Polypeptide Semicarbazide Glass Slide Microarrays: Characterization

Bioconjugate Chem. 2004, 15, 317−325

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Polypeptide Semicarbazide Glass Slide Microarrays: Characterization and Comparison with Amine Slides in Serodetection Studies Xavier Duburcq,†,‡ Christophe Olivier,‡ Re´mi Desmet,§ Matej Halasa,§ Olivier Carion,§ Bruno Grandidier,# Thomas Heim,# Didier Stie´venard,# Claude Auriault,‡ and Oleg Melnyk*,§ UMR CNRS 8527, Biological Institute of Lille, 1 Rue du Pr Calmette 59021 Lille, France, Sedac-Therapeutics, Le Gale´nis, Baˆt. B, 85 Rue Nelson Mandela 59120 Loos, France, UMR CNRS 8525, Biological Institute of Lille, 1 Rue du Pr Calmette 59021 Lille, France, and IEMN, Cite´ Scientifique, Avenue Poincare´, 59652 Villeneuve d’Ascq Cedex, France. Received July 10, 2003; Revised Manuscript Received December 12, 2003

We have described in the accompanying article the preparation of peptide-protein semicarbazide microarrays and their use for the simultaneous serodetection of antibodies directed against different pathogens. Here, we present a comparative study between semicarbazide and amine glass slides in an immunofluorescent serodetection assay using HIV (Gp120, Gp41), HCV (mix-HCV, core, NS3, and NS4), and HBV (HBs) recombinant antigens. Amine and semicarbazide surfaces displayed the same sensitivity for antibodies detection just after printing. However, the reactivity of protein antigens changed rapidly upon aging on amine slides but not on semicarbazide slides. Peptide or protein semicarbazide microarrays were found to be remarkably stable for months. Additional data concerning the characterization of the semicarbazide surface (homogeneity of the slides, chemical stability, contact angle measurements, atomic force microscopy studies, reproducibility of serodetection results) are also presented and discussed.

INTRODUCTION

The parallel detection of antibodies in complex biological samples have a wide range of potential applications in the diagnosis of allergies, autoimmune, and infectious diseases as well as in epitope mapping studies and vaccines development. The need for high-throughput screening systems have stimulated the development of microfabricated analytical devices. In the preceding paper, we have described the preparation of novel peptide-protein microarrays based on the utilization of glass slides functionalized by a semicarbazide layer. This surface can be used for the site-specific immobilization of glyoxylyl-peptidic antigens through R-oxo semicarbazone ligation or for the immobilization of recombinant proteins through physisorption. These peptide-protein arrays could be used for the capture of antibodies in the sera of infected individuals using an immunofluorescent assay. Owing to the miniaturization, very small quantities of antigens, of sera (1 µL), and of tetramethylrhodamine labeled secondary antibodies (1 µg) could be used. The microarrays displayed high levels of sensitivity and specificity for the detection of HIV, HCV, HBV, EBV, and syphilis antibodies as revealed by the screening of a collection of sera referenced against these pathogens. We disclose in this article a comparative study between semicarbazide and amine microarray glass slides, the later surface being often used for the preparation of * To whom correspondence should be addressed. Phone: 33(0)3 20 87 12 14. Fax: 33(0)3 20 87 12 35. E-mail: oleg.melnyk@ ibl.fr. † UMR CNRS 8527, Biological Institute of Lille. ‡ Sedac-Therapeutics. § UMR CNRS 8525, Biological Institute of Lille. # IEMN.

protein microarrays (1, 2). In particular, we have examined the performance of the two surfaces in the serodetection of HBV, HIV, and HCV infections and the evolution of the serodetection results during an accelerated aging of the arrays. The reproducibility of antibody detection with semicarbazide microarrays was documented. We have also carried out contact angle measurements with reference liquids to determine the sensitivity of both surfaces to air pollution. Finally, the semicarbazide glass slides were analyzed by atomic force microscopy to give a picture of the surface topography and to measure the height of the silane layer. MATERIALS AND METHODS

