Engineering Bioactive Surfaces with Fischer Carbene Complex

ARTICLE pubs.acs.org/bc

Engineering Bioactive Surfaces with Fischer Carbene Complex: Protein A on Self-Assembled Monolayer for Antibody Sensing Piyali Dutta,† Sudeshna Sawoo,† Namrata Ray,† Othman Bouloussa,‡ and Amitabha Sarkar*,† † ‡

Department of Organic Chemistry, Indian Association for the Cultivation of Science, Kolkata-700032, India Institut Curie, Laboratoire Physico-Chimie Curie (UMR CNRS 168), 26 rue d’Ulm, 75248 Paris cedex 05, France

bS Supporting Information ABSTRACT: A Fischer carbene complex was grafted onto selfassembled monolayers (SAMs) on gold or glass by a copperfree “click” reaction. Pendant lysine residues of protein A obtained from Staphylococcus aureus rapidly reacted with the electrophilic metal complex on SAM effecting a covalent attachment of protein A with the surface. The protein A coated surface further led to bioaffinity immobilization of rabbit IgG in an oriented manner, a feature that also permits its purification from rabbit serum. Rabbit IgG could be removed from protein A coated surface by pH adjustment. The regenerated protein A surface was reused three times without loss of activity.

’ INTRODUCTION Research in current years has revealed a huge potential of organometallic bioconjugates1 in radiopharmaceuticals, novel drugs, and even organometallic bioanalysis. Coupling of an organometallic carboxylic acid to amino acids and peptides via amide formation is probably the most extensively studied conjugation reaction.2,3 Biomolecules that have pendant primary amino or unhindered secondary amino groups react instantly and often quantitatively with electrophilic alkoxy Fischer carbene complexes of general formulas (CO)5WdC(OR1)R2 to yield corresponding amino carbene complexes that are stable Fischer carbene bioconjugates.4 Progress of this reaction can easily be monitored by the typical shift of W—CO stretch in the region 1900
2010 cm
1 which is transparent to most organic functional groups and water.5 By adopting appropriate techniques, it is possible to place a Fischer carbene complex at the external terminus of a densely packed, stable, self-assembled monolayer (SAM) on a flat (e.g., glass slide) or a curved (e.g., colloidal gold) solid surface.6
8 The exposed Fischer carbene complex on the molecular film can then be utilized to covalently graft a protein to the SAM on glass or gold as reported earlier.6,8 One of the most advanced applications of SAMs on solid surface is the design of biosensors.9
14 Immobilization of proteins with retention of their activity on highly ordered monolayers is of paramount importance in diagnostic, affinity separation, and biomaterials technology.15
17 The key factor behind the development of the immunosensor is the efficient immobilization of an antibody on the solid surface of assay device.18,19A number of techniques have been developed to immobilize antibodies on solid surfaces: physicochemical adsorption20
26 on poly(L-lysine) coated glass, plastic, or nitro-cellulose membrane, Langmuir
Blodgett methods,27,28 and r 2011 American Chemical Society

covalent attachment.29
36 Physical adsorption proves to be the easiest of procedures, but the method often suffers from relative instability, random orientation, and denaturation of attached antibodies, yielding poor reproducibility. Covalent attachment yields more stable, functional monolayers, but the orientation of proteins is often uncontrolled. Bioaffinity interactions, being much more specific in structural recognition features, can guarantee a certain mode of orientation during binding of one protein with another. Protein A binds specifically to the Fc region of immunoglobulin molecules derived from various mammal species.37
43 If protein A is covalently bound to a surface and its interaction with IgG is affinity controlled (hence potentially reversible by altering pH conditions), one can have a bioactive surface that can sense IgG protein and can be regenerated after each run. The present paper describes the results of such an approach. We describe the following sequence: (a) formation of a highly ordered, densely packed monolayer on gold using C11-chains with a triethylene glycol spacer and terminal Fischer carbene complex; (b) covalent immobilization of protein A by aminolysis reaction with the help of Fischer carbene termini on SAM; (c) bioaffinity immobilization of rabbit IgG and its detection on glass surface by fluorescence microscopy; and, finally, (d) recycling of the protein A slide for iterative purification. Atomic force microscopy (AFM) and ATR-IR were the two surface analytical techniques primarily used for following the chemistry and topology of SAM on gold surfaces.

