Nanoporous Silicon as a Support for High-Performance

Nov 8, 2003 - Small spot area, good spot reproducibility, and homogeneous spot profiles were ... The dispensing was performed using an in-house-develo...
0 downloads 0 Views 215KB Size
Download PDF
Anal. Chem. 2003, 75, 6968-6974

Macro-/Nanoporous Silicon as a Support for High-Performance Protein Microarrays Anton Ressine,† Simon Ekstro 1 m,† Gyo 1 rgy Marko-Varga,‡ and Thomas Laurell*,†

Department of Electrical Measurements, Lund Institute of Technology, Lund University, P.O. Box 118, S-221 00 Lund, Sweden, and Department of Analytical Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden

The present work demonstrates the possibilities of using macroporous silicon as a substrate for highly sensitive protein chip applications. The formation of 3D porous silicon structures was performed by electrochemical dissolution of monocrystalline silicon. The fabricated macroporous silicon network has a rigid spongelike structure showing high uniformity and mechanical stability. The microfluidic properties of the substrates were found to be essential for a good bioassay performance. Small spot area, good spot reproducibility, and homogeneous spot profiles were demonstrated on the substrates for immobilized aRIgG. Water contact angles were measured on the porous surface and compared to that of planar silicon, silanized glass, and ordinary microscope glass slides. The effect of the porous surface on the performance of a model IgG-binding immunoassay is presented. aRIgG was microdispensed onto the chip surface forming a microarray of spots with high affinity for the target analyte. The dispensing was performed using an in-house-developed piezoelectric flow-through dispenser. Each spot was formed by a single droplet (100 pL) at each position. The macroporous silicon allowed a high-density microarraying with spot densities up to 4400 spots/cm2 in human plasma samples without cross-talk and consumption of only 0.6 pmol of antibodies/1-cm2 array. Antigen levels down to 70 pM were detected. The Human Proteome Organisation (HUPO, www.hupo.org) emerged as an initiative to consolidate international and regional proteome activities and to assist in the coordination of public proteome initiatives. The task of identifying structure, function, and expression of proteins in a given species is the next major milestone within life science research. In line with the efforts of deciphering the proteome, new and more efficient biotechnology concepts for improved protein expression mapping are constantly being sought. Protein array technology is one such approach that is considered to have a great potential for global analysis of protein expression in a high-throughput fashion.1-5 It is clear that the * Corresponding author. E-mail: [email protected]. † Department of Electrical Measurements. ‡ Department of Analytical Chemistry. (1) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; et al. Science 2001, 293, 21012105. (2) de Wildt, R. M. T.; Mundy C. R.; Gorick B. D.; Tomlinson I. M. Nat. Biotechnol. 2000, 18 (9), 989-994.

6968 Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

impact of screening for gene and protein recruitment will be the next revolution within diagnostics.6 This array technology, in a chip format analogous to DNA chips, has several advantages over conventional immunoassays. The chip-based assay enables rapid analysis of a large number of samples in a single parallel experiment. The amount of material needed is commonly very small. Reaction volumes can be hundreds to a thousand times lower than the amount that is generally used in conventional microtiter plate assays.7 Several research groups and commercial vendors are developing protein chip separation technologies that may rival 2D PAGE as the method of choice for proteome analysis. Protein chips are commonly based on fundamental properties of molecular interaction, such as, ion exchange, hydrophobic/ hydrophilic functionalities or biospecific binders, that is, antibodies, receptor ligand functionalities, DNA/RNA, etc., tethered in array format on surfaces designed to capture the proteins of interest.8 Harvard Institute of Proteomics Research is developing the FLEX (full length expression) Repository to provide researchers with the complete set of known genes and open reading frames in a robot-accessible array of cDNA clones. With their system, high-throughput parallel screens of proteins for the creation of protein microarrays and the facilitation of structural determinations can be designed.9 Housemann et al. 10 presented a protein biochip for quantitative analysis of protein kinase activity. The principle was based on the immobilization of the peptide substrate for the Src kinase on a self-assembled monolayer (SAM) of alkanethiolates on gold surfaces. The monolayer helped to prevent nonspecific binding of proteins to the biochip. The group demonstrated that the Src kinase specifically phosphorylated the peptide substrates on the chip and subsequently quantified the effect of three known Src kinase inhibitors on the enzyme’s activity. Eventually, researchers may be able to adapt this technology to high-throughput phosphoproteomics studies. Snyder’s group used biochips to conduct biochemical assays of 119 (3) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (4) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2 (2), R4.1R4.13. (5) Fung, E. T.; Thulasiraman, V.; Weinberger, S. R.; Dalmasso, E. A. Curr. Opin. Biotechnol. 2001, 12 (1), 65-69. (6) Service, R. F. Science 2003, 300, 236-239. (7) Cahill, D. J. J. Immunol. Methods 2001, 250, 81-91. (8) Merchant, M.; Weinberger, S. R. Electrophoresis 2000, 21 (6), 1164-1177. (9) Brizuela, L.; Richardson, A.; Marsischky, G.; Labaer, J. Arch. Med. Res. 2002, 33, 318-324. (10) Housemann, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270-274. 10.1021/ac034425q CCC: $25.00

