New 3-D microarray platform based on macroporous polymer monoliths

Analytica Chimica Acta 644 (2009) 95–103

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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

New 3-D microarray platform based on macroporous polymer monoliths M. Rober a , J. Walter b , E. Vlakh a , F. Stahl b , C. Kasper b , T. Tennikova a,∗ a b

Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoy pr. 31, 199004 St. Petersburg, Russia 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 2008 Received in revised form 3 April 2009 Accepted 6 April 2009 Available online 17 April 2009 Keywords: Macroporous monolithic materials Three-dimensional protein microarray Bioanalysis

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 are suggested as operative matrices at the development of three-dimensional (3-D) microarrays. For this purpose, several rigid macroporous copolymers differed by reactivity and hydrophobic–hydrophilic properties 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 dimethacrylate (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-coethylene 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 pair was used to demonstrate the potential of developed test-systems, as well as to optimize microanalytical conditions. The offered microarray platforms were applied to detect the bone tissue marker osteopontin directly in cell culture medium. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The microarrays represent independent and quite promising analytical tool with great potential at different practical fields, such as molecular biology, medicine, analytical biotechnology, pharmacology, ecology and bioinformatics. Ekins and coworkers explained in ambient analyte theory that microspot assays have to be much more sensitive that conventional ligand-binding assays due to the highest fractional occupancy of the captured antibodies and highest signal per area achieved in microspots [1,2]. The application of biological test-systems for high sensitive spot-analysis is based on a specific binding of target molecule (protein, DNA, RNA, peptide, etc.) to its biocomplementary ligand immobilized on a solid surface. The efficiency of processes based on a principle of biorecognition (bioaffinity interactions) directly

Abbreviations: GMA, glycidyl methacrylate; EDMA, ethylene dimethacrylate; GDMA, glycerol dimethacrylate; HPIEAA, N-hydroxyphthalimide ester of acrylic acid; CEMA, 2-cyanoethyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; CyOH, cyclohexanol; DoOH, dodecanol; PEG, poly(ethylene glycol); 2-D and 3D, two- and three-dimensional, respectively; BSA, bovine serum albumin; IgG, immunoglobulin G; OPN, osteopontin. ∗ Corresponding author. Tel.: +7 812 323 04 61; fax: +7 812 323 68 69. E-mail address: [email protected] (T. Tennikova). 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.04.015

depends on ligand immobilization that, in turn, is dramatically influenced by properties of used solid support. General approaches applied to the matrix functionalization with, for example, proteins are physical adsorption, covalent attachment via specific surface interaction and bioaffinity (oriented) immobilization [3,4]. It is a real problem to define the best way of protein immobilization for all developed systems, because the result will depend both on the properties of biomolecule to be immobilized and used solid matrix, that is optimal experimental conditions 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-called planar two-dimensional supports. In this case, the substance of interest forms a specific pair with a ligand located on a rigid monolayer representing microarray’s surface. Such devices based on glass slides, non-porous synthetic polymers and metals provide the uniform signal and satisfactory results reproducibility [7]. At the same time, 3-D microarrays are expected to reduce the steric hindrance problems, to offer the higher affinity capacity and, consequently, to increase a sensitivity of analysis, as well as to provide similar to solution conditions for immobilized biomolecules and, therefore, to prevent the loosing of its binding activity. One of the first examples of 3-D microarrays was polyacrylamide gel pads attached to the glass surface which were developed by