Received: February 7, 2011 Revised: April 26, 2011 Published: May 17, 2011 1202

dx.doi.org/10.1021/bc200073r | Bioconjugate Chem. 2011, 22, 1202–1209

Bioconjugate Chemistry

’ EXPERIMENTAL PROCEDURE General Methods and Materials. Thin-layer chromatography was performed on TLC Silica gel 60 F254 purchased from Merck and visualized by UV and I2 adsorption. Column chromatography was typically performed using 230
400 mesh silica gel. NMR spectra were recorded on Bruker Advance 200 or 300 MHz spectrometers at room temperature with CDCl3 as solvent and TMS as internal reference. ATR-IR spectra were recorded on Nicolet 380 spectrometer. Contact angles were measured with a digidrop from GBX (Romans sur Is
ere, France). The thickness of self-assembled monolayers (SAMs) on gold slides was investigated by an ellipsometer (Sentech SE 500) operating at a 70° incidence angle. Fluorescence images were acquired using a Leica DM 3000 Upright Trinocular Research Microscope with Leica DFC 425 Scientific digital camera and LAS software. The topography of the protein coated surface was investigated by atomic force microscopy (diCP-II) with DI Company SPMlab Analysis software. Protein A from Staphylococcus aureus (MW 42 KDa),44 IgG from rabbit serum (Reagent grade, > 95%, MW 150 KDa),45,46 and gold coated slides (coated successively with a 20-Å-thick layer of titanium and a 100-Å-thick layer of gold) were purchased from Sigma-Aldrich. Gold chloride and BSA were obtained from SRL. Solvents like ethanol, DMF, and toluene were purchased from SRL and dried using standard procedure. Reagents were purchased from Sigma-Aldrich and were used as received. S-11-(2-(2-(2-Bromoethoxy)ethoxy)ethoxy)undecylethanethioate (4). Synthesis of 2-(2-(2-(undec-10-enyloxy)ethoxy)ethoxy)ethanol (2) and S11-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)undecylethanethioate (3) from 11-bromoundecene (1) was carried out following a procedure reported in literature.7 To a solution of S-11-(2-(2-(2hydroxyethoxy)ethoxy)ethoxy)undecylethanethioate (3) (2 g, 5.2 mmol) in dry DCM (16 mL) under argon atmosphere, carbon tetrabromide (2.1 g, 6.3 mmol) was added slowly. The reaction mixture was cooled to 0 °C and triphenyl phosphine (2 g, 7.6 mmol) in dry DCM (4 mL) was added dropwise to it. After stirring for 6 h, the solvent from the reaction mixture was evaporated out. The product was extracted with petroleum ether (3  15 mL), the combined organic layers were evaporated under vacuum, and the product was purified by flash column chromatography (30% ethyl acetate/petroleum ether). Product obtained was 2 g (86%). 1H NMR (CDCl3, 300 MHz): 1.21
1.26 (m, 14H), 1.49
1.56 (m, 4H), 2.30 (s, 3H), 2.84 (t, J = 6 Hz, 2H), 3.44 (q, J = 6 Hz, 4H), 3.55
3.67 (m, 8H), 3.80 (t, J = 6 Hz, 2H). 13 C NMR (CDCl3, 75 MHz): 26.16, 27.14, 28.89, 29.18, 29.24, 29.53, 29.57, 29.63, 29.71, 30.36, 30.72, 70.15, 70.70, 70.80, 71.31, 71.64, 196.17. HRMS (ESI) m/z [MþNa]þ, calcd for C19H37BrO4SNa 463.1494; found 463.1498. 11-(2-(2-(2-Bromoethoxy)ethoxy)ethoxy)undecane-1-thiol (5). S-11-(2-(2-(2-Bromoethoxy)ethoxy)ethoxy)undecylethanethioate (4) (2 g, 4.5 mmol) was dissolved in dry methanol (15 mL) under argon atmosphere. To it 0.1 mL of concentrated HCl was added and the reaction mixture was heated under reflux for 5 h. Then, the crude reaction mixture was cooled and the solvent was concentrated under reduced pressure. Flash column chromatography (25% ethyl acetate/petroleum ether) afforded 1.6 g (89%) of compound (5). 1H NMR (CDCl3, 300 MHz): 1.23
1.31 (m, 14H), 1.51
1.61 (m, 4H), 2.48 (q, J = 6 Hz, 2H), 3.42 (q, J = 6 Hz, 4H), 3.54
3.63 (m, 8H), 3.78 (t, J = 6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): 24.72, 26.17, 28.45, 29.14, 29.55, 29.57, 29.63, 29.72, 30.38, 34.13, 70.15, 70.65, 70.70, 70.80, 71.30, 71.62. HRMS (ESI) m/z [MþNa]þ, calcd for C17H35BrO3SNa 421.1388; found 421.1380.