© 2003 American Chemical Society Published on Web 11/08/2003

protein kinases cloned from yeast.11 These proteins represent nearly all of the 122 yeast kinases predicted by bioinformatics database mining based on gene sequence homology. The kinases were expressed, immobilized in the wells of the biochip, and tested for in vitro kinase activity in 17 different assays. The results of this study suggested that more kinases actually exist in yeast than predicted by the sequence homology studies. Another promising concept for protein separation is to develop a library of recombinant antibodies (scFv) for each protein in a cell and then pattern these antibodies (Abs) onto different spots on a protein chip. An example of this approach has been presented earlier.12,13 The surface of the chip plays an important role in ensuring the immobilization of the capture elements and providing the reproducible detection of a ligand-binding event. The anchoring of the capture elements could be done via either covalent or noncovalent bonds to reduce the possibility of dissociation during the washing stages and also to prevent protein denaturation. Different types of surfaces have already been explored for arraying proteins,14,15 and the search for new supports giving superior characteristics is still a challenge. The following types of microarray surfaces are currently available: 1. Filters and membranes, e.g., nitrocellulose or PVDF,16,17 are readily derivatized for covalent attachment and are low cost and reusable. However, a disadvantage is that they allow only a limited spot density as each sample tends to spread out. 2. Derivatized glass substrates1,3,4 are compatible with most commercial microarrayers and are low cost and readily derivatized for covalent attachment. But it could introduce concentration effects that are nonuniform spot intensity profiles caused by localized aggregation on the spot and also the well-known “coffee stain effect”/the dried ring. 3. Gel pads and agarose films18,19 provide reduced evaporation rate from the surface and high sample capacity, but are more expensive due to the photolithography process in the fabrication and may require longer washing steps. 4. Gold/aluminum-coated substrates derivatized with dextrans or SAMs can immobilize proteins via cysteines or other amino acid residues.14 Over the past years porous silicon, fabricated in the surface of monocrystalline silicon wafers,20-22 have emerged as an interesting support for high localized immobilization of proteins (e.g., enzymes).23 The rising interest for porous silicon in mi(11) Zhu, H.; Klemic, J. F.; Chang, S.; Bertone, P.; Casamayor, A.; Klemic, K. G.; Smith, D.; Gerstein, M.; Reed, M. A.; Snyder, M. Nat. Genet. 2000, 26, 283-289. (12) Borrebaeck, C. A. K.; Ekstro¨m, S.; Malmborg Hager, A. C.; Nilsson, J.; Laurell, T.; Marko-Varga, G. Biotechniques 2001, 30, 1126-1133. (13) Wingren, C.; Ingvarsson, J.; Lindstedt, M.; Borrebaeck, C. A. K. Nat. Biotechnol. 2003, 21 (3), 223. (14) O’Connor, D. C.; Pickard, K. In Microarrays and Microplates: Applications in Biomedical Science; Ye, S., Day, I. N. M., Eds.; BIOS Scientific Publishers: Oxford, 2003; pp 65-72. (15) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55-63. (16) Ge, H. Nucleic Acids Res. 2000, 28 (2), e3. (17) Walter, G.; Bussow, K.; Cahill, D.; Lueking, A.; Lehrach, H. Curr. Opin. Microbiol. 2001, 250, 81-91. (18) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278, 123-131. (19) Afanassiev V.; Hanemann, V.; Wolfl, S. Nucliec Acids Res. 2000, 28, e66. (20) Lehmann, V.; Gosele, U. Appl. Phys. Lett. 1991, 58 (8), 856-858. (21) Smith, R. L.; Collins, S. D. J. Appl. Phys. 1992, 71 (8), R1-R22. (22) Canham, L. Properties of porous silicon; Inspec Publication: London, 1997.