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Mirzabekov and coworkers [8,9]. These devices provided increased affinity capacity, reduced protein denaturation and limited nonspecific protein binding. However, it was reported by Mirzabekov and coworkers [8], as well as by Kusnezow et al. who tested a hydrogel surface from Perkin Elmer [10], that three-dimensional gel structure played a role of additional barrier for diffusion of large molecules and required very long incubation times or some additional experimental tricks to accelerate diffusion of macromolecules, such as, the use of electrophoresis or mixing the solutions with a micropump within the microarray chamber [8]. Microarrays based on nitrocellulose membranes are another example of non-planar microunits. Due to their enormous binding capacity they provide much higher signal intensity in comparison with 2-D microarrays [11]. Nevertheless, it has some disadvantages, such as significant non-specific protein binding which makes nitrocellulose surfaces not suitable for analysis of complex protein mixtures, as well as quite high autofluorescense. Colorizing of a membrane in black colour was suggested as an original solution of the last problem [12]. In the early 1990s a new class of continuous media based on a rigid macroporous copolymer of glycidyl methacrylate and ethylene dimethacrylate, or poly(GMA-co-EDMA), synthesized by a bulk polymerization and commonly called later as monolith, was developed and introduced to the World market as fast and efficient chromatographic stationary phase [13,14]. Similarly to the macroporous polymer beads, monoliths are characterized by fixed porous properties, maintained constant even in a dry state. Their internal structure represents a system of interconnected polymer microglobules separated by pores, and their structural rigidity is secured by extensive cross-linking. Today monolithic materials are successfully used in a wide range of fast interphase dynamic processes, such as high-performance liquid chromatography [15–18], bioconversion processes (flow-through enzyme reactors) [19,20], capillary electrochromatography [21] and solid phase extraction [22]. Recently a new type of 3-D microarrays intended for analytical detection of influenza virus and based on a rigid macroporous poly(GMA-co-EDMA) monolithic layers, have been developed by our scientific group [23]. In this paper for the first time a quantitative comparison of affinity pair formation at flow-through (affinity chromatography) and non-flowing (microarray) conditions was carried out. The matter of present study was the application of new macroporous monolithic materials for 3-D microarray construction, as well as a comparison of their bioanalytical potentials. For these purposes, five developed and thoroughly characterized macroporous monolithic copolymers were used: glycidyl methacrylate-co-ethylene dimethacrylate (poly(GMA-co-EDMA)), glycidyl methacrylate-co-glycerol dimethacrylate (poly(GMA-coGDMA)), N-hydroxyphthalimide ester of acrylic acid-co-glycidyl methacrylate-co-ethylene dimethacrylate (poly(HPIEAA-coGMA-co-EDMA)), 2-cyanoethyl methacrylate-co-ethylene dimethacrylate (poly(CEMA-co-EDMA)) and 2-cyanoethyl methacrylate-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate (poly(CEMA-co-HEMA-co-EDMA)). Original functional groups in the structure of synthesized copolymers made possible realization of one-step surface modification with protein ligands. The optimization of analytical procedure was performed using model protein affinity pair, namely, mouse IgG—goat antimouse IgG conjugated with Alexa Fluor 555. The detection limit found for developed devices was compared with that observed for 2-D microarrays based on aldehyde-modified glass slides. To demonstrate the efficiency of suggested microarrays for determination of target proteins in complex biological liquids, the procedure of qualitative detection of well-known bone tissue marker osteopontin in cell culture medium has been developed.

2. Materials and methods 2.1. Reagents Glycidyl methacrylate (GMA, 97% pure), ethylene dimethacrylate (EDMA, 98% pure), cyclohexanol (99% pure), glycerol dimethacrylate (GDMA, 85% pure), 2-hydroxyethyl methacrylate (HEMA, 98% pure), 2-hydroxy-2-methylpropiophenon (Darocur1173, 97% pure), 3-(trimethoxysilyl) propyl methacrylate, PEG600, 1,4-dioxan were purchased from Sigma–Aldrich GmbH (Taufkirchen, Germany). N-hydroxyphthalimide ester of acrylic acid (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 tetrahydrofuran (99% pure, Sigma–Aldrich GmbH (Taufkirchen, Germany)) according to the published method [24]. 2-Cyanoethyl methacrylate was purchased from Yarsinthez (Yaroslavl’, Russia). Purified mouse IgG was obtained from Zytomed (Berlin, Germany), goat anti-mouse IgG conjugated with Alexa Fluor 555 was purchased from Molecular Probes (Eugene, USA). TopBlock was purchased from Fluka (Buchs, Switzerland). Human osteopontin, monoclonal mouse anti-osteopontin IgG and polyclonal mouse biotinylated anti-osteopontin IgG were purchased from R&D Systems (Wiesbaden, Germany). Cy3-conjugated streptavidin was obtained from Jackson ImmunoResearch Laboratories (Suffolk, UK). The reaction of silanization was carried out in toluene (Vecton Ltd, Russia). Following buffers were used for microanalytical manipulations: sodium borate buffer (1.25 × 10−2 M Na2 B4 O7 × 10H2 O, 8.8 × 10−3 M NaOH), PBS (0.137 M NaCl, 2.7 × 10−3 M KCl, 4.3 × 10−3 M Na2 HPO4 , 1.4 × 10−3 M KH2 PO4 ), SSC (3 M NaCl, 0.3 M Na3 C6 H5 O7 ) containing 2% sodium dodecyl sulfate. All buffers were prepared by dissolving the analytical grade salts in distilled water and were additionally purified by filtration through a 0.45 ␮m Milex Millipore microfilter (Wien, Austria). The glass slides with dimensions 25 mm × 75 mm × 1.2 mm were obtained from BioVitrum (St. Petersburg, Russia). The glass aldehyde slides were purchased from CEL Associates Inc. (Pearland, USA). 2.2. Apparatus The 125 W mercury lamp (Philips, Netherlands) of wide radiation spectrum and constant intensity was used for free-radical polymerization. The monolith morphology was studied using scanning electron microscope JSM-35 CF produced JEOL (Tokyo, Japan). The mean pore size and pore size distribution were determined by mercury intrusion porosimetry using PASCAL 440 Thermoquest Instrument (Rodano, Italy). For spotting of proteins to be immobilized on a microarray surface sciFLEXARRAYER S3, Scienion (Berlin, Germany) was used. The washing procedure was carried out using a Thermomixer Comfort (Eppendorf, Germany). The special secure seal hybridization chambers (Grace Biolabs, Bend, USA) were used to perform the coupling of immobilized mouse IgG with goat anti-mouse IgG conjugated with Alexa Fluor 555. Microarrays were scanned using Scanner GenePix 4000 B (Axon Instruments, USA). GenePix 6.0 software was used to analyze the scan data. 2.3. Methods 2.3.1. Microarray fabrication The first step of microarray construction was glass surface etching with hydrofluoric acid (HF) using special mask. The process was