ARTICLE

Preparation of SAM with Compound 5. The gold surface (11 mm  11 mm) was immersed in a mixture of concentrated H2SO4 and 30% aq. H2O2 (70:30 v/v) for 1 min, then washed with distilled water and dried under N2. The cleaned gold surfaces were dipped in a freshly prepared 1 mM solution of compound 5 in dry ethanol for 36 h. The slides were then rinsed thoroughly with absolute ethanol in order to remove excess absorbate and dried under argon atmosphere. Surface Modification by SN2 Reaction from Bromide to Azide SAM. Nucleophilic displacement of bromide by azide was carried out in DMF with sodium azide. The bromo terminated gold substrate was incubated in a saturated solution of sodium azide in DMF and stirred for 48 h. The surface was then washed with water and dried under nitrogen. Grafting of Fischer Carbene Complex (6) via Cu Free “Click” Cycloaddition Reaction. The alkynyl Fischer carbene complex 6 was synthesized using a reported procedure.6 The gold slides covered with azido terminated monolayers were dipped in a 10 mM solution of Fischer carbene complex 6 in dry toluene under argon atmosphere at 50 °C for 4 h. It was then washed with dry toluene followed by absolute ethanol in order to ensure complete removal of excess Fischer carbene complex. Finally, the surface was dried under argon. Covalent Immobilization of Protein A. Gold surfaces coated with Fischer carbene was immersed in a solution of protein A (10 mg/L) in phosphate buffer (pH 8.0) for 10 min at room temperature. The substrates were rinsed thoroughly with buffer solution (thrice), then with milli-Q water (thrice), and dried under argon atmosphere. The residual Fischer carbene complexes were blocked by treatment with 1 M ethanolamine (pH 9.0) for 30 min. Finally, the surfaces were treated with 1% (w/v) BSA in phosphate buffer (pH 8.0) for 2 h to block nonspecific binding. Then, the surfaces were extensively rinsed with milli-Q water and dried under argon. Recognition of Rabbit IgG. The recognition experiment was carried out as following: protein A coated gold surfaces were incubated in a solution of rabbit IgG (50 μg/mL) in phosphate buffer (pH 8.0) for 1 h. The surfaces were extensively washed with phosphate buffer containing 0.05% Tween (v/v), followed by milli-Q water, and dried under argon atmosphere. Regeneration of Protein A Coated Surface after Extraction of Rabbit IgG. The rabbit IgG coated surface was incubated in citrate buffer (pH 3.5) for 15 min. The slide was then immersed in phosphate buffer (pH 9.0) for 15 min to recover the protein A coated slide that was reused.

’ RESULTS AND DISCUSSION Synthesis of Bromo Terminated Long Chain Thiol with TEG Spacers. The bromo terminated long chain thiol was

synthesized from triethylene glycol (TEG) according to the reaction sequence represented in Scheme 1. The long chain thiol comprises four distinct parts: (i) thiol group for chemisorptions on Au surface, (ii) long hydrocarbon chain for creating a densely packed monolayer, (iii) a hydrophilic TEG spacer for maximum availability of the functional tether extending in water, and (iv) bromo group at the terminus of chain to be converted to the Fischer carbene moiety. TEG was first converted to monoalkene-TEG 2 by treatment with 11bromoundecene in the presence of sodium hydroxide. Radical mediated addition of thiolacetic acid across the double bond in 2 followed by treatment with carbon tetrabromide and 1203

dx.doi.org/10.1021/bc200073r |Bioconjugate Chem. 2011, 22, 1202–1209

Bioconjugate Chemistry

ARTICLE

Scheme 1. Synthesis of 11-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)undecane-1-thiol (5)

Scheme 2. Grafting of Bromo Terminated Long Chain Thiol (5) and Sequential Chemical Modification of the Terminal Functional Group

triphenylphosphine afforded the brominated product 4. The compound 4 was then hydrolyzed with catalytic amount of conc. HCl to obtain the desired thiol 5 in good yield. Each compound was purified by column chromatography and characterized by standard spectroscopic analyses. Formation of Fischer Carbene Terminated SAM on Gold Surface. A monolayer of bromo terminated thiol 5 was selfassembled on gold surface (surface a) following usual procedure. After thorough washing with ethanol, the gold surface was analyzed by ATR-IR spectroscopy. The presence of strong absorption bands at 2960.0 cm
1 and 2840.0 cm
1 (aliphatic C
H stretching) clearly indicated the presence of bromo terminated chains on the monolayer. The bromo terminated surface was then treated with saturated solution of sodium azide in DMF for 48 h at room temperature leading to surface b. The presence of a typical band at 2118.9 cm
1 established nucleophilic displacement of the bromine atom by azido group. The gold substrate was then treated with a solution of alkoxy Fischer carbene complex 6 in toluene at 50 °C for 4 h. Since the triple bond is highly activated due to the presence of strong electron withdrawing tungsten pentacarbonyl moiety, no Cu(I) salt is required as catalyst for the 1,3-dipolar cycloaddition reaction.47 After thorough washing with toluene, it was analyzed by ATR-IR. The asymmetric stretch of the azido group at 2118.9 cm
1 was no longer visible. Instead, new absorption bands appeared at 2065.1 cm
1and 1930.6 cm
1 for W—CO stretching. These clearly revealed that the Fischer carbene moiety was now covalently bound to the SAM (surface c). Chemical modifications on the gold surface were also monitored by ellipsometry. Thickness of the initially formed bromo terminated monolayer was 2.87 nm. After nucleophilic substitution reaction with azido group, the thickness reduces to 2.74 nm, and after the “click” cycloaddition reaction with the activated