Peptide Synthesis. HCV core p21 15-45, HCV NS4 1925-1947, and EBV VCA p18 153-176 glyoxylyl peptides were synthesized in an automated peptide synthesizer (Pioneer, Perseptive Biosystems Inc., MA) using the Fmoc/tert-butyl strategy (3) and an isopropylidene tartrate based linker (4-6) as described elsewhere (7). Proteins. HCV core (1.5 mg/mL), HCV NS3 (0.5 mg/ mL), HCV NS4 (0.4 mg/mL), HCV mixture of core, NS3, NS4, and NS5 antigens (1/1/1/1 by weight, 2 mg/mL overall), HIV-I Gp120 (0.5 mg/mL), and HIV-I Gp41 (2 mg/mL) were purchased from Beijing Hepatitis Research Institute or from Henan Lily Bio-Products Company (Beijing, China). All these proteins were supplied in a pH 9.6 0.05 M carbonate/bicarbonate buffer. HBs antigen (1 mg/mL) was purchased from Advanced ImmunoChemical (Long Beach, CA) and was supplied in a 0.01 M pH 7.2 PBS solution. All these proteins were diluted before printing in the buffer used for their storage. Protein A (51 mg/mL) was purchased from SigmaAldrich (Saint Quentin Fallavier, France) and was supplied in water. Protein A was diluted with a 0.1 M pH 5.5 sodium acetate buffer before printing.

10.1021/bc034118r CCC: $27.50 © 2004 American Chemical Society Published on Web 02/28/2004

318 Bioconjugate Chem., Vol. 15, No. 2, 2004

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Table 1. Contact Angle Measurements on Semicarbazide or Amine Glass Slides γ,a mJ/m2

contact angle, deg (standard error) semicarbazide amine

w

dim

fad

tot

LW

acid

base

∆Gsws,b mJ/m2

∆Gsw,c mJ/m2

36.3(0.5) 33.1(0.5)

30.9(0.2) 31.5(0.6)

15.6(0.9) 21.5(0.7)

56.4 54.0

43.9 43.8

1.2 0.7

33.9 39.8

4.6 13.7

-131.5 -133.7

a The surface tension parameters of diiodomethane (dim), water (w), and formamide (fad) were taken from the literature (20). b Interfacial interaction free energy of the surface in water. c Free energy of binding of water to the surface.

Preparation of the Glass Slides. Amine Glass Slides. Microscope slides (ESCO precleaned microslide, frosted) were cleaned using a freshly prepared piranha solution (H2SO4/H2O2, 1/1 by volume, overnight). The slides were washed with water (3 × 3 min) and methanol (3 min) and treated with 3% 3-aminopropyltrimethoxysilane in methanol/water, 95/5 by volume (30 min, under sonication). The slides were then washed with methanol, water (two times), and methanol and annealed for 15 min at 110 °C. Semicarbazide glass slides were prepared and characterized as described elsewhere (8). Rapidly, the amine slides prepared as described above were treated with triphosgen/diisopropylethylamine in 1,2-dichloroethane and then Fmoc-NHNH2 (9) in DMF containing 1% EtOH (by volume) to install the semicarbazide functions. Finally, removal of the Fmoc groups was performed with piperidine/1,8-diaza-bicyclo[5.4.0]undec-7-ene (DBU) in N,N-dimethylformamide (DMF, 30 min). Four slides were used for the quality control. Three slides were immersed for 1 h at 37 °C in a 10-4 M solution of Rho-Lys-Arg-NH(CH2)3NH-CO-CHO (synthesized according to refs 7 and 8, where Rho is (5)-6-carboxytetramethylrhodamine). One slide was treated with the control peptide Rho-Lys-ArgNH2. The fluorescence intensity of the slides was quantified using an Affymetrix 418 array scanner (MWG, Santa Clara, CA). Amine and semicarbazide slides were stored at room temperature in air in a closed box. Printing of the Arrays. The slides were printed using a 4-pin Affymetrix 417 arrayer (MWG, Santa Clara, CA) or a 32-pin manual arrayer (Microarray printer XMM 47832, Xenopore, Hawthorne). The printed slides were incubated overnight at 37 °C in a humid chamber (60% relative humidity) and stored until use at room temperature. The number of replicates is indicated in the figures or tables. Human Sera. Sera were collected from the clinical laboratory of the Centre Hospitalier Re´gional (Lille, France). HCV. The reactivity of HCV referenced sera was determined with the AxSYM HCV version 3.0 Abbott ELISA kit (30 HCV-negative and 100 HCV-positive individuals). For some sera an additional RIBA HCV 3.0 SIA test (Ortho-Clinical Diagnostics) or a DECISCAN HCV PLUS test (BIORAD) was performed. HIV. The reactivity of HIV referenced sera (90 HIVpositive and 20 HIV-negative individuals) was determined with DADE BEHRING Enzygnost HIV Integral and with BIORAD GENSCREENPLUS HIV tests. The positivity was confirmed with BIORAD New Lav Blot I and II. HBs. The reactivity of HBs referenced sera (40 HBsnegative and 60 HBs-positive individuals) was determined with the BIORAD MONOLISA anti-HBs PLUS test. Detection of Antibodies. The phosphate-buffered saline (PBS) used for the preparation of the saturation, incubation, and washing solutions was 0.01 M pH 7.2. PBS-A contained 0.1% of Tween 20 (by volume) and 2.5%