crosensor and microsystem application areas are explained by several factors: its extraordinary material properties such as a high surface area-to-volume ratio (hundreds of square meters per cubic centimeter), which makes it highly efficient for biomolecule immobilization; a pore geometry, morphology, and density that can easily be adjusted and varied during the fabrication process; optical properties of the porous layer that can be tuned during the fabrication process; and new technologies for porous silicon fabrication compatible with standard microelectronic and MEMS techniques.24,25 Another example of applications of porous silicon in the analysis of biomolecules is the use of porous silicon as an immobilization matrix in microstructured enzyme reactors.26,27 The shifts in the Fabry-Perot fringe pattern of reflected light during an analyte binding event on functionalized porous silicon surface was used as the basis for optical interferometric biosensing.28 The application of porous silicon for biomolecular screening with encoded porous-silicon photonic crystals was also described.29 This paper reports the development of a new substrate for protein chips, pore chip protein array (PCPA), based on the use of macroporous silicon as the matrix for immobilized Abs. The use of porous silicon (PS) can give several advantages over conventional supports, e.g., high surface area enlargement, flexibility to change interfacial properties of the porous silicon surface, low background interference and potential for co-integration with silicon microfabricated devices. Porous silicon is fabricated by means of anodic dissolution in hydrofluoric acid. Since the morphology and microstructure of PS is strongly governed by a large number of etching parameters such as HF concentration, current density, anodization time, temperature, illumination, crystal orientation, silicon type, and doping level, various porous matrixes with different physical properties can easily be obtained in a controllable and reproducible way. The PCPA concept presented herein enables high-density arraying of over 4000 spots/cm2. Furthermore, the surface provides a low nonspecific background signal and in a standard aRIgG/RIgG assay specific binding events were detected at a level of 70 pM, i.e., 10 ng/mL. In our opinion, it is clear that new developments within protein microarrays may play a key role in aligning the disparity between genomics and proteomics.30,31 EXPERIMENTAL SECTION Materials and Reagents. Silicon wafers were purchased from Topsil Semiconductor Materials A/S (Frederikssund, Denmark). Ethanol and 45% aqueous solution of HF were obtained from Merck AG (Darmstad, Germany). Silanized glass slides (Si(23) Laurell, T. In Sensors, Update 10; Baltes, H., Fedder, G. K., Korvink, J. G., Eds.; Wiley-VCH: Weinheim, 2002; pp 3-32. (24) Foll, H.; Christophersen, M.; Carstensen, J.; Hasse, G. Mater. Sci. Eng. R. 2002, 39 (4), 93-141. (25) Stewart, M. P.; Buriak, J. M. Adv. Mater. 2000, 12 (12), 859-869. (26) Drott, J.; Rosengren, L.; Lindstrom, K.; Laurell, T. Thin Solid Films 1998, 330, 161-166. (27) Drott, J.; Rosengren, L.; Lindstrom, K.; Laurell, T. Mikrochim. Acta 1999, 131, 115-120. (28) Tinsley-Bown, A. M.; Canham, L. T.; Hollings, M.; Anderson, M. H.; Reeves, C. L.; Cox, T. I.; Nicklin, S.; Squirrell, D. J.; Perkins, E.; Hutchinson, A.; Sailor, M. J.; Wun, A. Phys. Status Solidi A 2000, 182, 547-553. (29) Cunin, F.; Schmedake, T. A.; Link, J. R.; Li, Y. Y.; Koh, J.; Bhatia, S. N.; Sailor, M. J. Nat. Mater. 2002, 1, 39-41. (30) Payk, K. 5th Siena Meeting from Genome to Proteome: Functional; Siena, Italy, 2-5 September 2002. (31) Albala, J. S.; Humphery-Smith, I. Curr. Opin. Mol. Ther. 1999, 1 (6), 680684.

