Anal. Chem. 2006, 78, 7392-7396
Articles
Improved Sensitivity and Physical Properties of Sol-Gel Protein Chips Using Large-Scale Material Screening and Selection Soyoun Kim,*,†,‡ Youngdeuk Kim,§ Philseok Kim,§ Jeongmin Ha,§ Kyunyoung Kim,§ Mijin Sohn,| Jin-San Yoo,| Jungeun Lee,†,‡ Jung-ah Kwon,⊥ and Kap No Lee⊥
Chemistry Department, Dongguk University, Seoul, Korea, LG Chem Ltd., Research Park, Daejeon, Korea, LG Life Sciences, Research Park, Daejeon, Korea, Diagnosis Division, Kuro Korea University Hospital, Seoul, Korea, and NanoBio Technology Lab, National Research Laboratory (NRL), Ministry of Science and Technology, Seoul, Korea
Protein chips are a powerful emerging technology with extensive biomedical applications. However, the development of optimal, economical surface materials capable of maintaining the activity of embedded proteins is a challenge. Here, we introduce a new optimized, low-cost, solgel biomaterial for use in protein chips with femtogramlevel sensitivity. A novel protein chip material with significantly improved physical properties and sensitivity was produced using unique screening and selection methods. Using this platform, the sensitive, specific detection of the interactions between an HIV antigen and its antibody and between a cyclin-kinase protein pair was observed. This study is the first to demonstrate the detection of protein-protein interactions on sol-gel microarrays and describes an important improvement in the physical properties of sol-gel-derived protein chip materials for biomedical research. The development of protein chip technology has provided a novel tool for examining the complex proteins interactions (i.e., protein-protein, protein-nucleic acid, and protein-small molecule) with a high sensitivity and throughput using small sample volumes.1-4 However, the major challenge for the advancement of protein chip technology is the requirement of optimized chip materials that can maintain the native conformation of the embedded proteins.5-8 Unlike other biomolecules, such as DNA, * To whom correspondence should be addressed. Tel: 82-2-2260-3840. Fax: 82-2-2268-8204. E-mail:
[email protected]. † Dongguk University. ‡ National Research Laboratory (NRL). § LG Chem Ltd. | LG Life Sciences. ⊥ Kuro Korea University Hospital. (1) 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. (2) MacBeath, G. Genome Biol. 2001, 2 (comment2005). (3) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2 (research0004). (4) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (5) Cahill, D. J. J. Immunol. Methods 2001, 250, 81-91. (6) MacBeath, G. Nat. Genet. 2002, 32 Suppl, 526-532.
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proteins tend to unfold when immobilized, leading to a loss of activity and binding sites that are dependent on the threedimensional structure.7-9 In order for protein chip technology to compete with more standard protocols (e.g., enzyme-linked immunosorbent assay), the support materials need to be easy to handle and store, be suitable for mass production, and be capable of yielding high-quality, reproducible results.10-12 Sol-gel materials, the room-temperature synthesized silica glasses, have been studied extensively for use in the entrapment of proteins and have been shown to maintain the protein activity over months or even longer.13,14 The immobilization of proteins within sol-gel-derived materials is an intriguing possibility for use in protein chips because these materials do not require affinity captured agents or tagged recombinant proteins, thereby enabling the entrapment of a wide variety of proteins in their native state.14 The use of various silicates or additives can allow the optimization of materials for the best biocompatibility and activity of the immobilized proteins.13,14 In addition, sol-gel-derived materials are relatively inexpensive and compatible with almost every support chip substrate, which makes mass production and seamless integration with the current technology feasible.14 Despite these advantages, the development of sol-gel-derived protein chips for medical applications has been unsuccessful.15-20 (7) Angenendt, P.; Glokler, J.; Sobek, J.; Lehrach, H.; Cahill, D. J. J. Chromatogr., A 2003, 1009, 97-104. (8) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55-63. (9) Kasemo, B. Surf. Sci. 2002, 500, 656. (10) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278, 123-131. (11) Wiese, R.; Belosludtsev, Y.; Powdrill, T.; Thompson, P.; Hogan, M. Clin. Chem. 2001, 47, 1451-1457. (12) Price, C. P. Clin. Chem. 2001, 47, 1345-1346. (13) Livage, J.; Coradin, T.; Roux, C. J. Phys. Condens. Matter 2001, 13, R673R691. (14) Gill, I. Chem. Mater. 