of half-fat milk (by weight). PBS-B contained 0.05% of Tween 20 (by volume). The saturation of the printed slides was performed under sonication in PBS-A during 60 min. After four washings with PBS-C, 1 µL of human serum diluted in 49 µL of PBS-A was incubated during 45 min at 37 °C under cover-glass. After four washings with PBS-B, 50 µL of tetramethylrhodamine-conjugated goat antibodies to human IgG-A-M (Jackson ImmunoResearch Laboratories, Baltimore, MD) diluted 1/100 in PBS-A were incubated for 45 min at 37 °C under cover-glass. The slides were then washed three times with PBS-B, with distilled water, and with absolute ethanol (ACROS Organics, Geel, Belgium) and dried in air. Data Collection and Analysis. The slides were scanned with an Affymetrix 418 array scanner (MWG, Santa Clara, CA). The scanner sensitivity is indicated in the figures or tables. The data were analyzed using the Affymetrix Jaguar software (MWG, Santa Clara, CA). Statistical Analyses. Statistical analyses were performed using the nonparametric Mann-Whitney U-test (two samples) or the Kruskal-Wallis test for more than two samples (10). Contact Angle Measurements. Contact angle measurements were performed at 20 °C with a Digidrop goniometer (GBX, Romans sur Ise`re, France) and water, formamide, and diiodomethane as reference liquids. For each liquid, at least nine drops of 1 µL were used for the determination of the angle (median, interquartile range). The Lifshitz-van der Waals and Lewis acid and base surface tension parameters (van Oss theory (22); see Table 1) were determined using the Young’s equation. The interfacial interaction free energy between the surface in water (∆Gsws) and the free energy of binding of water to the surface (∆Gsw) were calculated as described in ref 22. AFM Studies. The thickness of the amine or semicarbazide silane layer was obtained by local reactive ion etching of the surface followed by atomic force microscopy (AFM) analysis of the step between the silicon oxide and semicarbazide regions. Reactive Ion Etching (RIE). A 200 nm layer of polystyrene was first deposited on the glass surfaces by spincoating (v ) 3000 rpm, acc ) 2500 rpm/s during 10 s). The samples were dried by a rapid thermal annealing (120 °C, 1 min) and covered with a 1.6 µm photolithographic resin layer (S18S18, Chimie Tech Service, Anthony, France). The irradiation of the resin by a UV light (365 nm, 5 mW/cm2, 12 s) was performed through an optical mask with square-shaped patterns with lateral dimensions of 9 µm. The irradiated regions were removed by a MF319 solution (Chimie Tech Service, Anthony, France). The polystyrene layer in the square-shaped pattern was decomposed with an oxygen plasma (Plasmalab 80 plus, Oxford instrument; 0.1 Torr; 20 sccm; 100 W) during 7 min. The engraving speeds for S18S18 and for polystyrene thickness were 200 and 170 nm/min, respectively (the silicon oxide substrate was not affected by the oxygen plasma). Finally, the samples were soaked with

Polypeptide Semicarbazide Glass Slide Microarrays

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Figure 1. Fabrication of amine and semicarbazide functionalized glass slides used in this study.

sonication in a series of solvents to remove the unreacted parts (toluene, isopropyl alcohol, and water, 10 min each). AFM Measurements. AFM (atomic force microscopy) was carried out with a Nanoscope IIIa controller (Digital Instrument) using intermittent contact mode at room temperature under ambient conditions. Processing of the AFM images was achieved by removing only local planes to the images. The height of the silane layer was measured from averaged profiles (so-called profile line) obtained along lines that run through the square-shape patterns. To characterize the substrate homogeneity, the root-mean-squared surface roughness (Rrms) was measured in the different layers. RESULTS AND DISCUSSION

