Oriented immobilization of antibodies by a self-assembled monolayer of 2-(biotinamido)ethanethiol for immunoarray preparation

Colloids and Surfaces B: Biointerfaces 47 (2006) 107–111

Short communication

Oriented immobilization of antibodies by a self-assembled monolayer of 2-(biotinamido)ethanethiol for immunoarray preparation Se-Hui Jung a , Ha-Young Son b , Jong Seol Yuk a , Jae-Wan Jung a , Kyung Hwan Kim a , Chang-Hee Lee b , Hoon Hwang b , Kwon-Soo Ha a,∗ a

Department of Molecular and Cellular Biochemistry and Nano-Bio Sensor Research Center, Kangwon National University College of Medicine, and Chunchon, Kangwon-Do 200-701, South Korea b Department of Chemistry, Kangwon National University, Chunchon, Kangwon-Do 200-701, South Korea Available online 6 September 2005

Abstract Orientation of antibodies is very important in the preparation of immunoarrays to keep the activity of antibodies on solid surfaces. Thus, we synthesized a new bifunctional compound, 2-(biotinamido)ethanethiol, and investigated whether the thiol compound is useful to analyze antibody–antigen interactions on immunoarrays with a spectral SPR biosensor. The synthesized organic thiol was characterized by nuclear magnetic resonance spectroscopy and mass spectrometry. 2-(Biotinamido)ethanethiol formed a monolayer on a gold surface and properly immobilized antibodies via streptavidin and biotinylated protein G. Optimal molar ratio of 2-(biotinamido)ethanethiol and mercaptohexanol for antigen–antibody interactions was 1:2. Thus, 2-(biotinamido)ethanethiol is an useful bifunctional linker in the preparation of immunoarrays on gold surfaces. © 2005 Elsevier B.V. All rights reserved. Keywords: 2-(Biotinamido)ethanethiol; Bifunctional linker; Immunoarrays; Spectral SPR biosensor

1. Introduction Microarray technology has become an essential tool for high-throughput analysis of biomolecules including nucleic acids and proteins [1]. DNA microarrays have been used as a powerful tool in gene expression profiling and mutation mapping. There has been a great demand toward protein arrays, since protein arrays are very powerful tools in analyzing functions of multiple proteins in a parallel format [2]. However, proteins may undergo denaturation during being arrayed on a solid substrate and show non-specific binding in the analysis of multiple biomolecular interactions [3]. Thus, it is essential to develop methods to reduce denaturation and non-specific interaction in preparing protein arrays. Abbreviations: GST, glutathione S-transferase; MALDI-TOF, matrixassisted laser desorption/ionization-time of flight; NMR, nuclear magnetic resonance; SPR, surface plasmon resonance ∗ Corresponding author. Tel.: +82 33 250 8833; fax: +82 33 250 8807. E-mail address: [email protected] (K.-S. Ha). 0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2005.07.007

Proteins can be arrayed onto solid supports by physical adsorption [1,4,5], covalent coupling [6,7], and oriented immobilization using streptavidin-coated surface or Ni2+ chelating surface [3,8]. Proteins are easily immobilized onto solid surfaces by physical adsorption, but the immobilized proteins can be susceptible to the reduction of biological activity by an inappropriate orientation caused by physical adsorption [1]. More specific and stronger attachment of proteins can be obtained by covalent modification [1,6,7]. Covalent modification requires a bifunctional cross-linker, which has one functional group that reacts with a base support, and another group that interacts with an active group of proteins [2,9,10]. However, protein immobilization by covalent modification may results in the random orientation of biomolecules and loss of their biological activities. Thus, orientated immobilization of protein or antibodies is the most important issue in the preparation of protein arrays or immunoarrays to reduce non-specific interaction and to increase the sensitivity of biosensors. There have been

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a number of coupling methods for oriented immobilization of biomolecules, including immobilization of biotinylated biomolecules onto streptavidin-coated surfaces [3], immobilization of his-tagged proteins onto Ni2+ -chelating surfaces [8,11,12], and immobilization of glutathione S-transferase (GST)-fusion proteins onto GSH surfaces [1,3]. In addition, antibodies can be arrayed by using protein A or G from Staphylococcus that specifically bind to the Fc region of antibodies [13]. In this study, we present a new bifunctional compound, 2-(biotinamido) ethanethiol in preparing immunoarrays. We synthesized the thiol compound and characterized by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The thiol of 2-(biotinamido)ethanethiol interacts with gold surfaces to form a biotin monolayer, and the biotin part binds streptavidin, which in turn immobilize biotinylated proteins or antibodies. The formation of a 2(biotinamido)ethanethiol monolayer on gold arrays and interactions of antigens with antibodies on the thiolated biotin surface were analyzed by a spectral surface plasmon resonance (SPR) biosensor.