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

6969

lanePrep), anti-rabbit IgG (aRIgG), and rabbit IgG-FITC (RIgGFITC) were from Sigma (St. Louis, MO). Angiotensin-1 homologue (Ang1h-FITC) was synthesized at Innovagen AB (Lund, Sweden). Human plasma samples were obtained from healthy volunteers. PCPA Fabrication. Macroporous silicon supports were fabricated by anodic dissolution of p-type silicon wafers in HF solution. This process was performed in a two-compartment cell. Current was passed through the wafer to initiate and progress the porous silicon layer formation. Macroporous silicon support surfaces were formed on p-type 20-70 Ω‚cm silicon wafers of (110) crystal orientation. The backside of the wafer was illuminated during the complete anodization period using a 100-W halogen lamp (Osram) at a distance of 2 cm from the transparent sapphire glass (Melles Griot BV) window on the backside of the electrochemical cell. A current density of 10 mA/cm2 was applied for 10 min. The electrolyte was a 1:1 mixture by volume of 45% HF and 95% ethanol. The porosified silicon wafer with the macroporous layer on the surface was diced into small pieces sized ∼1 cm2 forming the PCPA chips. Antibody Binding Assay Protocol. To evaluate immobilization efficiency of the macroporous silicon chips, a model antibody binding bioassay was performed. The capture antibodies aRIgG were arrayed onto the macroporous silicon chip followed by a washing step in 10 mM PBS (pH 7.4) containing 0.05% v/v polyoxyethylene sorbitan monolaurate (PBS-Tween). Arrayed chips were blocked with PBS-Tween containing 5 wt % nonfat milk for 20 min. After washing in PBS, the chips were incubated in solutions containing target antibody-/RIgG-FITC for 1 h. Following the antibody incubations, the chips were washed twice in 10 mM PBS and then in pure water in order to remove salt from the surface. Microarraying. Microarrays were formed by spotting aRIgG in 58-µm droplets onto the macroporous silicon using an in-housedeveloped chip-based piezoelectric microdispenser32 and a computercontrolled arraying station. The diameter of the droplets was measured by stroboscopic imaging, and the volume was calculated to be ∼100 pL. In all experiments, microarrays were formed by dispensing one droplet per spot in order to keep the spot area at minimum. Microarray Imaging. Fluorescence microscope imaging was performed in order to evaluate the level of bioactive capture antibodies immobilized onto the macroporous silicon. Images were grabbed with a CCD camera (Orca-ER, Hamamatsu, Japan) mounted onto the fluorescence microscope. Mean fluorescence intensities of the spots were measured and compared for different amounts of immobilized capture antibodies and for different concentrations of probe molecule solution. Study of Macroporous Silicon Chip Properties. Fabricated chips were studied using scanning electron microscopy to characterize morphology and geometry of the macroporous silicon layers. For comparative studies, contact angle measurements were performed on 2-nL water droplets dispensed on macroporous silicon, hydride-terminated planar silicon, glass slides, and aminosilanized glass slides. Subsequently digital images of the deposited droplets on the surfaces were grabbed with a CCD camera and contact angles were derived. (32) Laurell, T.; Wallman, L.; Nilsson, J. J. Micromech. Microeng. 1999, 9 (4), 369-376.

6970 Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

Figure 1. Chemical pathway of silicon anodization and schematic drawing of the principle for the formation of the nano-/macroporous silicon network. Formation process includes following stages: (A) migration of charge carriers in the bulk silicon to the electrolyte interface, (B) random pore nucleation, (C) macropore formation and propagation, and (D) nanoporous side branching.

RESULTS AND DISCUSSION Processing and Characterization of Macroporous Silicon. Formation of 3D porous silicon surfaces with a high surface-tovolume ratio was performed by electrochemical dissolution of a crystalline silicon wafer in HF solution. Different types of morphologies and geometries could be obtained depending strongly on anodization conditions, HF concentration, and dopant level of the Si wafer.22,24 This gives a high flexibility to form 3D PCPA supports with different pore sizes, shapes, orientations, densities, and levels of branching. Depending on the characteristic pore size, porous silicon layers can be separated into three general categories: microporous, mesoporous, and macroporous.24 This is the simplest classification regarding the average pore diameter. Microporous silicon has pore diameters less than 10 nm, mesoporous with pore sizes in the range of 10-50 nm, and macroporous with pore sizes larger than 50 nm. Both geometry (characteristic sizes of pores and distances between adjacent pores) and morphology of the porous layer (pore shape, orientation, level of branching) affect the physical properties of the protein chip and thus its performance characteristics. The general demands on the porous supports for protein immobilization are as follows: good uniformity and mechanical stability of the porous layer, low intrinsic fluorescent background, and low wetting ability, i.e., a high liquid contact angle. The conditions described in the Experimental Section were chosen to make porous silicon chips matching these demands. The simple model for pore formation in silicon is based on the transport of minority charge carriers, i.e., holes, through the bulk silicon. The chemical anodization mechanism as described by Lehmann and Go¨sele20 and the stages of nano-/macroporous silicon network fabrication are schematically shown in Figure 1. According to this reaction pathway, the silicon surface continuously vacillates between hydride- and fluoride-terminated states. It is suggested that hydride bonds passivate the silicon surface