triple bond in alkynyl Fischer carbene, the thickness of SAM increases to 3.43 nm reflecting the introduction of a much larger group on the SAM terminus. Contact angle measurements were also carried out on the gold-coated surfaces. While the contact angle of the bromo terminated SAM was found to be 99.8 ( 0.2°, it decreased to 85.6 ( 1.5° on displacement with azido group, which further decreased to 82.3 ( 1.1° on cycloaddition with Fischer carbene complex. These results are consistent with the changes brought about on the functionality of SAM on the gold surface. Covalent Attachment of Protein A on SAM Leads to Bioaffinity Immobilization of Rabbit IgG. In immunoaffinity systems, one of the most important steps is immobilization of the antibody on the substrate surface, which can then be used for bioaffinity separation or biorecognition. Staphylococcal protein A is known to bind the Fc region of immunoglobulin molecules with high affinity; as a result, the Fab fragments point away from the surface, in order to allow for easy antigen
antibody binding.48
50 This affinity between protein A and IgG is also extensively used for the development of an affinity system for antibody purification.51 Accordingly, we sought to immobilize IgG molecules in an oriented fashion on surface-bound protein A. Protein A consists of a single polypeptide chain of molecular weight 42 000. The three-dimensional structure of the protein A molecule is very stable to heat, denaturing agent, and a large pH interval (0.99
11.8) (see Supporting Information).52 High frictional ratio of 2.1
2.2 and intrinsic viscosity of 29 mL/g suggest that protein A is not a typical globular protein but rather has a markedly extended shape having approximate dimensions of 2.5  25 nm.44,53 At first, the Fischer carbene coated surface was treated with protein A solution in phosphate buffer (pH 8) at room temperature for 10 min (surface d, Scheme 3). The primary amino groups of the pendant lysine residues of protein A reacted 1204

dx.doi.org/10.1021/bc200073r |Bioconjugate Chem. 2011, 22, 1202–1209

Bioconjugate Chemistry

ARTICLE

Scheme 3. Immobilization of Protein A and Rabbit IgG on Gold Surface

Figure 1. AFM topographic image of (A) Fischer carbene terminated SAM and (B) protein A immobilized by reaction with Fischer carbene complex.

Figure 2. (A) ATR-IR spectrum and (B) AFM topographic image of rabbit IgG coated surface.

rapidly with the electrophilic carbene carbon resulting in the formation of new C
N bonds and thereby anchoring the protein covalently to the surface. This protocol was perceived as a mild, rapid, and reliable procedure for protein immobilization that would minimize multilayer formation. Physically adsorbed protein A molecules were then removed by repeated washing with the buffer solution and milli-Q water. The slides were then dried under a stream of argon.

The protein A coated surface was characterized by ATR-IR and AFM. After aminolysis, the absorption peaks for W—CO stretching show a decrease of 8 cm
1. For AFM imaging, a noncontact mode was employed. Figure 1B represents the image of the surface after incubation in protein A solution for 10 min. A dense coverage was observed throughout the protein-coated surface, whereas the Fischer carbene terminated SAM did not show any significant undulation of the surface (Figure 1A). The 1205

dx.doi.org/10.1021/bc200073r |Bioconjugate Chem. 2011, 22, 1202–1209

Bioconjugate Chemistry

ARTICLE

Figure 3. AFM topographic image of (A) protein A slide and (B) rabbit IgG after second cycle, (C) protein A slide, and (D) rabbit IgG coated slide after third cycle.