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carried out at intensive interfusion (reaction time was varied from 15 to 70 min) and resulted in a formation of special wells of desirable shape. After that the manufactured slides were washed three times with water, boiled in 0.1 M NaOH for 40 min, washed three times with distilled water and, finally, dried at 100 ◦ C. To introduce double bonds necessary for further triple copolymerization, glass slides were held in 15% solution of 3(trimethoxysilyl) propyl methacrylate in toluene for 17–20 h at room temperature [25]. To avoid a photochemical destruction of used silane, a reactor was preliminarily wrapped up in aluminum foil. The functionalized glasses were consequently washed three times with toluene, acetone and ethanol and stored in ethanol in a dark. Directly before use, the glasses were dried for 1.5 h at 35 ◦ C. Preliminary optimization of polymers synthesis was carried out: poly(GMA-co-EDMA) [26], poly(GMA-co-GDMA) [27], poly(HPIEAA-co-GMA-co-EDMA) [28], poly(CEMA-co-EDMA) and poly(CEMA-co-HEMA-co-EDMA) [29]. The mixture containing monomers, porogens and 2-hydroxy-2-methylpropiophenon (Darocur-1173) as initiator was applied for polymer macroporous layers preparation. The ratio functional monomer(s): crosslinker was equal to 60:40 vol%. The ratio porogens: monomers for all copolymers, except HPIEAA-containing one, was also chosen to be 60:40 vol%, whereas for mentioned terpolymer it was 75:25 vol%. The ratios HPIEAA:GMA = 13.3:53.3 mol% and CEMA:HEMA = 22:46 mol% were used. Initiator concentration 1% in related to the mass of monomers was used for all five polymers production. All reagents for polymerization were mixed and the solution was purged with nitrogen for 5 min. Preliminary prepared and functionalized wells on a glass surface were filled with reaction mixture and polymerization was allowed to proceed under UV-lamp at room temperature in nitrogen medium for 30 min. 2.3.2. Optimization of microanalytical conditions using model bioaffinity system 2.3.2.1. Immobilization of mouse IgG on monolith surface. To establish optimal conditions for immobilization process, the following parameters were varied: pH and concentration of mouse IgG solution, volume of sample spotted onto a monolith surface, immobilization temperature. Mouse IgG always was spotted on a monolith surface in 10 replicates in one column. The sample volume was varied from 100 to 700 pL. Two printing buffers were used for immobilization of mouse IgG, namely, 0.01 M sodium borate buffer, pH 9.5, and 0.01 M PBS, pH 7.5. The slides containing spotted probes of IgG were incubated at 4 ◦ C and 37 ◦ C. Following reaction times were used for different slides: 17 h for poly(GMA-co-EDMA) and poly(GMA-co-GDMA), 2 h for poly(HPIEAA-co-GMA-co-EDMA), 4 h for poly(CEMA-co-EDMA) and poly(CEMA-co-HEMA-co-EDMA). 2.3.2.2. Washing procedures and surface blocking. After immobilization process was stopped, the slides were consequently washed with 0.01 M PBS, 2 M NaCl and 0.01 M PBS; the time of washing was varied from 5 to 60 min for each solution. Then the slide’s surface was blocked with 1% BSA in PBS for 45 min at slight stirring and ready-to-use microarray was finally washed with PBS for 5 min and then twice with water for 5 min each time. For comparison, the same experiment without blocking step was also carried out. 2.3.2.3. Coupling with anti-mouse IgG and detection of pair formation. The solutions of goat anti-mouse IgG conjugated with Alexa Fluor 555 with concentrations 20 ng mL−1 and 200 ng mL−1 were prepared by dilution of commercial probe with concentration 2 mg/mL in 0.2% TopBlock solution in PBS. Slides with immobilized mouse IgG were immersed into obtained protein solution and incubated at 25 ◦ C and 300 rpm for 2 h. After coupling the slides were washed using following washing buffers: 2× SSC (3 M sodium chloride and