Figure 2. SEM images of the porous silicon network. (a) Cross sections and (b) top views of the rigid spongelike porous silicon network structure.

until the holes reach the interface. Dissolution of silicon in the form of SiF62- ion subsequently occurs, and molecular hydrogen is released. The porous silicon formation process is also schematically illustrated in Figure 1. A. Initiation of Charge Carrier Movement under Applied Voltage. As the voltage is applied to the electrochemical cell, the charge carriers inside the bulk silicon, i.e., holes, migrate toward the cathode. B. Random Macropore Nucleation. When holes reach the interface between the silicon and electrolyte, the pore nucleation is initiated and macropore growth starts. C. Macropore Formation. Due to the enhanced electrostatic field at the pore tips, holes are attracted to this location, which results in a limited transport of holes to the side wall of the generated pore. Hence, the pore is found to propagate faster at the tip. D. Development of the Porous Silicon Network. With time, side branching nanopores emerge and adjacent macropores become interconnected through the pore walls, forming a rigid spongelike porous silicon network. The fabricated macroporous layers on the silicon wafers showed good uniformity and mechanical stability. The scanning electron microscope (SEM) study showed the thickness of the layers to be ∼100 µm and the characteristic pores size to be in the range of 1-4 µm. Macropores are located at the silicon interface and oriented perpendicularly to the silicon wafer surface, Figure 2a (lower left inset). Adjacent macropores, Figure 2a (upper right inset), are also interconnected with each other with tiny nanopore channels running through the pore walls and forming a nanopore substructure (this nanopore channel structure is not visible on the presented SEM images). These morphologies resemble the polymeric superhydrophobic surfaces reported recently.33 The macroporous skeleton and the nanoporous substructure together form a rigid spongelike porous silicon network. The homogeneity of the porous layer is well illustrated in Figure 2b, showing a top view of the porous surface. Microarraying and Microfluidic Properties of the PCPA. In this study, the anchoring of the capture antibodies was (33) Erbil, H. Y.; Demirel A. L.; Avci, Y.; Mert, O. Science 2003, 299, 13771380.

performed via physical adsorption. This immobilization method was chosen since it is simple and straightforward, not requiring multiple processing steps. The capture molecules were microdispensed onto the chip surface forming an array of spots with high affinity for the target analyte. The idea of the capture protein chip concept is to create microarrays where each unique spot has an affinity to a certain target protein. Microarraying was performed in a single drop mode where each spot was formed by only one droplet of ∼100-pL volume. Microfluidic properties of the support surface are essential for the performance of the PCPA. When capture antibodies are dispensed on the chip, the spot area strongly depends on the wetting properties of the chip surface. A low wetting ability of the surface leads to a better drop confinement on the chip and thus to a lower spot area. This gives several significant benefits. First, higher immobilization densities of antibody can be obtained on nonwetting surfaces, i.e., surfaces with a high value of the water contact angles, since the antibody is distributed over a smaller spot area. Second, the possibility to create microarrays with higher density of spots per square centimeter emerges, as smaller spot sizes inherently allow higher density arraying. Another important advantage is that the binding equilibrium is reached more rapidly with a reduced spot size, since the rate at which the capture proteins become occupied with a target molecule is inversely proportional to the size of the protein spot.34 In this study, we compared the microfluidic properties of the following: (a) ordinary microscope glass slides, (b) glass slides derivatized with aminoalkylsilane, (c) hydride-terminated flat silicon surfaces, and (d) porous silicon surfaces. Contact angle measurements were performed, and the values were found to be 16, 53, 84, and 110 deg, respectively, suggesting that macroporous silicon could be a good candidate surface for microarraying according to the reasoning above. The low wetting properties of the macroporous silicon support is a quite useful phenomenon in the perspective of high-density protein microarraying. The presence of a macropore network with a microstructured topology on the top of the silicon wafer changes the wetting behavior of the surface drastically (from hydrophilic with a contact angle below 90 deg on a planar hydride-terminated (34) Ekins, R. P. Clin. Chem. 1998, 44 (9), 2015-2030.