2001, 13, 3404-3421. (15) Gill, I.; Ballesteros, A. Trends Biotechnol. 2000, 18, 282-296. (16) Kim, Y. D.; Park, C. B.; Clark, D. S. Biotechnol. Bioeng. 2001, 73, 331337. (17) Park, C. B.; Clark, D. S. Biotechnol. Bioeng. 2002, 78, 229-235. (18) Cho, E. J.; Bright, F. V. Anal. Chem. 2002, 74, 1462-1466. (19) Besanger, T. R.; Easwaramoorthy, B.; Brennan, J. D. Anal. Chem. 2004, 76, 6470-6475. 10.1021/ac0520487 CCC: $33.50
© 2006 American Chemical Society Published on Web 09/27/2006
The development of sol-gel-based protein chips hinges on optimizing the following factors: (1) the size of the nanoporous structure in the sol-gel matrix must be fit to the size of the encapsulated biomolecule;21-24 (2) the gelation time should be long enough to process spotting before gelation; (3) the adhesion of the spot to the support surface needs to be strong enough to endure harsh washing; (4) a neutral pH of the sol-gel is needed for the maximal protein activity; and (5) the morphology of each spot needs to be uniform, which is essential for automated computer image analysis.25 In this study, unique screening and selection methods were used to obtain the best formulation for a novel three-dimensional sol-gel chip with optimal physical properties, including strong spot adhesion and good protein activities. The selected formulations were then used to detect protein-antibody interactions and protein-protein interactions on the sol-gel-derived protein chips. EXPERIMENTAL SECTION Protein Preparations. The bovine serum albumins (BSA) protein and anti-BSA antibodies were purchased from SigmaAldrich. The HIV P24 protein, anti-P24 antibodies, and Cy3-labeled secondary antibodies (goat, rabbit, and mouse) were purchased from Abchem. The human CyclinT and Cdk9 proteins 26 were cloned and expressed using the pET28 system (Novagen). The labeled peptides, 4-1-BB proteins, were obtained from LG Life Sciences. Protein labeling was performed using the fluorescent labeling kit according to the standard protocol (Molecular Probes). Material Preparation. Solutions containing 5-25% silicate monomers [tetramethyl orthosilicate (TMOS; Gelest), n-methyltrimethoxysilane (MTMOS; Aldrich), tetraethyl orthosilicate (TEOS; Sigma), ethyltriethoxysilane (Gelest), tetramethoxysilicate (TMS; Sigma), methyltrimethoxysilicate (MTMS; Sigma), or 3-aminotrimethoxysilane (3-ATMS; Gelest)], 5-15% of intermediates [polyglycerylsilicate (PGS)15,27 or diglycerylsilane20], and 2.5-15% of additives [3-glycidoxpropyltrimethoxysilane (GPTMOS; Sigma), N-triethoxysilylpropyl-O-poly(ethylene oxide) urethane (PEOU; Gelest), glycerol (Sigma), poly(ethylene glycol (PEG: Sigma), PEG400 (Sigma), or PEG8000 (Sigma)] were mixed in various combinations with 10 mM HCl. Different concentrations (0-500 mM) of buffers with pHs ranging from 4 to 9 containing various salts (potassium sulfate and ammonium phosphate, sodium phosphate) were mixed with each formulation to yield more than 100,000 formulations. These formulations were arrayed individually onto poly(methyl methaacrylate) (PMMA; SPL) plates using an microarrayer according to the manufacturer’s protocol (GeneMachine, Genomic Solutions). Screening Assays. The following assays were made for the spots from 100,000 formulations. (20) Rupcich, N.; Goldstein, A.; Brennan, J. D. Chem. Mater. 2003, 15, 18031811. (21) Boury, B.; Corriu, R. J. Adv. Mater. 2000, 12, 989-992. (22) Fujii, T.; Yano, T.; Nakamura, K.; Miyawaki, O. J. Membr. Sci. 2001, 187, 171. (23) Gogotsi, Y.; Nikitin, A.; Ye, H.; Zhou, W.; Fischer, J. E.; Yi, B.; Foley, H. C.; Barsoum, M. W. Nat. Mater. 2003, 2, 591-594. (24) Suh, C. W.; Kim, M. Y.; Choo, J. B.; Kim, J. K.; Kim, H. K.; Lee, E. K. J. Biotechnol. 2004, 112, 267-277. (25) Copeland, S.; Siddiqui, J.; Remick, D. J. Immunol. Methods 2004, 284, 99106. (26) Price, D. H. Mol. Cell. Biol. 2000, 20, 2629-2634. (27) Gill, I.; Ballesteros, A. J. Am. Chem. Soc. 1998, 120, 8587.