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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 the same gain for all slides. Signal intensity (signal mean, SM), relative signal intensity (signal mean–background mean, SM–BM) and signal to noise ratio ((signal mean − background mean)/standard deviation of background, SNR) were calculated and compared. 2.3.2.4. 2-D glass microarray. Experiment with functionalized glass slide was carried out according to the protocol optimized earlier. Immobilization of mouse IgG solutions in PBS on the surface of aldehyde-bearing glass slides was performed for 1 h at 4 ◦ C. Washing procedure was carried out using following scheme: 0.01 M PBS for 5 min; 2 M NaCl for 5 min; 0.01 M PBS for 5 min. 1% solution of BSA in PBS was used for surface blocking that was performed for 45 min at slight stirring. The concentration of goat anti-mouse IgG conjugated with Alexa Fluor 555 and used for coupling was 200 ng mL−1 . It was established that this concentration represents the detection limit for this type of 2-D glass microarrays. Coupling and washing procedures were carried out as it was described for 3-D monolith-based microarray. 2.3.3. Analysis of osteopontin (OPN) in cell culture medium Monoclonal anti-osteopontin IgG solution of concentration of 1 mg mL−1 was spotted on monolith surface (sample volume was equal 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 for 40 min. Surface blockage was performed as it was described above for model experiments. After blocking, the slides were washed with PBS containing 0.05% Tween 20 (3 × 5 min). The microarrays obtained were treated with standard solution of osteopontin. The concentration of osteopontin in PBS containing 0.2% TopBlock was varied from 0.001 to 1 ␮g mL−1 . The reactions were carried out for 2 h at 25 ◦ C and 300 rpm. After the reaction proceeded the slides were washed 3 times with 0.01 M PBS containing 0.05% Tween 20 for 5 min. The same washing scheme was used also after reactions with biotinylated antibodies and labelled streptavidin. 1 ␮g mL−1 solutions of biotinylated polyclonal antibodies against OPN in PBS containing 0.2% TopBlock was used for following coupling that was carried out for 2 h at 25 ◦ C. Afterwards, the slides were washed with 2× SSC (3 M sodium chloride and 0.3 M sodium citrate) containing 2% sodium dodecyl sulfate (pH 7.0) for 5 min, with 1× SSC (pH 7.0) for 5 min, with 0.5× SSC (pH 7.0) for 5 min. To detect affinity complex formation, the microarray surface was covered with 1 ␮g mL−1 solution of streptavidin–Cy3 conjugate with PBS containing 0.2% TopBlock and the reaction was left to run for 30 min at 25 ◦ C. After the reaction was stopped, the slides were washed according to the procedure described above. Finally, the slides were dried and scanned. The described procedure was applied to all monolith-based microarrays developed in this study. After preliminary experiments with standard osteopontin were performed and the limit of sensitivity was established, two samples of cell culture medium were analyzed. One of them was a supernatant of SAOS-2 cell line from human osteosarcoma obtained after 5 days of cultivation. The second sample was obtained also after 5 days of cultivation of primary mesenchymal stem cells derived from human adipose tissue. 3. Results and discussion 3.1. Preparation of polymer-inorganic platforms The present study is devoted to the development of new type of platforms for construction of microarray based on macroporous monolithic materials.

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Table 1 Characteristics 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) Poly(GMA-co-GDMA) Poly(HPIEAA-co-GMA-co-EDMA) Poly(CEMA-co-EDMA) Poly(CEMA-co-HEMA-co-EDMA)

1.5 1.2 1.9 1.1 1.4

25 29 14 36 28

4.0 4.0 0.8a 4.1 1.2

a

Only HPIE-groups were considered as functional ones.

Because of significant fragility of thin monolithic layers, the macroporous copolymers have to be attached to the inert solid surface. Solid support used for monolith attachment should be stable at different organic solvents and inorganic buffers. Glass matrices are very often used for such purposes and undoubtedly satisfy all above-mentioned requirements. Besides that, the possibility of manufacturing of operative wells at glass surface using relatively simple procedures of its treatment (with, for example, hydroflu-

oric acid, or by careful processing with thin milling cutter) also represents an important advantage of this inorganic support. The process of polymer-inorganic microarray platforms construction included two steps: (1) manufacturing of operative wells on a glass surface and (2) polymer layer preparation. Thus, the first step of monolith-based microarray platform fabrication was a preparation of operative wells with carefully defined geometry, as well as a glass surface activation providing the cova-

Fig. 1. Dependence of signal intensity on pH of immobilized protein solution: (a) slides images after microanalysis performance: (A) poly(GMA-co-EDMA), (B) poly(GMAco-GDMA), (C) poly(CEMA-co-HEMA-co-EDMA), (D) poly(CEMA-co-EDMA); (b) graphical presentation of the dependence of signal intensity on pH of immobilized protein solution. Conditions: immobilization temperature 37 ◦ C; PMT gain = 450 for poly(GMA-co-EDMA), poly(GMA-co-GDMA) and poly(HPIEAA-co-GMA-co-EDMA); PMT gain = 250 for poly(CEMA-co-HEMA-co-EDMA) and poly(CEMA-co-EDMA).

