- Email: [email protected]
The selective adsorption of human serum albumin on N-isobutyryl-cysteine enantiomers modified chiral surfaces
Biochemical Engineering Journal 69 (2012) 155–158
Contents lists available at SciVerse ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
The selective adsorption of human serum albumin on N-isobutyryl-cysteine enantiomers modified chiral surfaces Qiao Chen, Juan Zhou, Qian Han, Yonghua Wang, Yingzi Fu ∗ Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
a r t i c l e
i n f o
Article history: Received 2 May 2012 Received in revised form 18 August 2012 Accepted 8 September 2012 Available online 13 September 2012 Keywords: N-isobutyryl-cysteine Human serum albumin Adsorption Amino acids Protein Sensors
a b s t r a c t The stereoselective interaction between human serum albumin (HSA) and N-isobutyryl-cysteine (NIBC) enantiomers was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical responses of the NIBC enantiomers modified surfaces were varied along with the inserted time and the concentration of HSA. The results showed that HSA molecules displayed different adsorption behaviors on NIBC enantiomers modified surfaces and preferred to be adsorbed on the l-NIBC modified surface. That is to say, l-NIBC modified surface had stronger interaction with HSA molecules than d-NIBC modified surface, suggesting that the molecular configuration of l-NIBC had matched better with HSA than the case of d-NIBC. The investigation of the interaction between protein and chiral molecules may not only help to understand the high selectivity of chirality in biosystems, but also provide a reference for studying chiral drugs. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Chiral recognition is one of the most fundamental and crucial properties of various natural systems, which plays a very important role in understanding the interactions of biological molecules [1,2]. Living systems contain large numbers of biological macromolecules, and most of them show natural enantioselectivity [3]. For example, cells and DNA can sense the surface chirality and exhibit different adhesion and activation behaviors on enantiomorphous surfaces [4–6], human serum albumin (HSA) modified indium tin oxide (ITO) electrodes can selectively adsorb tryptophan enantiomers [7] and ␥-gluoblin can selectively recognize mandelic acid enantiomers [8]. Therefore, the study on the interaction between biomacromolecules and chiral molecules is valuable and fascinating in understanding the properties of biomacromolecules and the high selectivity of chiral molecules in biosystems [9]. Serum albumin is the most abundant proteins in the circulatory system and maintains the normal plasma osmotic pressure [10]. HSA which contains 585 amino acid residues is 67,000 Da molecular mass and it is a valuable biomarker of many diseases and widely used in clinical treating of diseases [11–13]. At the same time, HSA plays an important role in the transport of drugs, metabolites, and endogenous ligands [14].
Thiol molecules modified surfaces have received steadily increasing attention [15,16]. Chiral thiol molecules have been widely studied and applied in chiral recognition [17]. For example, l-homocysteine modified electrode has been used to selectively recognize amino acids enantiomers in the presence of copper ions [18], and penicillamine enantiomers functionalized gold nanoparticles can enantioselectively recognize 3,4-dihydroxyphenylalanine [19]. Protein-based biosensor, prepared by the interaction between a self-assembled functional monolayer film and protein molecules, has wide applications [20]. N-isobutyryl-cysteine (NIBC) enantiomers, one of the derivatives of cysteine, have been reported to selectively recognize amino acids [21]. Here, the adsorption behaviors of HSA on NIBC enantiomers modified gold surfaces have been studied. To date, various methods have been developed to study the interaction between protein and chiral molecules [22–24]. In this work, the stereoselective interaction between HSA and N-isobutyryl-cysteine enantiomer modified chiral surfaces was investigated via electrochemical methods. The results shown that larger response signal was observed from the interaction between the l-NIBC modified gold surface and HSA molecules than the case of d-NIBC modified gold surface. 2. Experimental 2.1. Materials and methods
∗ Corresponding author. Tel.: +86 023 68252360; fax: +86 023 68253195. E-mail address: [email protected] (Y. Fu). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.09.008
Human serum albumin, N-isobutyryl-l(d)-cysteine (l- or dNIBC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA)
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Fig. 1. (A) Cyclic voltammograms and (B) electrochemical impedance spectroscopy of different electrodes in 5 mM [Fe(CN)6 ]4−/3− solution (pH 6.7) (a) l-NIBC-Au, (b) d-NIBC-Au, after (c) d-NIBC-Au and (d) l-NIBC-Au interacted with 0.1 mM HSA for 1 h.