2.3. Synthesis of 2-(biotinamido)ethanethiol [5-(2-oxohexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid (2-mercapto-ethyl)-amide] (2) 0.10 g compound (1) (0.255 mmol) and 0.049 g 2amino ethane thiol (0.429 mmol) were added to 4 ml of N,N-dimethylformamide, mixed with 0.06 ml triethylamine (0.0429 mmol) and the solution was stirred for 6 h at 20–30 ◦ C. The reaction mixture was combined with 10 ml of water and extracted with CH2 Cl2 . Following treating the organic layer with Na2 SO4 to remove water, the solvent was evaporated under a reduced pressure. The remaining solid was purified by a column chromatography on silica gel (eluent: CH2 Cl2 /MeOH = 9/1) and product yield was 31% (0.0236 g); 1 H NMR (CDCl3 ) δ 5.11 (s, 1H, NH), 4.82 (s, 1H, NH), 4.50–4.54 (m, 1H, bridgehead), 4.30–4.35 (m, 1H, bridgehead), 3.14–3.20 (m, 1H, S-CH2 ), 2.91–2.97 (two d, 1H, J = 5.0 Hz, 12.8 Hz, S-CH), 2.72 and 2.76 (two s, 1H, SCH2 ), 2.34 (t, 2H, J = 7.3 Hz, NHCH2 CH2 ) 2.01 (m, 1H, NH), 1.42–1.75 (m, 10H, CH2 ). CI-MS calcd for C12 H21 N3 O2 S2 303.11, found 303.1. 2.4. Surface modification of gold arrays

2. Materials and methods 2.1. Chemical reagents Octadecyltrichlorosilane, mercaptohexanol, tetracarbone chloride, hexadecane were obtained from Sigma (St. Louis, MO). Monoclonal anti-rhoA and anti-rac1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated protein G, polyclonal anti-haptoglobin, monoclonal anti-hemoglobin, hemoglobin and haptoglobin were purchased from Sigma (St. Louis, MO). GST, GST-rhoA and GST-rac1 were prepared by expressing the genes in E. coli (BL21) according to the procedures of Leem et al. [14]. 2.2. Synthesis of 5-(2-oxo-hexahydro-thieno[3,4d]imidazol-4-yl)-pentanoic acid 2,3,5,6-tetrafluoro-phenyl ester (1) 0.5 g biotin (2.05 mmol) and 0.34 g tetra-fluorophenol (2.05 mmol) were added to 5 ml of SOCl2 , stirred for 3 h at room temperature and mixed with 10 ml of CH2 Cl2 . The mixture was stirred for 3 days and the solvent of SOCl2 was evaporated under a reduced pressure. The remaining solid was purified by a column chromatography on a silica gel (eluent: CH2 Cl2 /MeOH = 9/1), and the product yield was 16 % (0.126 g); 1 H NMR (CDCl3 ) δ 6.95–7.06 (m, 1H, ArH), 5.41 and 5.51 (two s, 1H, NH), 5.02 (s, 1H, NH), 4.50–4.56 (m, 1H, bridge head), 4.31–4.37 (m, 1H, bridgehead) 3.15–3.22 (m, 1H, S-CH2 ), 2.90–2.98 (m, 1H, S-CH2 ), 2.69–2.77 (m, 1H, S-CH), 2.35 (t, 1H, J = 7.4 Hz, COCH2 ), 1.55–1.87 (m, 7H, CH2 ). This product was directly used for the next step without further purification.