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lent binding of polymer monolith to inorganic matrix. The operative wells were made to be rectangular of 18 mm × 60 mm well per standard microscopic glass slide. Varying etching reaction time from 15 to 70 min it was managed to obtain the 0.08–0.35 mm depth wells. The depth 0.2 mm was finally found to be optimal from the point of view of further analytical procedure development. The forming thickness of synthesized macroporous monolithic support seems to be enough to provide, on the one hand, the sufficient amount of immobilized ligands, and, consequently, the sufficient adsorption capacity of tested antibody which can be easily detected in spite of non-transparency of polymer support. On the other hand, this thickness allows avoiding significant printed zone spreading that is observed for the case of thinner polymer layers. In order to obtain the wells with mentioned rectangular geometry, the glass was incubated with HF for 30 min. Both after etching with hydrofluoric acid and mechanical treatment, the glass slides were incubated with NaOH to convert Si–F– and Si–O–Si–, respectively, into Si–OH-groups that was necessary for the next matrix activation step (silanation). 3-(Trimethoxysilyl)propyl methacrylate was used as a coupling agent. The last step of the microarray platform fabrication was the photo-initiated synthesis of macroporous polymer layers directly on activated surface of wells performed. As it was mentioned above, macroporous flow-through poly(GMA-co-EDMA) devices (disks, rod columns, capillaries) are widely used in the processes based on dynamic interphase mass transition (chromatography, flowing enzyme reactors, etc.). Such stationary phases are demonstrated the numerous advantages comparing to the packed columns. Their unique features [30] are quite useful for all discussed applications, but especially for those based on a principle of biorecognition (affinity chromatography) [31,32]. Obviously, all positive features of latter could be transferred to modern and impetuously developing bioanalytical field, namely, microarray technology. The main difference between affinity chromatographic separations and microarray analysis is the distinctive mechanism of interphase mass exchange. While in chromatography on monoliths this process is governed by fast convection, in the case of microarray, the mass transfer mechanism is based on slow molecular diffusion. However, the optimized porous structure of polymer monolithic materials along with very small thickness of operative layer allow obtaining much better results in comparison, for example, with gel supports. As it was declared, four new polymethacrylate materials were developed for microarray platform fabrication. The characteristics of used monoliths are presented in Table 1. Despite all positive and very well described in numerous papers and reviews features of poly(GMA-co-EDMA) (including easy one-step biofunctionalization of polymer matrix), the reaction between epoxy groups of a sorbent and amino-bearing ligands is characterized by slow kinetics. It is known that immobilization capacity reached its maximum only after 16–18 h [33]. Therefore, to avoid any undesirable changes in bound biomolecule, we met a problem to introduce more reactive functional groups into polymer structure. Besides, the porous structure of new materials must be similar to that of recently successfully tested poly(GMA-co-EDMA). Poly(HPIEAA-co-GMA-co-EDMA) and poly(CEMA-co-EDMA) containing highly reactive activated ester groups which allow fast covalent attachment of proteins, were developed and tested as a base for discussed microarrays. In the case of terpolymer, GMA is not considered as a reactive comonomer since the interaction between HPIE-groups of material and ligand amino groups proceeds much faster, namely, within 2 h, versus the reaction between amino and epoxy groups. Poly(GMA-co-GDMA) monolith and poly(CEMA-co-HEMA-co-EDMA) were used as the examples of monoliths with enhanced hydrophilicity that can be very important in bioanalysis.

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

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

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Fig. 4. Influence of surface blocking SNR: (a) SNRs obtained without and with surface blocking; (b) images of monolith’s surface after microanalysis performance without and with surface blocking. Conditions: mouse IgG concentration 1 mg/ml; pH 7.5; immobilization temperature 37 ◦ C; poly(GMA-co-EDMA) material.

