from a humanosteosarcoma, and a supernatant obtained after cultivation of pri-mary mesenchymal stem cells derived from human adipose tissue.To compare the signal intensities obtained with cell culture media

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M. Rober et al. / Analytica C

ith analogous results established for standard osteopontin solu-ions, 16-well monolith-based platforms were used. The presencef osteopontin was easily detected in cell supernatant of SAOS-2,hile for cell supernatant obtained after mesenchymal stem cells

ultivation the target protein was not found. Fig. 7 demonstrates aomparison of SNRs obtained for microarrays based on developedolymers. As it was expected the highest SNRs were obtained foroly(CEMA-co-EDMA) and poly(GMA-co-GDMA) due to high reac-ivity of activated ester groups of the former and higher hydrofilicityf the later compared with poly(GMA-co-EDMA).

. Conclusions

In present study the new platforms for 3-D microarrays con-truction based on macroporous polymer monolithic materialsere developed. Each step of microanalytical device fabrication

manufacturing of operative wells, introduction of double bondsnto the well surface and photo-initiated copolymerization of

onomers) was carefully investigated and optimized. Along withell known poly(GMA-co-EDMA), four new monolithic materialsere tested and compared as microarray supports.

It was demonstrated that the use of monoliths containing reac-ive monomers with activated ester groups, as well as a hydrophilicross-linker, led to the increasing of analytical efficiency inomparison with poly(GMA-co-EDMA). The best analytical poten-ial was determined for poly(CEMA-co-EDMA) material. Besideshat, monolith-based microarrays were compared with aldehyde-earing 2-D glass slides and the difference between the detection

imits of all tested devices was established. All developed materialsre characterized with high spot quality and excellent sensitivity. Itas found that all polymer devices had at least one order of mag-itude advantage regarding to sensitivity in comparison with 2-Dicroarray (glass slide). As a practical example, a detection of bone

issue marker, namely, osteopontin was developed using suggestedicroanalytical system based on poly(GMA-co-EDMA). The detec-

ion limit was found to be equal to 0.01 �g mL−1 that correspondedo 0.306 pmol mL−1.

cknowledgements

This work was supported by DFG grant (Grant No. KA 1784/4-1;oordinators: Prof. T. Tennikova, IMC RAS and Dr. C. Kasper, ITC UH)nd RBRF grant (08-08-00876-a, Supervisor Prof. T. Tennikova). Theuthors are grateful to German Academic Exchange Service (DAAD)

or providing the scholarship for PhD student Marina Rober for four-

onth work at the Institute of Technical Chemistry, University ofannover (ITC UH). The authors are also grateful to Prof. Thomascheper (ITC, Hannover University) and Prof. Valery Krasikov (IMCAS) for organizing help and fruitful discussions.

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a Acta 644 (2009) 95–103 103

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