Gold arrays were fabricated and modified according to the procedures of Yuk et al. [15]. Briefly, gold arrays with 50 spots (diameter of each spot, 2 mm) were fab˚ films on pyrex ricated by depositing Ti/Au (50/450 A) glasses by a RF-magnetron sputtering apparatus and were cleaned with a cleaning solution of NH4 OH:H2 O2 :H2 O (1:1:5, v/v/v) at 70 ◦ C for 10 min. Then, gold arrays were incubated with a mixture of hexadecane/tetracarbone chloride/octadecyltrichlorosilane (20:5:0.04, v/v/v) for 20 min, and washed with a mixture of hexadecane/tetracarbone chloride (20:5, v/v) at 45 ◦ C, tetracarbone chloride at 45 ◦ C and ethanol at room temperature in order. Following cleaning with the cleaning solution at 70 ◦ C for 10 min, the surface of gold array was modified with the mixtures of 1mM 2(biotinamido)ethanethiol and various concentrations (0, 1, 2 and 4 mM) of mercaptohexanol. And the arrays was incubated with 2 ␮l of streptavidin (500 ␮g/ml) in phosphate buffer (8.1 mM Na2 HPO4 , 1.2 mM KH2 PO4 , pH 7.4) for 10 min, and then further incubated with 2 ␮l of biotinylated protein G (100 ␮g/ml) in phosphate buffer for 10 min. 2.5. Preparation of immunoarrays and analysis with a spectral SPR biosensor Monoclonal antibodies against hemoglobin, GST, rhoA, rac1 and polyclonal anti-haptoglobin (100 ␮g/ml in phosphate buffer, pH 7.4) were applied to the modified surface of gold arrays for 10 min. The immunoarrays were blocked with 10 mg/ml of bovine serum albumin and incubated for 10 min with proteins in phosphate buffer, such as hemoglobin, haptoglobin, GST, GST-rhoA and GST-rac1. The arrays were washed twice with 0.1% Tween 20 in the phosphate

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buffered saline (8.1 m Na2 HPO4 , 1.2 mM KH2 PO4 , 2.7 mM KCl, 138 mM NaCl, pH 7.4) for 5 min, rinsed with dH2 O, flushed with N2 gas, and immediately analyzed by a wavelength interrogation-based SPR biosensor (self-developed) [9]. The analysis of protein interactions on immunoarrays was performed by the line-scanning mode of a spectral SPR biosensor (self-assembled) according to the previous report [9].

3. Results and discussion 3.1. Synthesis and characterization of 2-(biotinamido)ethanethiol In order to develop a new surface to orient proteins or antibodies on gold arrays, we have synthesized a new linker, 2-(biotinamido)ethanethiol [5-(2-oxohexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid (2mercapto-ethyl)-amide], by thiolation of biotin and characterized the synthetic compound by NMR spectroscopy and mass spectrometry. 2-(Biotinamido)ethanethiol can be used as a bifunctional linker, which has a thiol group to immobilize the linker onto gold surface and a biotin moiety to interact with streptavidin. As shown in Fig. 1, synthesis of 2-(biotinamido)ethanethiol was accomplished in two steps with a moderate overall yield. Activation of biotin by esterification with 2,3,5,6-tetrafluorophenol is crucial to achieve a desired chemo-selectivity. Briefly, biotin was treated with thionyl chloride and 2,3,5,6-tetrafluorophenol in methylene chloride to afford biotin ester (1) in 16% isolated yield. Then, (1) was converted into its amide derivative (2) in 31% yield in the presence of aminoethanethiol and triethylamine in N,N-dimethylformamide. The synthesized 5(biotinamido) ethanethiol was characterized by NMR and a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometers. As shown in Fig. 2, a proton NMR spectrum showed the presence of ethanethiol group. The newly formed amide N–H signal appeared at 2.03 ppm in a broad triplet. In addition, MALDI-TOF spectrum clearly showed the molecular ion peak at 303.1 mass unit. Thus, it was suggested that 2-(biotinamido)ethanethiol was properly synthesized to be used as a bifunctional linker for protein immobilization. 3.2. Layer formation of anti-rac1 and rac1 on the self-assembled monolayer of 2-(biotinamido)ethanethiol We have investigated whether 2-(biotinamido)ethanethiol can form a monolayer on a gold surface and properly immobilize proteins and antibodies. To study that, gold surface was modified by a self-assembled monolayer of 2(biotinamido)ethanethiol and successively incubated with streptavidin, biotinylated protein G, anti-rac1 and GST-rac1. Modification of gold surface by the successive incubation

Fig. 1. Synthesis of 2-(biotinamido)ethanethiol. (1) 5-(2-Oxo-hexahydrothieno[3,4-d]imidazol-4-yl)-pentanoic acid 2,3,5,6-tetrafluoro-phenyl ester; (2) 5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid (2mercapto-ethyl)-amide [2-(biotinamido)ethanethiol].