3.2. Optimization of ligand covalent binding to monolith-based microarrays As it was mentioned above, a model bioaffinity system consisting of mouse IgG and goat anti-mouse IgG conjugated with fluorescent label Alexa 555 was used to demonstrate the potential of developed platforms to be applied for protein microarrays. It is well known that efficiency of solid-phase analysis based on affinity interactions of complementary biological molecules significantly depends on a quantity of immobilized ligand. Therefore, the influence of immobilization conditions (including immobilization temperature, concentration of a protein ligand to be immobilized and pH) on microanalytical results was investigated. In order to determine the optimal ligand’s concentration and pH of reaction, sodium borate buffer (pH 9.5) and PBS (pH 7.5) were compared. Sodium borate buffer was chosen since the reaction between epoxy, activated ester and amino groups proceed with highest rate at alkaline conditions. Besides, from numerous publications it is well known that protein immobilization on the surface of poly(GMA-co-EDMA) at pH ∼9.0 results in highest reaction yield, e.g. the highest amount of immobilized protein [34]. Moreover, the Ka value of ␧-aminogroup of lysine equal to 10 allows supposing its high activity at covalent binding to any mentioned reactive group of a solid support. PBS was tested since physiological mild conditions are preferable from the point of view of maintaining of protein structure responsible for its further specific binding to the protein to be analyzed. It was established that, in general, for all tested polymer materials both pHs could be used for protein immobilization. However, the mean signal intensity (SM) determined in following bioanalytical experiments was 2–3 times higher when PBS was applied as a buffer for protein immobilization (Fig. 1). The main reason of higher signal intensity when PBS is used as

printing buffer is a retention of active protein conformation at pH ∼7.5. Besides that, the ligand density also plays an important role. It is interesting, but this data are opposite to those obtained for affinity chromatography with poly(GMA-co-EDMA). In affinity chromatography, the use of pH 7.5 seemed to be non-optimal value because of resulting in lower ligand surface concentration that followed by decreasing complement’s adsorption capacity. Probably, the tendency observed in microarray format can be explained by

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

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Fig. 6. Comparison of maximal SNRs obtained for developed monolith surfaces at analyzed protein concentration equal to 20 ng/ml. Conditions: pH 7.5; immobilization temperature 37 ◦ C; for poly(GMA-co-EDMA), poly(GMA-co-GDMA) and poly(HPIEAA-co-GMA-co-EDMA) was used PMT gain 450, for poly(CEMA-co-EDMA) and poly(CEMA-co-HEMA-co-EDMA) was used PMT gain 250.

significant increasing of immobilized ligand amount at higher pH values provided by much more higher reactivity of ester groups. In turn, high ligand density might complicate the affinity complex formation and, consequently, lead to analytical efficiency reduction. Besides that, pH 7.5 is friendlier for protein’s native conformation contrary to alkaline conditions. Thus, for the next experiments PBS has been chosen as a ligand printing buffer for all discussed macroporous polymer supports. To reveal the optimal temperature used for ligand immobilization, the slides were incubated at 4 ◦ C, as it was often used in biological practice, and 37 ◦ C, that should be preferable in terms of a rate of the reaction. The main reason of reaction performance at 4 ◦ C is the apprehension to loose the binding activity of immobilized protein because of, the same as in the case of elevated pH, some probable distortion of protein conformation. However, it is known that any multiple-bond covalent immobilization of protein molecule inside three-dimensional porous space leads to its stabilization regarding the preservation of molecular conformation. Therefore, in spite of a risk of partial protein denaturation, we registered higher SM values for all tested polymer materials where ligand immobilization was performed at 37 ◦ C. Fig. 2 illustrates one example of a dependence SM on reaction temperature. The quantity of immobilized mouse IgG necessary for obtaining of high signal intensity was also optimized by variation of its concentration from 0.2 up to 1.0 mg mL−1 (Figs. 1a and 3). It was shown that in the case of poly(GMA-co-EDMA) monolith, SM value reached its maximum at 1.0 mg mL−1 of mouse IgG concentration used for printing, whereas for poly(GMA-co-GDMA) the optimal ligand concentration lied in interval 0.6–1.0 mg mL−1 that corresponded to maximal level of signal intensity. In the case of poly(HPIEAA-co-GMA-co-EDMA), high SM value was already achieved at 0.2 mg mL−1 and only slightly raised at further IgG con-