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

6971

Figure 3. Performance of the IgG binding assay and fluorescent images (lower insets) of single spots with immobilized antibodies incubated in a solution with labeled target analyte for (1) the macroporous silicon chip and (2) the aminoalkylsilane-derivatized glass. The upper insets show the capture antibody droplets on their corresponding surfaces immediately after deposition, illustrating the obtained spot sizes due to the different wetting properties.

silicon to hydrophobic, having a contact angle value higher than 90 deg on a porosified surface). This phenomenon occurs as the area of the surface that actually is in physical contact with the fluid is reduced to very small submicrometer well-spaced contact points. The effect is more commonly known as the “Lotus flower effect”.35 More fundamental descriptions on the effect on surface wetting properties when micro- and nanostructures are introduced in the surface are given by Cassie and Baxter.36 As stated by the Cassie-Baxter model, an enhanced hydrophobicity of a surface can be achieved by roughening the surface by making it porous. Thus, the observed increase in contact angle on macroporous silicon compared with a planar hydride-terminated silicon surface is in good agreement with this model. Another important issue raised in microarray applications is the homogeneous coverage of the spot in the antibody deposition step.37 For example, for glass slides (the substrates most commonly used), it is known that sample drops drying on the surface result in unwanted ringlike structures, so-called “coffee stain” or “doughnut effect”. This effect is caused by the evaporation-driven convection mechanism, which moves the fluid from the center of the droplet to the surface contact line, where evaporation is faster. The “doughnut-shaped” intensity profile is a big disadvantage in the perspective of quantitative determinations. Introduction of a microstructured topology on the top of the silicon wafer by electrochemical etching allows a substantial reduction of this undesired spot profile. To demonstrate the effects of reduced spot size and improved spot homogeneity, an IgG binding assay was performed on two types of surfaces with different wetting properties: (1) the macroporous silicon chip and (2) an aminoalkylsilane-coated glass slide as outlined in Figure 3. The amino-terminated glass slides were chosen for comparison as they are one of the commonly used supports for biomacromolecule microarraying, which provide (35) Barthlott, W.; Neinhuis, C. Planta 1997, 202, 1-8. (36) Cassie, A. B. D.; Baxter, S. Trans. Faraday Soc. 1944, 40, 546-551. (37) Blossey, R. Nat. Mater. 2003, 2 (5), 301-306.

6972

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

chemically “mild” conditions for the immobilized molecules. It is known that strong hydrophobic surfaces such as octadecylsilanemodified surfaces, may alter the protein configuration and thereby the bioaffinity properties of the capturing proteins bound to the surface. This is the motivation why this approach was not investigated further. As an alternative to a C-18 modification, we rather propose the low wetting properties that we obtain on the porous silicon surfaces since this maintains the properties of good spot confinement and yet a chemically hydrophilic surface, which is more suitable for the biomacromolecule affinity binders that we use. Antibody anchoring to the macroporous PCPA and aminosilanized glass surface was done by dispensing 140 amol of capture aRIgG/spot. The difference in wetting properties of the two surfaces led to the fact that the obtained spot diameter was about half the size on the macroporous support than on the aminosilanized glass surface, which corresponds to a 4-fold decrease in spot area, Figure 3, drop images (top inset), and spot fluorescence intensity image (low inset). Thus, the antibody densities predicted from the amount deposited on these supports differ by a factor of 4 yielding approximately 7.2 and 1.8 pmol/cm2 for macroporous PCPA and amino-silanized glass, respectively. Following the antibody anchoring, the IgG binding assay was performed according to the protocol described in the Experimental Section. Results of the fluorescent imaging of the RIgG-FITC specifically bound to the aRIgG immobilized on the surface are presented in Figure 3 (lower insets). The average diameter of the fluorescent spots obtained on the macroporous PCPA was found to be about 55 µm, and 110 µm on the amino-silanized glass supports. The macroporous surface demonstrated better fluorescence signal than the silanized glass, which is attributed to the increased antibody density. Spots on the macroporous supports are highly uniform, showing homogeneous intensity profiles. No concentration effects are observed, which is common for the derivatized glass supports, enabling a better quantitative evaluation of the spot signal. We have also observed that the porous silicon surfaces display stable wetting properties over longer period of time. This, however, is highly dependent on the type/morphology of porous silicon that is studied. The macroporous silicon investigated in this paper displays very stable performance over several weeks, whereas several microporous silicon surfaces tested in our preliminary studies displayed a rapid change in contact angles over hours (data not shown). These observations demonstrate that a proper tuning of the pore morphology may provide surfaces possessing stable hydrophobic states possibly over months. To investigate the stability of the porous silicon regarding corrosion in the buffer solutions used, the macroporous silicon surfaces were also investigated by SEM imaging, showing no evidence of changed morphology. Optical Properties of PCPA in Immunoassays. The porous silicon has not been widely used as an immobilization support for fluorescence applications. One reason may be the fact that the intrinsic fluorescence of freshly prepared porous silicon is commonly very high and thus a high background fluorescence in protein chip applications could be anticipated. An important observation in our study, however, revealed that the treatment of fabricated macroporous silicon during the bioassay procedure, i.e., the blocking and incubation, caused a dramatic suppression of