Preparation of the Glass Slides. The functionalization of glass slides by a semicarbazide sol-gel layer was discussed in detail elsewhere and is rapidly described in Figure 1 (8). Commercially available glass slides were silanized with 3-aminopropyltrimethoxysilane. The amine groups were treated successively with triphosgene and Fmoc protected hydrazine to install the semicarbazide functions. The correct functionalization of the slides is controlled using a glyoxylyl probe labeled with tetramethylrhodamine, which is able to react chemoselectively with the surface through the formation of an R-oxo semicarbazone linkage. This aspect will be discussed later, since this quality control was used to document the chemical stability of the semicarbazide glass slides upon aging. Amine glass slides used for the comparative study were the same as those utilized for the synthesis of semicarbazide glass slides (Figure 1). Fabrication of Protein Microarrays on Semicarbazide or Amine Glass Slides. Comparison in a Serodetection Study. The preparation of protein microarrays by adsorption of proteins on surfaces is an appealing strategy because no chemical modification of the proteins is required. As shown in the accompanying paper, recombinant proteins were found to adsorb efficiently on semicarbazide glass slides. The corresponding microarrays permitted the highly sensitive and specific detection of antibodies in human sera and were used for the simultaneous serodetection of multipathogen infections. Alternatively, proteins are known to adsorb on amine-modified surfaces. For this reason, amine glass slides were often utilized for the preparation of protein microarrays (11, 12). For example, Mezzasoma et al. have recently described the fabrication of antigen microarrays for the serodiagnosis of infectious diseases using aminosilane-derivatized slides (13). Robinson et al. have printed antigens or antibodies on poly-L-lysine for the detection of their cognate ligands (14). Both semicarbazide and amine glass slides present NH2 groups on the surface. However, the pKa values of semicarbazide and alkylamines are completely different (15). Indeed, the pKa of semicarbazide is 3.65 at 25 °C, whereas the pKa of methylamine is 10.65 at 25 °C. Thus, the semicarbazide surface is expected to be uncharged at physiological pH, whereas in the same conditions amine slides are protonated and thus cationic surfaces. Given the different physicochemical properties of these

Figure 2. Adsorption of protein A (A) or HBs antigen (B) on amine or semicarbazide slides. For protein A, the arrays were incubated 1 h with a human serum (diluted 1/50). For HBs, the arrays were incubated 2 h with a HBs positive referenced serum (diluted 1/50). Detection of captured antibodies was performed with tetramethylrhodamine labeled goat antihuman IgG-A-M antibodies. n ) 6, L35, PMT50. The interquartile range is indicated at the top of the bar (median).

surfaces, we have decided to print recombinant proteins on both surfaces in a microarray format and to evaluate their ability to detect antibodies in sera. In addition, we have studied the stability of the printed arrays upon accelerated aging. Despite the importance of this aspect for the development of protein microarrays, no long-term stability studies have been reported to our knowledge. In a preliminary study, we have printed protein A and the surface antigen of hepatitis B virus (HBs) at 0.04 and 0.1 mg/mL, respectively (Figure 2). Each printed slide was incubated first with a human serum (ELISA referenced HBV positive serum for HBs microarrays) and then with goat antihuman secondary antibodies labeled with tetramethylrhodamine. The arrays were analyzed using the Cy3 channel of a fluorescence microarray scanner. As shown in Figure 2, both proteins behaved similarly on the two surfaces, both in term of signal intensity and background level. These results show that adsorption of proteins occurs similarly on amine and semicarbazide surfaces, even if the mechanism of immobilization is probably different. These preliminary data in hand, we have selected other antigens derived from HCV to confirm the previous observations. We used recombinant core, NS3, and NS4 antigens and a mixture of core, NS3, NS4, and NS5 antigens named mix-HCV antigen. The HCV antigen microarrays were incubated with a collection of ELISA referenced HCV sera (16+, 4-) just after the printing step. The determination of the cutoff values of positivity is described in detail elsewhere (16). As shown in Figure 3, both types of microarrays displayed 100% of sensitivity and specificity with this collection of sera. Usually, the signal displayed by amine slides was equal or superior to the signal obtained with semicarbazide supports. However, for mix-HCV and core antigens, some sera led to higher signals on semicarbazide slides. Stability Study of Amine and Semicarbazide Microarrays, Protein Antigens. These data set the stage for the stability study, which was performed using both surfaces. We used the previous HCV antigen microarrays and HIV antigen microarrays composed of two HIV recombinant proteins Gp 120 (0.5-0.05 mg/mL) and