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centration increasing. When poly(CEMA-co-HEMA-co-EDMA) was used, the mean signal intensity increased linearly with increasing of protein concentration. At the same time, for poly(CEMA-co-EDMA) it was found that SM increased with increasing of mouse IgG concentration from 0.2 to 0.8 mg mL−1 and significantly decreased at 1 mg mL−1 (Fig. 3b). Evidently, the reason of this phenomenon is abundant density of immobilized mouse IgG which leads to steric hindrances for affinity complex formation. The reason of described effects of different IgG concentrations on registered SM, the most probably, can be explained by different content of reactive groups and their binding activity in synthesized monolithic polymers. Thus, the conclusion is that such parameter as protein to be immobilized concentration has to be selected for each support individually. To optimize analytical conditions, it was necessary to establish a sufficient sample volume used for spotting procedure. The variation of this parameter from 100 to 700 pL was tested to make a right choice. It was found that sample volume equal to 500 pL appeared to be sufficient to reach the saturation of polymer surface with protein ligand. It is common for microarray-based analytical protocols to carry out the procedure of surface blocking after ligand immobilization. BSA-containing buffer is widely used for this purpose. Since in our study very big protein (IgG) with molecular mass ∼150 kDa was used as immobilized ligand, the application of smaller BSA as a blocking agent looks acceptable. To confirm the necessity of surface blocking, two experiments carried out under the same conditions were performed. At one of them, the slide with blocked by 1% BSA in PBS surface was incubate with fluorescently labelled goat anti-mouse IgG, while another one was carried out analogously but without blocking procedure. It is clear from Fig. 4 that the blocking of monolith’s surface leads to higher SNR that testifies a better spot quality. The same result was observed for all monolithic supports under discussion. The last step of discussed investigation was devoted to very important establishment of optimal time of slide washing after ligand immobilization step. Washing time was varied form 5 to 60 min. It was shown that the longer time the slides were washed, the higher SM was registered. I was found that only after 40 min washing the mean signal intensity became to be constant. First of all, this result can be explained by slow diffusional mobility of unbound big antibodies that seriously limited their exit from monolith’s pores with sufficiently wide size distribution. A necessity of stabilization of bound protein conformation can be considered as a second reason of results obtained. 3.3. Comparison of efficiency of monolith-based 3-D microarrays and 2-D glass slides The microanalytical results obtained using different macroporous monolithic materials as operative supports and aldehydebearing glass slides were compared. It was impossible to compare registered signal values at the same instrumental gain for all slides, because the scanning of poly(CEMA-co-EDMA) and poly(CEMA-co-HEMA-co-EDMA)

Table 2 Times of immobilization procedures, durations of analysis, intrafield and interfiled coefficients of variation, limit of detection for all compared slides. Type of microarray surface

Time of immobilization (h)

Duration of analysis (h)

Intrafield coefficient of variation (%)

Interfield coefficient of variation (%)

Limit of detection (pg mL−1 )

Aldehyde-modified glass slide Poly(GMA-co-EDMA) Poly(GMA-co-GDMA) Poly(HPIEAA-co-GMA-co-EDMA) Poly(CEMA-co-EDMA) Poly(CEMA-co-HEMA-co-EDMA)

1 16 16 2 4 4

3.5 5.25 5.25 5.25 5.25 5.25

17 8 8 17 10 10

14 8 10 19 11 11

0.200 0.020 0.020 0.020 0.002 0.020

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Fig. 7. Results of OPN detection in cell culture medium obtained for some of developed polymer surfaces. Conditions: pH 7.5; immobilization temperature 37 ◦ C; for poly(GMAco-EDMA) was used PMT gain 450, for poly(GMA-co-GDMA) and poly(CEMA-co-HEMA-co-EDMA) was used PMT gain 350, for poly(CEMA-co-EDMA) was used PMT gain 300.

microarrays demonstrated better resolution at lower gain. Therefore, the values of maximum SNR obtained at different gains optimal for each test-system were used for their comparison. As it seen from Fig. 5, the highest SNR was obtained for poly(CEMAco-EDMA). The second nice result was detected for hydrophilic poly(GMA-co-GDMA). The sensitivity of test-system based on hydrophilic poly(GMA-co-GDMA) was two times higher relating to conventional poly(GMA-co-EDMA) that probably can be explained by better penetration of water-surrounding protein molecules into a three-dimensional porous space of hydrophilic material. The microarrays based on terpolymers, namely, poly(CEMA-coHEMA-co-EDMA) and poly(HPIEAA-co-GMA-co-EDMA), showed the comparable results and, moreover, very close to that obtained with poly(GMA-co-EDMA) material. Such result is explained by low content of functional groups in terpolymers (Table 1) that, in its turn, leads to the low ligand’s surface density. It should be noted that the content of functional monomer in the poly(CEMAco-HEMA-co-EDMA) is three times lower in comparison with poly(CEMA-co-EDMA). For the case of poly(HPIEAA-co-GMA-coEDMA), the amount of activated ester groups is one and a half time lower comparatively to CEMA-containing terpolymer. Finally, it was found that the sensitivity of analysis on microarray based on poly(CEMA-co-HEMA-co-EDMA), poly(HPIEAA-co-GMA-co-EDMA) and poly(GMA-co-EDMA) was practically the same, whereas the reaction of ligand immobilization taking place for two terpolymers was much faster than for GMA-bearing copolymer. This fact can be counted as a great practical advantage of these devices in spite of demonstrated modest sensitivity. A comparison of developed microarray based on macroporous polymer monoliths with widely used glass biochips was also performed. A detection limit for test-system based on macroporous supports, except poly(CEMA-co-EDMA), was found to be 20 ng mL−1 (0.133 pmol mL−1 ), while for glass slide this value came to 200 ng mL−1 (1.333 pmol mL−1 ) for used affinity system, e.g. the difference of one order of magnitude was observed. The sensitivity limit was established according to a concentration of a solution of complementary labelled antibody to which the slides with immobilized ligand were immersed to reach affinity pair formation. Fig. 6 demonstrates SNR for monolith-based microarrays obtained using concentration of goat anti-mouse IgG-Alexa Fluor 555 20 ng mL−1 . As it seen from the figure, SNR of poly(CEMA-co-