average height and the average roughness (Ra) of the Fischer carbene terminated gold surface were 4.23 and 0.61 nm, respectively. After protein A immobilization, the height and Ra increased to 7.13 and 0.95 nm, respectively (Figure 1B). The increase in height by 2.9 nm is consistent with the reported size of protein A molecule. The protein A coated slide was then incubated in rabbit IgG solution in phosphate buffer (pH 8.0) for 1 h at room temperature.54 Excess IgG was removed by washing with buffer and milli-Q water several times. Surface e in Scheme 3 represents the substrate surface carrying oriented IgG molecules on the selfassembled monolayer. ATR-IR spectrum of the IgG coated surface showed that the amino carbene band remained at the same position 2064.5 cm
1 and 1922.2 cm
1 which established that Fischer carbene complex did not undergo significant decomposition (Figure 2A). Figure 2B displays the AFM image of the IgG immobilized on protein A bonded surface. From AFM images, it was evident that the average height of the immobilized protein A (Figure 1B) increased from 7.13 to 15.78 nm and Ra changed to 1.59 nm after incubation in rabbit IgG. The change in height by 8.65 nm roughly corresponds to the height of IgG (8.5 nm).55 The section analysis of the gold surfaces modified with Fischer carbene complex, protein A, and rabbit IgG are included in the Supporting Information. Recycling of the Protein A Coated SAM on Flat Gold Surface. The interaction between protein A and rabbit IgG is affinity-driven. The immobilized IgG can be debased by changing the pH of the buffer medium. This ought to give rise to a free protein A surface which can be recycled. As described below, this expectation was indeed realized. The initial average height of the protein A coated slide was found to be 7.13 nm by AFM (Figure 1B). After incubation with

Figure 4. Fluorescence image of rabbit IgG coated slide, incubated in solution of FITC tagged goat antirabbit IgG in phosphate buffer.

rabbit IgG, the height increased to 15.78 nm (Figure 2B). After removing the rabbit IgG, by treating the gold surface with a typical elution buffer like citrate buffer (pH 3.5) for 15 min the average height decreased to 7.11 nm (Figure 3A). After that, the slide was immersed in a neutralizing buffer like phosphate buffer (pH 9.0) for 15 min to restore original protein conformation. When it was incubated again in rabbit IgG solution, the average height increased to 15.81 nm (Figure 3B). On repeating the process for the third time, we observed that the height changed from 7.06 nm (Figure 3C) to 15.69 nm (Figure 3D). Thus, in all three cycles the change in height with immobilization of rabbit IgG is consistent with the height of IgG. 1206

dx.doi.org/10.1021/bc200073r |Bioconjugate Chem. 2011, 22, 1202–1209

Bioconjugate Chemistry

ARTICLE

Figure 5. Fluorescence microscopy image of rabbit IgG coated slide after incubation in FITC tagged goat antirabbit IgG solution in phosphate buffer after (A) second cycle and (B) third cycle.

min. The citrate buffer containing the rabbit IgG was readily neutralized with Tris-HCl buffer (pH 9.0) and concentrated. The purified IgG was analyzed by SDS-PAGE gel after chemical reduction using 2-mercaptoethanol. IgG has a molecular mass of 150 kD and contains two heavy chains of 50 kD and two light chains of about 25 kD. By reduction with 2-mercaptoethanol, the heavy and the light chains were resolved. This is depicted in Figure 6. The spot at 49 kD represents the heavy chain and the spot at 26 kD represents the light chain. The cycle was repeated three times. No contaminants are visible in the purified IgG (Figure 6). Figure 6. Picture of SDS-PAGE analysis of rabbit IgG fraction obtained by chemical reduction with 2-mercaptoethanol. Molecular weight standard is on lane (PM) and chemically reduced IgG after first cycle (lane 1), second cycle (lane 2), and third cycle (lane 3).

Detection of Rabbit IgG on Glass Using Fluorescence Microscope. The presence of rabbit IgG can also be proven

visually by fluorescence microscopy techniques. These experiments were performed on glass slides using 11-bromoundecyltrichlorosilane as the primary chain. The bromo terminated surface was converted to the Fischer carbene terminated surface as depicted in Scheme 1. The Fischer carbene coated slide was subsequently treated with protein A solution and then with rabbit IgG solution as described before. After thorough washing by buffer, the IgG coated slide was immersed in a solution of FITC tagged goat anti rabbit IgG in phosphate buffer (pH 8). After careful washing with buffer and milli-Q water, the slide was observed under fluorescence microscope. Bright fluorescent spots were observed (Figure 4). Fluorescence microscopy was also used to verify the regeneration of protein A after extraction of IgG on glass slides. The FITC tagged goat antirabbit IgG was used and fluorescence spots of nearly equal distribution of density were observed across several cross sections of the slide indicating high reproducibility (Figure 5). Since protein A binds specifically with the Fc region of rabbit IgG, this property allows the use of surface-bound protein A for purification of rabbit IgG from rabbit serum. We used protein A coated microscopic glass slides and immersed it in a solution of rabbit serum (50 μg/mL) in phosphate buffer (pH 8.0) for 1 h. After thorough washing with buffer and milli-Q water, the slides were immersed into eluting buffer (citrate buffer pH 3.5) for 15