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Figure 3. Detection of antibodies using amine or semicarbazide slides and HCV core, NS3, NS4, and mix-HCV antigens. HCV referenced sera (for ELISA, AxSYM HCV version 3.0, Abbott; for Western blot, RIBA, CHIRON RIBA HCV 3.0 SIA, Ortho-Clinical Diagnostics). The cutoff values of positivity (mean ( 3 SD) were determined using a collection of 30 HCV ELISA negative sera. The normality of the distribution was verified using the Shapiro-Wilk test. (A) mix-HCV, cutoff: 161 ( 36. (B) NS3 recombinant protein, cutoff: 28 ( 8. (C) NS4 recombinant protein, cutoff: 31 ( 9. (D) Core recombinant protein, cutoff: 23 ( 7. n ) 6, L35, PMT50. The interquartile range is indicated at the top of the bar (median).

Figure 4. Stability of HCV core, NS3, NS4, and mix-HCV antigens on amine or semicarbazide surfaces. HCV referenced positive serum (for ELISA, AxSYM HCV version 3.0, Abbott; for Western blot, RIBA, CHIRON RIBA HCV 3.0 SIA, Ortho-Clinical Diagnostics). The microarrays were stored at 38 °C in a humid chamber. n ) 9, L35, PMT50. The interquartile range is indicated at the top of the bar (median).

Gp41 (2-0.1 mg/mL). For each study, one ELISA referenced positive serum was selected. A series of microarrays were used for antibody detection just after printing. The other slides were placed in a humid chamber (60% relative humidity) at 38 °C to accelerate the aging. The data corresponding to the HCV stability study are presented in Figure 4. The specificity of antibody detection was verified using HCV ELISA negative sera (data not shown). After 1 month, the fluorescence signal was found to drop significantly for all the tested antigens on amine slides: -37% for mix-HCV, -50% for NS3, -16% for NS4, and -44% for core. Alternatively, the largest variation observed with semicarbazide slides was +7.5%

for NS4. Interestingly, the signals displayed by NS3 or core antigens were found to be stable on the semicarbazide surface (-0.8% and +2.7%, respectively, nonsignificant at p < 0.05), whereas the loss of reactivity on amine slides upon aging was as high as 45-50%. This high difference in stability was confirmed with other sera (data not shown). The data corresponding to the HIV study are collected in Figures 5 and 6. For amine microarrays (Figure 5), the signals displayed by Gp120 and Gp41 antigens changed significantly (p < 0.05) upon aging for all the tested concentrations. The figure for Gp41 was complex and depended on both concentration and time of aging



Figure 7. Stability of peptide arrays on semicarbazide slides. HCV P21 and NS4 peptides, EBV VCA p18 peptide, HCV and EBV positive referenced sera. Peptides were printed at 10-4 M. n ) 6, L35, PMT50. The interquartile range is indicated at the top of the bar (median).

Figure 5. Stability of HIV Gp41 (A) and Gp120 (B) antigens on amine surfaces. HIV positive referenced serum, n ) 9, L35, PMT 50. The confidence interval (p ) 0.05, t ) 2.26) is indicated at the top of the bar (mean).

Figure 6. Stability of HIV Gp41 (A) and Gp120 (B) antigens on semicarbazide surfaces. HIV positive referenced serum, n ) 9, L35, PMT 50. The confidence interval (p ) 0.05, t ) 2.26) is indicated at the top of the bar (mean).