Table 1. Immobilization Efficiency for the Selected Materialsa immobilization efficiencyb (%) materials/assays moleculesc
for small (70% adhesion of the appropriate biomolecules after washing were selected (fluorescence intensity was measured and quantified using GenePix Pro 4.0 software, LAU/mm2). Activity Assays. The activity assay of the seven formulations suitable for protein immobilization involved adding BSA (500 ng/ µL) to formulations 0-VI and arraying the mixtures on PMMA slides. The slides were incubated with the Cy-3-labeled anti-BSA antibody for 30 min, washed heavily with 1× PBS containing 0.1% Tween (Sigma), and then dried. The resulting spots were scanned and analyzed using GenePix Pro 4.0 software (Axon Instruments) Analytical Chemistry, Vol. 78, No. 21, November 1, 2006
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Table 2. Comparison of the Sensitivity and Pore Sizes pores analysis of a spotb
0d I II III IV V VI VII
7 selected formulations plus 1 control formulation (0)
sensitivity activity assaya
pore sizes (nm)
density
adsorption assayc
25.5% TMOS, 12.5% MTMS 17.5% TMOS, 12.5% MTMS, 4% PGS 20.5% TMOS, 10.5% MTMS, 5% PEOU 25.5% TMOS, 12.5% MTMS, 3% PEG400 25.5% TMOS, 12.5% MTMS, 5% PEG8000 25.5% TMOS, 12.5% MTMS, 2.5% glycerol 25.0% TMOS, 7.5% MTMS, 5% GPTMOS 10% MTMS
1.1475 3.45084 6.35116 18.8969 37.43029 16.58053 3.45084 5.3421
12.4 21.9 21.3 27.8 21.5 21.9 17.6 20.4
0.07 0.188 0.102 0.349 0.259 0.139 0.053 0.119
1993.33 578.29 304.06 265.66 14.69 77.24 517.62 99.64
a The activity or sensitivity of the 8 materials were measured at least twice, as described in the Experimental Section (Activity Assays). b The surface image of the spots taken by SEM were analyzed and the sizes of the pores were measured at least twice (Pore Size Analysis of Microspot in the Experimental Section). c Adsorption tests were performed to test the nonspecific adsorption of additional antibodies through the channels, as previously described.24 Briefly, microspots without any immobilized proteins were incubated with the labeled antibodies for 20 min, washed, scanned, and quantified by subtracting the background signals (LAU-BGs/mm2). The signals (measured by scanner fluorescence unit, LAU) were averaged across more than 20 spots for each formulation (see Experimental section). D formulation O was used as a control without any additives (same formulation as IV except for PEG 8000).
in order to quantify the signals versus the backgrounds (see “sensitivity” in Table 2). Adsorption Assays. For the adsorption assays of the nonspecific interaction test, the slides of the spots without any protein immobilized were incubated with Cy-3-labeled anti-BSA antibodies, washed with 1× PBS containing 0.1% Tween, and then scanned. Those spots with fluorescence less than the background after being washed were selected. Pore Size Analysis of Microspots. The pore size and density of the spots were measured from surface scanning electron microscope (SEM) images. The surfaces of the selected spots were observed using SEM (S-4800 SEM, Hitachi).28 A conducting coating for SEM observations was applied after complete gelation using an ion beam sputter coater with an Ir target (∼20 nm thick).28 Image analysis software (Image-Pro Plus, MediaCybernetics) was used to count the number of pores in nine different areas (576 × 430 nm), and the average pore density was calculated as [total pore area]/[total area (576 × 430 nm)]. Sensitivity Measurements. The sensitivity and specificity of the antigen-antibody interactions in the sol-gel matrix was measured by immobilizing p24 protein in the formulation IV material and performing immunoassays on serial dilutions of serum containing HIV antibody against the p24 protein. The BSA proteins were also immobilized on the same chip to check for any nonspecific absorption of HIV antibodies in the sol-gel matrix. For the sensitivity tests of the native protein-protein interactions in formulation IV, the cyclinT proteins were immobilized on the slide, and the spots were incubated with the labeled Cdk9 proteins and scanned as described above. RESULTS AND DISCUSSION Screening and Selection for Optimal Sol-Gel Chip Materials. Scheme 1 shows the unique screening strategy first used to develop and optimize the sol-gel materials for protein chips. Approximately 100,000 formulations of TMOS-based sol-gel mixtures were generated, which were spotted individually on PMMA (28) Kusakabe, K.; Sakamoto, S.; Saie, T.; Morooka, S. Sep. Purif. Technology 1999, 16, 139.