EDMA) is quite higher and left the possibility to detect much lower protein amounts. The reproducibility of results obtained was estimated by intrafield and interfield coefficient of variation [29]: Coefficient of variation =

standard deviation mean of signal intensity

The intrafield and interfiled coefficients of variation for compared surfaces are presented in Table 2. The means of intrafield and interfiled coefficients of variation for glass slides are given according to data published elsewhere [35]. As it seen from Table 2, all monoliths under investigation show a better reproducibility compared to 2-D glass slides, except poly(HPIEAA-co-GMA-co-EDMA) demonstrating the highest values of intrafield and interfiled coefficients of variation. The reason of this fact is probably connected with low reproducibility of this kind of monoliths production. 3.4. Microarray-based detection of bone tissue marker osteopontin Osteopontin represents a phosphorilated bone matrix glycoprotein which has a wide area of tissue localization [36]. In vivo osteopontin is excreted to the blood plasma and its concentration reflects the increased turnover associated with bone destruction of aging, menopause and different conditions affecting bone metabolism. Preliminary experiments on determination of osteopontin were performed on poly(GMA-co-EDMA) microarray platform using standard system including monoclonal antiosteopontin, standard osteopontin solutions, biotinilated captures antiosteopontin and streptavidin conjugated with Cy3. To determine a detection limit, the concentration of osteopontin was varied from 0.001 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 human osteosarcoma, and a supernatant obtained after cultivation of primary mesenchymal stem cells derived from human adipose tissue. To compare the signal intensities obtained with cell culture media

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

with analogous results established for standard osteopontin solutions, 16-well monolith-based platforms were used. The presence of osteopontin was easily detected in cell supernatant of SAOS-2, while for cell supernatant obtained after mesenchymal stem cells cultivation the target protein was not found. Fig. 7 demonstrates a comparison of SNRs obtained for microarrays based on developed polymers. As it was expected the highest SNRs were obtained for poly(CEMA-co-EDMA) and poly(GMA-co-GDMA) due to high reactivity of activated ester groups of the former and higher hydrofilicity of the later compared with poly(GMA-co-EDMA). 4. Conclusions In present study the new platforms for 3-D microarrays construction based on macroporous polymer monolithic materials were developed. Each step of microanalytical device fabrication (manufacturing of operative wells, introduction of double bonds into the well surface and photo-initiated copolymerization of monomers) was carefully investigated and optimized. Along with well known poly(GMA-co-EDMA), four new monolithic materials were tested and compared as microarray supports. It was demonstrated that the use of monoliths containing reactive monomers with activated ester groups, as well as a hydrophilic cross-linker, led to the increasing of analytical efficiency in comparison with poly(GMA-co-EDMA). The best analytical potential was determined for poly(CEMA-co-EDMA) material. Besides that, monolith-based microarrays were compared with aldehydebearing 2-D glass slides and the difference between the detection limits of all tested devices was established. All developed materials are characterized with high spot quality and excellent sensitivity. It was found that all polymer devices had at least one order of magnitude advantage regarding to sensitivity in comparison with 2-D microarray (glass slide). As a practical example, a detection of bone tissue marker, namely, osteopontin was developed using suggested microanalytical system based on poly(GMA-co-EDMA). The detection limit was found to be equal to 0.01 ␮g mL−1 that corresponded to 0.306 pmol mL−1 . Acknowledgements This work was supported by DFG grant (Grant No. KA 1784/4-1; Coordinators: Prof. T. Tennikova, IMC RAS and Dr. C. Kasper, ITC UH) and RBRF grant (08-08-00876-a, Supervisor Prof. T. Tennikova). The authors are grateful to German Academic Exchange Service (DAAD) for providing the scholarship for PhD student Marina Rober for fourmonth work at the Institute of Technical Chemistry, University of Hannover (ITC UH). The authors are also grateful to Prof. Thomas Scheper (ITC, Hannover University) and Prof. Valery Krasikov (IMC RAS) for organizing help and fruitful discussions.

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