Figure 4. IgG binding assay performed on the PCPA. (A) Six microarrays with 140 amol of aRIgG dispensed per spot were incubated in solutions with varying RIgG-FITC concentrations ranging between 0.7 and 133 nM. (B) Microarrays with different amounts of aRIgG dispensed per spot were incubated in the following RIgG-FITC concentrations: 0.07, 0.7, 3.3, and 27.0 nM. Error bars indicates standard deviation of the spot intensities (n ) 10).

the intrinsic fluorescence (30 times lower when compared to the untreated porous silicon chip). After the whole assay was performed, the fluorescence background of macroporous silicon chips was found to be ∼2 times lower than the background signal obtained on the silanized glass slides (the measurements were made in the FITC spectral region). The intrinsic fluorescence quenching as a result of the bioassay treatment is an important observation and a property that enables the use of the macroporous silicon as a high surface area support with a low background signal for protein chip applications. The low wetting properties of the PCPA supports allow us to work in a single-drop dispensing mode yet obtaining high antibody array densities. These findings together make macroporous silicon an attractive protein chip surface. Model Antibody Binding Assay. To investigate the dynamic range of the assay performed on macroporous silicon supports and to determine the detection limit, several quantitative studies were performed. For every measuring point that composes the fluorescence data from the microarrays, 10 spots with identical aRIgG amounts were analyzed. For the first set of protein chip experiments, six PCPA microchips were arrayed with antibodies by dispensing 140 amol of aRIgG per spot position, Figure 4A. The IgG assay was performed according to the protocol described in the Experimental Section. The microarrays were bath incubated with different concentrations (0.7-133 nM; 100 ng/mL-20 µg/ mL) of RIgG-FITC. Increasing concentration of the labeled analyte in the incubation solution led to an increase in fluorescent signal. With increased concentration of RIgG-FITC in the incubation solution, the curve approaches the saturation region where all aRIgG binding sites are occupied. Bath incubation was performed by placing 200 µL of RIgG over the chip for 1 h. For the second set of experiments, four porous microarrays with varying amounts of aRIgG in each position on the chip were incubated in four different RIgG-FITC concentrations: 0.07, 0.7, 3.3, and 27.0 nM (10, 100, and 500 ng/mL and 4 µg/mL), Figure 4B. Compared to the first set of experiments, here both the concentration of the incubation solution and the amount of dispensed capture aRIgG per spot were varied. Decreasing the amount of capture molecules dispensed per spot led to reduction of the binding sites and thus to reduction of the signal intensity. An amount of 140 amol of aRIgG per spot was selected as a sufficient amount per spot for further studies as saturation is well

Figure 5. Reproducibility of the protein microarrays. Three PCPAs with different amounts of aRIgG dispensed per spot were incubated in a 27 nM RIgG-FITC solution. The chips were fabricated and the assay was run on different days.

reached for the concentration range investigated, Figure 4B. The binding events were possible to detect at a level of 70 pM. A reproducibility test was also performed on the macroporous silicon supports in order to quantitate the assay quality, Figure 5. For this purpose, three identical microarrays were made. Each microarray had varying amounts of dispensed aRIgG per spot as in the second set of experiments. The microarrays were incubated in 27 nM RIgG-FITC for 1 h. Immunoassay in Blood Plasma. To investigate the specificity of the immunoassay and the influence of other proteins binding competitively (specific or nonspecific), a protein binding assay in blood plasma was performed. Blood plasma (445 µL) was spiked with 5 µL of a 45 µg/mL solution of RIgG-FITC in PBS buffer in order to get a 3.5 nM concentration of RIgG-FITC in plasma. Then the PCPA chip was arrayed with 140 amol of aRIgG/spot (Figure 6A), and the bioassay was performed according to the protocol previously used. In a first investigation for false positive control, two macroporous PCPA with aRIgG immobilized were exposed to incubation with plasma samples spiked with Ang1h-FITC. Antigen concentrations of 350 and 1150 nM (0.5 and 2 µg/mL), corresponding to a 100and 328-fold higher antigen level than the RIgG-FITC-spiked plasma sample, were analyzed. No unspecific binding of the Ang1hFITC was observed. The same experimental conditions were used as described above (Experimental Section) for these crossreactivity investigations. Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