was monitored by a spectral SPR biosensor. A net shift of SPR wavelength represents the amount of biomolecules bound to the surface of arrays. The SPR wavelength of gold surface was 586.2 nm, which was determined by the 4thorder polynominal curve fitting technique from an average SPR spectrum of three time measurements [10]. Incubation with 2-(biotinamido)ethanethiol caused 2.8 nm of SPR wavelength shift, which indicated the layer formation of 2(biotinamido)ethanethiol. And further incubation with antirac1 and GST-rac1 caused 6.8 and 9.8 nm of SPR wavelength shift, respectively. These results suggested that 2(biotinamido)ethanethiol formed a monolayer on gold surface and properly immobilized anti-rac1 via streptavidin and biotinylated protein G, resulting in the interaction of anti-rac1 with GST-rac1. For oriented immobilization of antibodies, protein G has been frequently used, since the Fc region of antibodies specifically binds to the protein G [16]. And protein G binds to

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Fig. 2. Characterization of synthetic 2-(biotinamido)ethanethiol by NMR spectroscopy and mass spectrometry. (a) A proton NMR spectrum; and (b) a MALDITOF spectrum.

solid substrates by an avidin–biotin interaction that requires an addition bifunctional linker, such as mercaptoundecanoic acid [9]. However, when 2-(biotinamido)ethanethiol, a thiolated biotin, is used as a bifunctional linker, protein G

can be easily arrayed on the biotin surface of gold arrays. Thus, 2-(biotinamido)ethanethiol can be used as a very useful bifunctional linker to analyze antibody–antigen interactions on immunoarrays by SPR biosensors.

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Table 1 Resonance wavelength shift by antigen–antibody interactions on immunoarrays prepared on various mixtures of 2-(biotinamido)ethanethiol and mercaptohexanol Ratio of 2-(biotinamido)ethanethiol and mercaptohexanol 1:0 Hemoglobin Haptoglobin GST GST-rhoA GST-rac1

3.7 −1.1 0.3 4.7 7.1

± ± ± ± ±

0.6 0.6 0.8 3.8 1.5

1:1

1:2

5.2 ± 1.5 0.2 ± 0.4 1.2 ± 1.3 4.3 ± 3.1 7.1 ± 1.9

7.7 1.1 2.2 5.8 10.9

1:4 ± ± ± ± ±

2.6 0.5 1.1 1.6 2.3

5.4 0.1 1.3 5.1 10.1

± ± ± ± ±

1.9 0.2 0.2 1.4 2.7

The results are expressed as means of SPR wavelength shift ± S.D. from three separate experiments.

3.3. Analysis of antigen–antibody interactions on the surface of 2-(biotinamido)ethanethiol by a spectral SPR biosensor We investigated whether multiple antigen–antibody interactions can be analyzed on immunoarrays prepared on the surface of 2-(biotinamido)ethanethiol. It is reported that streptavidin is well immobilized on a mixture of mercaptoundecanoic acid and 1-octanethiol [17]. Thus, we prepared immunoarrays on various mixtures of 2(biotinamido)ethanethiol and mercaptohexanol, and determined an optimal molar ratio of two thiol compounds by analyzing antigen–antibody interactions. Gold arrays were modified using the monolayer of 2-(biotinamido)ethanethiol mixed with various concentrations of mercaptohexanol, and then incubated with streptavidin and biotinylated protein G to immobilize antibodies. The amount of immobilized protein or antibody was expressed by a net shift of SPR wavelength, which was determined by the line scanning mode of a spectral SPR biosensor. Antibodies bound to the protein G surface with different affinity, in which antihaptoglobin showed a higher affinity than other antibodies (data not shown). Antibody–antigen interactions on arrays were analyzed to determine the antibody activity by the line scanning mode of the SPR biosensor (Table 1). The highest interaction was observed by the ratio of 1:2 of 2(biotinamido)ethanethiol and mercaptohexanol, even though antibodies showed different affinity to their antigens. Thus, 2(biotinamido)ethanethiol is a good bifunctional linker to orient antibodies on the gold surface and the optimal molar ratio of 2-(biotinamido)ethanethiol and mercaptohexanol was 1:2. In addition, 2-(biotinamido)ethanethiol can be used to immobilize biotinylated proteins with a proper orientation via streptavidin. Thus, 2-(biotinamido)ethanethiol is a good bifunctional linker in the preparation of immunoarrays or protein arrays on gold surfaces.

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