(Figure 5A). For the highest concentrations (0.5-2 mg/ mL), the signal increased after 1 month and then decreased after 3 months, whereas for the lowest concentration (0.1 mg/mL) the signal always decreased. For Gp120 (Figure 5B), the signal was found to drop for the three highest concentrations. After 3 months, the signal represented only 20% of the initial value at 0.5 mg/mL and 10% at 0.25 mg/mL. In the meantime, Gp41 and Gp120 antigens were found to be stable on the semicarbazide surface (Figure 6). Thus, the stability study was continued up to 9 months. In the accompanying paper, we show that the highest concentrations used here for Gp41 and Gp120 (2 and 0.5 mg/mL, respectively) permitted the sensitive and

specific detection of antibodies against HIV. Interestingly, for these concentrations, the largest variation of the fluorescence intensity relative to the reference data obtained just after printing did not exceed 7%, illustrating the very good stability of both protein antigens on semicarbazide glass slides. Stability Study of Semicarbazide Microarrays, Peptide Antigens. In the accompanying paper, we have described the utility of semicarbazide glass slides for the fabrication of peptide-protein microarrays. Thus, it is also important to document the stability of the peptide probes during aging. The covalent attachment of small peptides to the glass surface through the formation of an R-oxo semicarbazone bond is important to avoid the loss of probe during the washing and incubation steps. The stability of the R-oxo semicarbazone linkage toward hydrolysis is expected to have a profound impact upon the results of the aging study. HCV core p21 15-45 (17), HCV NS4 1925-1947 (18), and EBV VCA p18 153-176 glyoxylyl peptides (19) were printed on semicarbazide slides. Figure 7 presents the evolution of the fluorescence intensity for these three peptidic antigens upon a 12-month stability study. For the three peptides, the differences were not found to be significant (p < 0.05), thus demonstrating the good stability of peptide R-oxo semicarbazone arrays. Reproducibility. Another important aspect of microarray technologies is the ability to reach a high level of reproducibility inside a slide and between slides of the same batch or of different batches. We have examined the reproducibility of semicarbazide microarray fabrication and antibodies detection using again Gp120 and 41 HIV antigens. The variation coefficients for Gp120 (5.0%) and Gp41 (5.5%) inside a slide were determined using 64 spots. These values are close to the variation coefficient obtained for the HCVcore p21 15-45 peptide (n ) 600). Next, two batches were tested (five slides per batch). The data presented in Figure 8 show that for a given antigen, the differences between the slides of a batch or between the two batches were not significant at p < 0.05, thus showing the good reproducibility of the assay. These results can be attributed to a quality control of the semicarbazide glass slides and to the simplicity of the microarray preparation and use. Chemical Stability of Semicarbazide Glass Slides. Recently, we have described a method allowing the control of the semicarbazide layer reactivity. This quality control utilized two peptides labeled with (5)-6-carboxytetramethylrhodamine. One peptide Rho-Lys-Arg-NH(CH2)3NH-CO-CHO (Rho-COCHO) was modified by a glyoxylyl group and thus was able to react chemoselec-


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Figure 8. Reproducibility study. The semicarbazide slides were printed with Gp41 (2 mg/mL) and Gp120 (0.5 mg/mL) and used for the detection of anti-HIV antibodies in a diluted human serum (1/50). n ) 6, L35, PMT50. The interquartile range is indicated at the top of the bar (median).

tively with the accessible semicarbazide groups on the surface, whereas the other peptide Rho-Lys-Arg-NH2 (Rho-CONH2) was a control lacking the glyoxylyl group for anchorage. The quality control was performed by immersing one slide 1 h at 37 °C in a 0.1 mM solution of Rho-COCHO and another slide in a solution of the control Rho-CONH2. Both slides were washed to remove the excess of peptide and analyzed using a fluorescence scanner. A large difference in fluorescence intensity between the two slides is indicative of a high level of surface functionalization. The variation coefficient of the fluorescence gives an indication of the homogeneity of the slide. This method of characterization was used to document the chemical stability of the semicarbazide slides upon aging. The slides of the same batch were analyzed just after preparation or following 3 months of storage at room temperature in a closed chamber. The data presented in Figure 9 show that the fluorescence intensity of the slides did not change over a 3-month period for both labeled peptides (p < 0.05). The variation coefficient of the fluorescence intensity was also unaffected in this experiment (4-8% for Rho-COCHO and 4-7% for Rho-CONH2). Thus, the chemical properties of the semicarbazide slides are conserved at least 3 months even if some air pollution could be detected after 30 days of storage by contact angle measurements (see later). Characterization of the Semicarbazide Surface by Contact Angle Measurements. In the previous