6973

Figure 6. (A) Automated microdispensing of antibodies via single 100-pL droplet delivery to each microarray position. The inset image shows a high-speed dispensing sequence at a droplet rate of 5 kHz. (B) Fluorescence image of the high-density protein microarray on the macroporous silicon substrate; 140 amol of aRIgG was immobilized per spot. The microarray was incubated with blood plasma spiked with RIgG-FITC to a level of 3.5 nM. The array density was 4400 spots/cm2.

It was found that the macroporous PCPA was able to selectively capture the protein of interest from a complex biological solution. Figure 6B shows a closeup of the resulting fluorescent image of the PCPA incubated in plasma spiked with RIgG-FITC. The high reproducibility of the protein spot fluorescent readout is clearly demonstrated. The protein spots have a size distribution of 55 ( 2 µm (N ) 50, for CI 0.95). The deposited protein spot sizes within, as well as between, array series are highly uniform. The inset image of the single spot also illustrates the high spot homogeneity, i.e., the absence of on-spot concentration and aggregation effects, and correspondingly this potentially opens the path for highperformance quantitative analysis. The reduced spot size obtained on the macroporous PCPA supports also gives the possibility to increase the spot density of the microarrays as compared to other supports, which typically show spot sizes in the range of 100 µm or larger. The typical spot size of 100-pL droplets are ∼55 µm in diameter on the macroporous surface. This safely allows spacing between the spots (center to center) down to 150 µm or even less without risking cross-talk between the spots. This should be compared to commercial spotters where 180-µm spot sizes at 250µm spacing distances are the lower limit for accurate sample delivery. On the macroporous PCPA, with spot sizes of 55 µm and spacing distance of 150 µm, array densities of 4400 spots/ cm2 can easily be obtained as shown in Figure 6B. This is an even higher array density than earlier reported by Pawlak et al. (3000 spots/cm2) who described microarray chips with an integrated microfluidic system based on planar waveguide technology utilizing the specific advantages of evanescent field fluorescence detection.38 The low consumption of capture antibodies per microarray is also one main issue for our microchip development. To reach this goal, the development of a homogeneous and well(38) Pawlak, M.; Schick, E.; Bopp, M. A.; Schneider, M. J.; Oroszlan, P.; Ehrat, M. Proteomics 2002, 2, 383-394.

6974 Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

defined surface for immunoreagent capture is mandatory. A low antibody consumption is a crucial part of making custom-made microchip arrays based on antibody libraries.12 The spots generated on the PCPA surface only required 140 amol of antibody on each spot for an assay sensitivity down to 70 pM. For the RIgG assay, this means that 1 µg of antibody will allow the analysis of ∼50 000 sample spots. CONCLUSIONS The fabrication of porous silicon is a quite flexible technique offering a possibility of developing 3D high surface area-to-volume ratio structures and controllable porous properties. By choosing the right anodization conditions, it is possible to vary the physical properties of the porous silicon layer to be formed (wetting ability, surface-to-volume ratio, intrinsic fluorescence background, mechanical stability). The suppression of the intrinsic fluorescence background during immobilization and subsequently blocking steps is an important finding that essentially opens the whole area for fluorescent bioassays on high surface area porous silicon supports. Together with the possibility of obtaining a hydrophobic behavior of the surface, macroporous silicon has proven to be a very promising surface for protein microchip applications. With a density of 4400 spots/cm2 the array format is well above the crucial range where sample cross-talk starts to cause a problem. Currently we are pursuing further development of the macroporous protein chip array principle to be used in human clinical samples for the analysis of inflammatory markers. ACKNOWLEDGMENT We acknowledge the financial support through SWEGENE. Received for review April 24, 2003. Accepted October 1, 2003. AC034425Q