’ CONCLUSION In conclusion, we have successfully applied a relatively mild protocol to modify Au/glass surface with Fischer carbene function that allows rapid immobilization of protein A on surface, which lead to oriented immobilization of rabbit IgG. The surfaces at different stages were characterized by ATR-IR and AFM. We also demonstrated that a protein A slide prepared by following our protocol can be used successively three times to purify rabbit IgG from rabbit serum. The work described above widens the scope of employing Fischer carbene complexes both in biological and biomedical studies. ’ ASSOCIATED CONTENT

bS

Supporting Information. Synthesis of compound 2, compound 3, and Fischer carbene complexes, ATR-IR spectrum, procedure for functionalization of glass slides, 1H and 13C NMR spectra of 4 and 5. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel: þ91 33 2473 4971 ext. 407. Fax: þ91-33-2473 2805.

’ ACKNOWLEDGMENT The authors wish to thank Dr. Santiswarup Singha and Prof. Anjan Kr. Dasgupta at Department of Biochemistry, University 1207

dx.doi.org/10.1021/bc200073r |Bioconjugate Chem. 2011, 22, 1202–1209

Bioconjugate Chemistry of Calcutta; R. Banik (AFM), P. P. Bhattacharya (IR) for technical supports at IACS; CSIR, New Delhi, for Research Fellowships (P.D. and N.R.); DST and IFCPAR, India, for financial support.

’ REFERENCES (1) Jaouen, G. (2006) Bioorganometallics: Biomolecules, labeling, Medicine, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (2) Moriuchi, T., and Hirao, T. (2010) Design of ferrocene-dipeptide bioorganometallic conjugates to induce chirality-organized structures. Acc. Chem. Res. 43, 1040–1051. (3) van Staveren, D. R., and Metzler-Nolte, N. (2004) Bioorganometallic chemistry of ferrocene. Chem. Rev. 104, 5931–5985. (4) Salmain, M., Licandro, E., Baldoli, C., Maiorana, S., Tran-Huy, H., and Jaouen, G. (2001) Transition metal (‘Fischer-type’) carbene complexes as protein labelling reagents. J. Organomet. Chem. 617
618, 376–382. (5) Samanta, D., Sawoo, S., Patra, S., Ray, M., Salmain, M., and Sarkar, A. (2005) Synthesis of hydrophilic Fischer carbene complexes as organometallic marker and PEGylating agent for proteins. J. Organomet. Chem. 690, 5581–5590. (6) Sawoo, S., Dutta, P., Chakraborty, A., Mukhopadhyay, R., Bouloussa, O., and Sarkar, A. (2008) A new bio-active surface for protein immobilization via copper-free ‘click’ between azido SAM and alkynyl Fischer carbene complex. Chem. Commun. 5957–5959. (7) Samanta, D., Faure, N., Rondelez, F., and Sarkar, A. (2003) Towards “designer” surfaces: functionalisation of self-assembled monolayer (SAM) on colloidal gold by alkene metathesis. Chem. Commun. 1186–1187. (8) Srivastava, P., Sarkar, A., Sawoo, S., Chakraborty, A., Dutta, P., Bouloussa, O., Methivier, C., Pradier, C.
M., Boujday, S., and Salmain, M. (2011) A versatile approach for the immobilization of amines via copper-free “click” reaction between azido self-assembled monolayer and alkynyl Fischer carbene complex. Application to the detection of staphylococcal enterotoxin A antibody. J. Organomet. Chem. 696, 1102–1107. (9) Samanta, D., and Sarkar, A. (2011) Immobilization of biomacromolecules on self-assembled monolayers: methods & sensor applications. Chem. Soc. Rev. 40, 2567–2592. (10) Choi, M. M. F. (2004) Progress in enzyme-based biosensors using optical transducers. Microchim. Acta 148, 107–132. (11) Sassolas, A., Leca-Bouvier, B. D., and Blum, L. J. (2008) DNA biosensors and microarrays. Chem. Rev. 108, 109–139. (12) Jelinek, R., and Kolusheva, S. (2004) Carbohydrate biosensors. Chem. Rev. 104, 5987–6015. (13) Campuzano, S., Galvez, R., Pedrero, M., Manuel de Villena, F. J., and Pingarron, J. M. (2003) An integrated electrochemical fructose biosensor based on tetrathiafulvalene-modified self-assembled monolayers on gold electrodes. Anal. Bioanal. Chem. 377, 600–607. (14) Malhotra, B. D., and Chaubey, A. (2003) Biosensors for clinical diagnostics industry. Sens. Actuators, B 91, 117–127. (15) Rusmini, F., Zhong, Z., and Feijen, J. (2007) Protein immobilization strategies for protein biochips. Biomacromolecules 8, 1775–1789. (16) Jonkheijm, P., Weinrich, D., Schr€oder, H., Niemeyer, C. M., and Waldmann, H. (2008) Chemical strategies for generating protein biochips. Angew. Chem., Int. Ed. 47, 9618–9647. (17) Wong, L. S., Khan, F., and Micklefield, J. (2009) Selective covalent protein immobilization: strategies and applications. Chem. Rev. 109, 4025–4053. (18) Saerens, D., Huang, L., Bonroy, K., and Muyldermans, S. (2008) Antibody fragments as probe in biosensor development. Sensors 8, 4669–4686. (19) Kurtinaitiene, B., Ambrozaite, D., Laurinavicius, V., Ramanaviciene, A., and Ramanavicius, A. (2008) Amperometric immunosensor for diagnosis of BLV infection. Biosens. Bioelectron. 23, 1547–1554. (20) Ramanavicius, A., Kurilcik, N., Jursenas, S., Finkelsteinas, A., and Ramanaviciene, A. (2007) Conducting polymer based fluorescence