Figure 9. Chemical stability of semicarbazides slides. The slides were analyzed after synthesis or after 3 months of storage in air using glyoxylyl Rho-COCHO or amide Rho-CONH2 peptides labeled with (5)-6-carboxytetramethylrhodamine as described elsewhere. The slides were scanned at L25, PMT40 using an Affymetrix 418 array scanner (MWG, Santa Clara, CA), and the data were analyzed with the Scanalyze software (Stanford University). Thirty measurements were performed per slide. The grid covered about 70% of the glass surface. The bars represent the mean and the confidence interval (p ) 0.05).

section, we have described a chemical method allowing a quality control of the semicarbazide surface reactivity. An interesting aspect of this method is the possibility characterizing the homogeneity of the surface viewed through the reactivity of solvent-accessible semicarbazide groups. A complementary method allowing the rapid, simple, and cheap characterization of surfaces is



Figure 10. Stability studies on semicarbazide (A) or amine (B) glass slides using contact angle measurements with formamide, water, or diiodomethane, 20 °C. The interquartile range is indicated at the top of the bar (median).

to perform contact angle measurements with liquids whose surface tension is well-known. R-Bromonaphthalene, dimethyl sulfoxide, and ethylene glycol were found to spread on semicarbazide slides. Diiodomethane, water, and formamide were found to be useful referenced liquids. Contact angle measurements performed just after the preparation of the surfaces can be considered as reference data for a quality control. These contact angles determined for amine and semicarbazide glass slides are collected in Table 1. The use of the Young’s equation permitted the determination of the Lifshitz-van der Waals (LW) and Lewis acid and base surface tension parameters according to the van Oss theory (Table 1) (20). In addition, we have calculated the interfacial interaction free energy of the surface in water (∆Gsws) and the free energy of binding of water to the surface (∆Gsw), which are a good indication of the hydrophilicity of a surface (21, 22). The contact angle of diiodomethane was found to be similar on both surfaces (p < 0.05, Table 1). The Lifshitzvan der Waals surface tensions of the surfaces were close to 44 mJ/m2, a typical γLW value for a large number of substances (22). The contact angle of water was found to be slightly greater on the semicarbazide surface (p < 0.05, Table 1), but the angle of formamide was found to be similar on both surfaces (p < 0.05, Table 1). Thus, the polar surface tension parameters for the two surfaces were also found to be similar. Taken together, it seemed that the amine and semicarbazide surfaces could not be distinguished by this technique. The fact that semicarbazide and amine surfaces have similar surface tension parameters could explain why proteins were found to adsorb similarly on both surfaces, at least if only interfacial interactions are considered. If we consider now the interfacial interaction free energy between the surfaces in water ∆Gsws, both surfaces displayed a positive value (Table 1). In addition, the free energy of binding of water to the surface ∆Gsw was found to be inferior to -113 mJ/m2, a value below which a substance is usually considered as hydrophilic (22). Evolution of Contact Angles on Semicarbazide or Amine Glass Slide upon Aging. Another interesting application of contact angle measurements is to follow the evolution of the surface upon aging. The silane layer covering the glass slide surface can be the subject of

Figure 11. Intermittent contact mode AFM image image (500 × 500 nm2) of the semicarbazide glass slide surface in air. The gray scale ranges from 0 (black) to 7 nm (white). The mean roughness Ra and the rms roughness were found to be 0.75 and 0.98 nm, respectively.

changes due to chemical transformation of surface functional groups (for example, air oxidation) or to the deposition of air contaminants. Air pollution of amine surfaces has been well studied (23). Contact angle measurements on amine surfaces obtained by silanization with 3-aminopropyltrimethoxysilane demonstrated a rapid pollution of the surface by air impurities. Thus, we have studied the evolution of the contact angles of the three referenced liquids diiodomethane, water, and formamide on both surfaces (Figure 10). The stability study was performed during 15 days for amine and 2 months for semicarbazide glass slides. In agreement with previous studies (23), the amine surface was found to be very sensitive to aging, as shown by the data obtained for the three referenced liquids and nonparametric statistical analysis (p < 0.05). The major change was observed during the first 8 days of aging. For semicarbazide glass slides, a significant change in the contact angles was observed after 30 days of aging for diiodomethane and 60 days for water (p < 0.05), thus suggesting that the semicarbazide glass slides are more stable toward air pollution than amine glass slides. Interestingly, pollution of the semicarbazide surface by