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Scheme 1. Novel Screening and Selection Strategy for Protein Chip Materialsa
a A total of 100 000 materials were screened using 5 screening assays described in the Experimental Section, resulting in 700 materials. The immobilization assay resulted in the selection of 17 materials, from which 7 materials were considered suitable for protein immobilization and were further selected on the basis of their activity, adsorption, and pore size.
slides using a microarrayer and screened for various critical physical properties. Five screening measurements were made for the spots from 100,000 formulations (see Screening Assays in Experimental Section). First, the adhesion was tested using a microarray washer, and those spots with a similar shape before and after washing were selected. Second, the morphology of the spots was observed by CLSM. The scan profile through the center of each spot showed the measured thickness and diameter, and spots that were flat, broken, or too large (>800 µm) were excluded. Third, the gelation time was measured both in a tube and on the spot by scratching the spots. Those spots with a gelation time of >4 h in the tubes and 20 min in the isolated spots were selected. Fourth, the optical transparency and low-level autofluorescence were detected with a laser scanner. Spots with fluorescence signals lower than the PMMA plate background were selected. These screening assays yielded 700 formulations with the desired characteristics. This pool of prescreened materials was then subjected to two successive selection protocols (Scheme 1) with the aim of identifying the best sol-gel formulations for protein chips. Based on the immobilization efficiency with the
Figure 1. Sensitivity of the sol-gel protein chips. (a) Sensitivity assays for the antigen (p24)-antibody interactions. The experiments were performed at least three times. Image analysis was carried out as described in the Experimental Section (Sensitivity). (b) Sensitivity assays for the protein (cyclinT)-protein (Cdk9) interactions.
labeled small molecules, proteins, and antibodies (see the Immobilization Efficiency Measurements in the Experimental Section), the selected formulations were subcategorized as being suitable for use with small molecules, moderate-sized proteins, and larger antibodies (Table 1).22 The sizes of the pores were similar on each spot made from the seven chip materials considered suitable for protein immobilization (Table 2). However, formulation IV exhibited higher protein-antibody binding activity and a lower signal-to-background ratio in the nonspecific adsorption test. Hence, formulation IV scored the highest (Table 2). Femtogram-Level Sensitive Detection Using the Selected Sol-Gel Protein Chip Material. Based on screening and selection, best formulation IV was used to generate a protein chip to test the sensitivity, which is the most important factor for a protein chip technology,1-8,29 The spots were immobilized with either HIV p24 antigens (200 ng/spot) or BSA, and the detection limit was tested using known amounts of the anti-p24 antigen antibody (see Sensitivity Assays in Experimental Section). The concentration of the antibody in 1 mL of serum was detected down to the femtogram level (Figure 1a). The BSA spots showed very low signals, even when the blocking step had been skipped. The nonspecific interactions with the material (no protein) and additional unrelated proteins (4-1BB) were quite low, but incubation with antibodies against the corresponding antigen (BSA or 4-1BB) produced specific interactions (data not shown). To confirm this finding in a different system, the proteinprotein interactions were measured using cyclinT and Cdk9 heterodimers 26 (Figure 1b). The results showed that the cyclinT immobilized in the sol-gel protein chip could detect the Cdk9 proteins down to the attogram level in 1 mL of PBS. This indicates that our novel material formulation held the embedded protein (cyclinT) in its native conformation, which is essential for proteinprotein interactions (Cdk9) 7-9,26. Also, this formulation has low (29) MacBeath, G. Nat. Biotechnol. 2001, 19, 828-829.