ARTICLE

quenching as a new approach to increase the selectivity of immunosensors. Biosens. Bioelectron. 23, 499–505. (21) Ouerghi., O., Touhami., A., Othmane., A., Ouada, H. B., Martelet, C., Fretigny, C., and Jaffrezic-Renault, N. (2002) Investigating antibody-antigen binding with atomic force microscopy. Sens. Actuators, B 84, 167–175. (22) Butler, J. E., Ni, L., Nessler, R., Joshi, K. S., Suter, M., Rosenberg, B., Chang, J., Brown, W. R., and Cantarero, L. A. (1992) The physical and functional behavior of capture antibodies adsorbed on polystyrene. J. Immunol. Methods 150, 77–90. (23) Wu, Z., Yan, Y., Shen., G., and Yu, R. (2000) A novel approach of antibody immobilization based on n-butyl amine plasma-polymerized films for immunosensors. Anal. Chim. Acta 412, 29–35. (24) Qian, W., Yao, D., Yu, F., Xu, B., Zhou, R., Bao, X., and Lu, Z. (2000) Immobilization of antibodies on ultraflat polystyrene surfaces. Clin. Chem. 46, 1456–1463. (25) Caruso, F., Rodda, E., and Furlong, D. N. (1996) Orientational aspects of antibody immobilization and immunological activity on quartz crystal microbalance electrodes. J. Colloid Interface Sci. 178, 104–115. (26) Chang, I. -N., Lin, J. -N., Andrade, J. D., and Herron, J. N. (1995) Adsorption mechanism of acid pretreated antibodies on dichlorodimethylsilane-treated silica surfaces. J. Colloid Interface Sci. 174, 10–23. (27) Owaku, K., Goto, M., Ikariyama, Y., and Aizawa, M. (1995) Protein A Langmuir-Blodgett film for antibody immobilization and its use in optical immunosensing. Anal. Chem. 67, 1613–1616. (28) Barraud, A., Perrot, H., Billard, V., Martelet, C., and Therasse, J. (1993) Study of immunoglobulin G thin layers obtained by the Langmuir-Blodgett method: application to immunosensors. Biosens. Bioelectron. 8, 39–48. (29) Liu, X., Wang, H., Herron, J. N., and Prestwich, G. (2000) Photopatterning of antibodies on biosensors. Bioconjugate Chem. 11, 755–761. (30) Jung, Y., Jeong, J. Y., and Chung, B. H. (2008) Recent advances in immobilization methods of antibodies on solid supports. Analyst 133, 697–701. (31) Kausaite-Minkstimiene, A., Ramanaviciene, A., Kirlyte, J., and Ramanavicius, A. (2010) Comparative study of random and oriented antibody immobilization techniques on the binding capacity of immunosensor. Anal. Chem. 82, 6401–6408. (32) Jain, S., Chattopadhyay, S., Jackeray, R., and Singh, H. (2009) Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte. Anal. Chim. Acta 654, 103–110.  Sullivan, C. K. (2003) (33) Susmel, S., Guilbault, G. G., and O Demonstration of labeless detection of food pathogens using electrochemical redox probe and screen printed gold electrodes. Biosens. Bioelectron. 18, 881–889. (34) Pei, R., Cheng, Z., Wang, E., and Yang, X. (2001) Amplification of antigen
antibody interactions based on biotin labeled protein
streptavidin network complex using impedance spectroscopy. Biosens. Bioelectron. 16, 355–361. (35) Su, X. -L., and Li, Y. (2004) A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157: H7. Biosens. Bioelectron. 19, 563–574. (36) Wadu-Mesthrige, K., Amro, N. A., and Liu, G. -Y. (2000) Immobilization of proteins on self-assembled monolayers. Scanning 22, 380–388. (37) Bae, Y. M., Oh, B. -K., Lee, W., Lee, W. H., and Choi, J. -W. (2005) Study on orientation of immunoglobulin G on protein G layer. Biosens. Bioelectron. 21, 103–110. (38) Kanno, S., Yanagida, Y., Haruyama, T., Kobatake, E., and Aizawa, M. (2000) Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization. J. Biotechnol. 76, 207–214. (39) Gao, D., McBean, N., Schultz, J. S., Yan, Y., Mulchandani, A., and Chen, W. (2006) Fabrication of antibody arrays using thermally responsive elastin fusion proteins. J. Am. Chem. Soc. 128, 676–677. 1208