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Figure 12. Determination of the amine (A) and semicarbazide (B) layer thickness by reactive ion etching and tapping mode AFM analysis. AFM images showing a dark area, where the silane layer was removed by RIE from the glass surface. The white spots corresponded to contaminants resulting from the cutting procedure of the glass slide. The curves below the AFM images show a representative line cut through the silane and the glass layers. The height of the step separating the two regions was 1.8-2.2 nm for both surfaces.

air contaminants did not alter the reactivity of the layer as shown previously in a 3 months of chemical stability study. AFM Characterization of Semicarbazide Glass Slides. Glass slides were cleaned using a piranha solution and silanized with 3-(aminopropyl)trimethoxysilane as described elsewhere. Some amine slides were used directly for AFM studies, and the others were treated as usual to install the semicarbazide groups. Thus, AFM measurements were performed on amine or semicarbazide slides coming from the same silanization bath. Figure 11 gives a typical aspect of a semicarbazide glass slide surface. The mean roughness Ra and the rms roughness were found to be, respectively, 0.75 and 0.98 nm. Similar results were obtained for the amine surface (data not shown). The thickness of the layer was determined by removing a defined region of the glass surface by reactive ion etching to get the original silicon oxide substrate. The height of the step at the frontier between the silicon oxide and semicarbazide surface was determined by AFM. As shown in Figure 12B, the thickness of the layer was ranging from 1.8 to 2.2 nm, a value superior to the length of one silane unit (about 1 nm in an extended conformation) (23). The same results were obtained for the amine surface (Figure 12A). These AFM studies demonstrate that the amine and semicarbazide layers are very similar in term of rugosity and thickness. We have shown previously that the two surfaces could not be distinguished by contact angle measurements. Nevertheless, the evolution of antigen reactivity on both surfaces was found to be drastically different. Additional studies are necessary to understand these differences. CONCLUSION

We have demonstrated in this article that semicarbazide functionalized glass slides allow the adsorption of proteins as efficiently as amine slides, with the important advantage of giving stable antibodies detection results.

In addition, semicarbazide glass slides were found to be less sensitive to air pollution than amine surfaces and chemically stable. Peptide microarrays prepared by R-oxo semicarbazone site-specific ligation were also found to give stable antibodies detection results, leading to the possibility of preparing high-quality peptide-protein microarrays. Both surfaces could not be distinguished by contact angle measurements or AFM studies. Thus, more experiments are needed to identify the origin of these large differences. ACKNOWLEDGMENT

We thank CNRS, Universite´ de Lille 2, Institut Pasteur de Lille, and Sedac-Therapeutics for financial support, Eric Diesis for technical assistance, and David Hot, Ludovic Huot, and Erwan Levillain for fluorescence measurements. We thank Pr Yves Lemoine for giving us an access to the Affymetrix 418 array scanner and Affymetrix 417 arrayer. LITERATURE CITED (1) Templin, M. F., Stoll, D., Schrenk, M., Traub, P. C., Vo¨hringer, C. F., Joos, T. O. (2002) Protein microarray technology. Drug Discovery Today 7, 815-822. (2) Lin, S. C., Tseng, F. G., Huang, H. M., Chieng, C. C. (2001) Microsized 2D protein array immobilized by micro-stamps and micro-wells for disease diagnosis and drug screening. Fresenius J. Anal. Chem. 371, 202-208. (3) Fields, G. B., Noble, R. L (1990) Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35, 161-214. (4) Melnyk, O., Fruchart, J.-S., Grandjean, C., Gras-Masse, H. (2001) Tartric acid-based linker for the solid-phase synthesis of C-terminal peptide R-oxo aldehydes. J. Org. Chem. 66, 4153-4160. (5) Fruchart, J.-S., Gras-Masse, H., Melnyk, O. (1999) A new linker for the synthesis of C-terminal peptide R-oxo aldehydes. Tetrahedron Lett. 40, 6225-6228. (6) Urbe`s, F.; Fruchart, J.-S.; Gras-Masse, H.; Melnyk, O. (2002) C-Terminal glyoxylyl peptides for densitive enzyme linked immunosorbent assays. Lett. Pept. Sci. 8, 253-258.

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Polypeptide Semicarbazide Glass Slide Microarrays: Characterization

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