Figure 2. Physical properties of selected spots. (a) Spot image taken with CLSM in standard reflection geometry mode (Spot Morphology in Experimental Section). The scan profile through the center of each spot revealed the measured thickness (∼63 µm) and diameter (∼380 µm). (b) The surface of the spot was observed using SEM (see Pore Size Analysis in Experimental Section). The bar graph shows the pore size distribution. The upper image corresponds to formulation O, which is the same formulation as formulation IV (lower image) except for the inclusion of PEG8000. Formulation IV showed the highest sensitivity.
background and noise levels (more than 10-fold) for the fluorescent signal, resulting in an even higher sensitivity for proteinprotein interactions. In addition, this material can hold up to 5 µg Analytical Chemistry, Vol. 78, No. 21, November 1, 2006
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of BSA proteins per spot, and still showed specific interactions with the Cy3-labeled anti-BSA antibody. Improved Physical Properties of the Selected Sol-Gel Protein Chip Material. The physical properties of this formulation were examined by imaging the protein chip spots by microscopy in standard reflection geometry mode (see Spot Morphology in Experimental Section). The spot showed good morphology with an average diameter and height of ∼380 and ∼63 µm, respectively (Figure 2a). The surface of the spot was observed using SEM (see Pore Size Analysis in Experimental Section). The observed sizes ranged from 15 to 60 nm, with a mean pore size of 21.5 nm (Figure 2b, lower image). The bar graph shows the pore size distribution. The upper image in Figure 2b corresponds to formulation O, which is the same formulation as in formulation IV except for the inclusion of PEG8000. This shows that adding PEG8000 increases the pore size. A comparison study was performed between the novel formulation (IV) of sol-gel-based microspots and that of a previous study.20 Each formulation was loaded with proteins (BSA) that were arrayed on slides, incubated with BSA antibodies, washed, and scanned. The spots from this study showed intense specific signals with a good morphology (Figures 1 and 2). On the other hand, the spots from the formulation reported by Rupcich et al.20 were broken, and the signal was could not be quantified (data not shown). In summary, a novel and intensive material screening and selection protocol was used to isolate 700 sol-based materials with the desired physical properties. Of these, 17 materials were selected for potential applications in drug screening, protein interaction assays, and immunoassays. From these materials, only seven materials were considered suitable for protein immobilization (Scheme 1). One of selected formulation (formulation IV) showed a large spot signal, no loss of activity for the reserved (30) Richalet-Secordel, P. M.; Poisson, F.; Van Regenmortel, M. H. Clin. Diagn. Virol. 1996, 5, 111-119.
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proteins, and low background noise after incubation with the antibody (Figure 1). This novel sol-gel-derived protein chip material (which showed the highest sensitivity) also showed an improved morphology and adhesion (Figure 2a) and large pore size and density (Figure 2b) for analysis. This novel chip was 1001000 times more sensitive than the protein chips previously reported 30 [compared in parallel with the protein chips and reagents from PerkinElmer (Hydrogel) and MSI (MSI modified glasses); data not shown]. CONCLUSIONS Our unique screening and selection system made it possible to develop high-sensitivity protein chip materials. This study is the first demonstration of protein-protein interactions detected on sol-gel-derived protein chip arrays and provides important information for the future use of sol-gel-based protein chips, which might replace the current proteomics technology. ACKNOWLEDGMENT S.K. acknowledges Drs. Jong-Kee Yeo and Jinyeong Yoo for their constant support during this project, and particularly President Yeo (CTO of LG Chem. Ltd.) for initiating the work. We thank Drs. Dong-ki Lee, Bonghyun Jung, Hyuck Yoo, Kyohan Ahn, and Byunghyeon Kim for their critical discussions regarding the manuscript, and Ms. J. Jang, Ms. H. Park, Mr. K. Chae, and Mr. J. Kim for their initial contributions to this project. This work was supported by a KRF grant (2003-042-E00117), and by the Protein Chip Technology Program (MOST). MOHW (Grant A050814) and KIEST (Grant 101-051-022) also supported this work. This work was supported by National Research Laboratory Program (M10600000251-06J0000-25110, MOST). S.K. dedicates this paper to the late Mr. Hee soo Kim, her beloved father. Received for review November 19, 2005. Accepted May 31, 2006. AC0520487