dx.doi.org/10.1021/bc200073r |Bioconjugate Chem. 2011, 22, 1202–1209

Bioconjugate Chemistry

ARTICLE

(40) Starodub, N. F., Pirogova, L. V., Demchenko, A., and Nabok, A. V. (2005) Antibody immobilisation on the metal and silicon surfaces. The use of self-assembled layers and specific receptors. Bioelectrochemistry 66, 111–115. (41) Oh, B.
K., Kim, Y.
K., Lee, W., Bae, Y. M., Lee, W. H., and Choi, J.
W. (2003) Immunosensor for detection of Legionella pneumophila using surface plasmon resonance. Biosens. Bioelectron. 18, 605–611. (42) Briand, E., Salmain, M., Comp
ere, C., and Pradier, C.
M. (2006) Immobilization of Protein A on SAMs for the elaboration of immunosensors. Colloids Surf., B 53, 215–224. (43) Demirel, G., C-aglayan, M. O., Garipcan, B., Duman, M., and Pis-kin, E. (2007) Oriented immobilization of IgG on hydroxylated Si(001) surfaces via protein-A by a multiple-step process based on a selfassembly approach. J. Mater. Sci. 42, 9402–9408. (44) Bj€ork, I., Petersson, B.
Å., and Sj€oquist, J. (1972) Some physicochemical properties of Protein A from Staphylococcus aureus. Eur. J. Biochem. 29, 579–584. (45) Ito, W., Nakamura, H., and Arata, Y. (1989) Protein-protein interactions on the surface of immunoglobulin molecules. J. Mol. Graphics 7, 60–63. (46) Strejan, G., and Campbell, D. H. (1971) Physicochemical, immunochemical and biologic properties of rabbit homocytotropic antibodies produced to crude extracts. J. Immunol. 106, 1363–1370. (47) Chakraborty, A., Dey, S., Sawoo, S., Adarsh, N. N., and Sarkar, A. (2010) Regioselective 1, 3-dipolar cycloaddition reaction of azides with alkoxy alkynyl fischer carbene complexes. Organometallics 29, 6619–6622. (48) Deisenhofer, J. (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of Protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20, 2361–2370. (49) Hjelm, H., Sj€ odahl, J., and Sj€oguist, J. (1975) Immunologically active and structurally similar fragments of Protein A from Staphylococcus aureus. Eur. J. Biochem. 57, 395–403. (50) Lu, B., Smyth, M. R., and O’Kennedy, R. (1996) Oriented immobilization of antibodies and its applications in lmmunoassays and immunosensors. Analyst 121, 29R–32R. (51) Hober, S., Nord, K., and Linhult, M. (2007) Protein A chromatography for antibody purification. J. Chromatogr., B 848, 40–47. (52) Sj€oholm, I. (1975) Protein A from Staphylococcus aureus spectropolarimetric and spectrophotometric studies. Eur. J. Biochem. 51, 55–61. (53) Ohnishi, S., Murata, M., and Hato, M. (1998) Correlation between surface morphology and surface forces of Protein A adsorbed on mica. Biophys. J. 74, 455–465. (54) Åkerstr€om, B., and Bj€orck, L. (1986) A physicochemical study of Protein G, a molecule with unique immunoglobulin G-binding properties. J. Biol. Chem. 261, 10240–10247. (55) Coen, M. C., Lehmann, R., Gr€oning, P., Bielmann, M., Galli, C., and Schlapbach, L. (2001) Adsorption and bioactivity of Protein A on silicon surfaces studied by AFM and XPS. J. Colloid Interface Sci. 233, 180–189.

1209

dx.doi.org/10.1021/bc200073r |Bioconjugate Chem. 2011, 22, 1202–1209