and stored in refrigerator. Other chemical reagents were analytical grade and used without further purification. All aqueous solutions were prepared with double distilled water. All experiments were completed at room temperature (25 ± 0.5 ◦ C). 2.2. Apparatus Electrochemical impedance spectroscopy (EIS) measurements were performed on Model IM6e (ZAHNER Elektrick Co., Germany). Cyclic voltammetry (CV) measurements were performed on a CHI 660D electrochemistry workstation (Shanghai Chenhua Instruments Co., China). Electrochemical experiments were performed in an unstirred electrochemical cell. A working electrode (the modified electrode or bare electrode), a platinum wire auxiliary electrode and a saturated calomel electrode (SCE) constituted the standard three-electrode system. 2.3. The formation of chiral surface The gold electrodes (Ø = 4 mm) were polished on microcloth carefully with 1.0, 0.3 and 0.05 m alumina slurries, respectively. Following that, the electrodes were sonicated in double distilled water, ethanol and double distilled water each for 5 min. Then, the cleaned electrodes were immediately immersed in 5 mM l- or dNIBC ethanol solution overnight at 4 ◦ C. The l- or d-NIBC modified chiral surfaces (l-/d-NIBC-Au) were obtained. 2.4. Experimental measurements The scan of CV was taken from −0.2 to 0.6 V (vs. SCE) at 100 mV s−1 in 5 mM [Fe(CN)6 ]4−/3− (0.1 M PBS, pH 6.73). The frequency range of EIS measurements from 0.1 to 105 Hz in a given open circuit voltage, amplitude was 0.22 V. The enantioselective detection was based on the difference of current response (I = I0 − I1 ) before and after the NIBC enantiomers modified surfaces immersed in HSA solution. The peak currents of the NIBC enantiomers modified electrodes were regarded as I0 , after it immersed in HSA solution, the peak current was regarded as I1.
3. Results and discussion 3.1. Characterization of the NIBC enantiomers modified surfaces As an effective and convenient method for investigating the feature of the electrode surface, CV was used to study the characterization of NIBC enantiomers modified surfaces. The bare gold electrode showed well redox peaks. A large decrease of peak current was observed after the gold electrode immersed in ethanol solutions of d- or l-NIBC overnight, suggesting d- or l-NIBC has successfully self-assembled on gold electrodes. 3.2. The selective adsorption of HSA on NIBC enantiomers modified surfaces 3.2.1. The adsorption characterization of HSA As shown in Fig. 1A, the electrochemical responses of lNIBC-Au (curve a) and d-NIBC-Au (curve b) were almost the same. The peak currents were decreased after l-NIBC and dNIBC modified surfaces interacted with 0.1 mM HSA about 60 min, hinting the adsorbed HSA insulated the interfacial electron transfer. However, the decreasing of peak current of l-NIBCAu (curve d) was larger than d-NIBC-Au (curve c), implying the adsorbed HSA molecules on l-NIBC-Au surface was higher. The adsorption of HSA has been greatly influenced by the surface chirality, and the interaction between HSA and l-NIBC-Au was larger, suggesting HSA prefers to adsorb on l-NIBC-Au surface. 3.2.2. EIS characterization of the adsorption of HSA As shown in Fig. 1B, the impedance spectra include a semicircle portion and a linear portion. The semicircle diameter equals the interfacial electron transfer resistance (Ret ). The l-NIBC-Au (curve a) and d-NIBC-Au (curve b) surfaces had almost the same Ret . After the modified surfaces inserted in HSA solution for 60 min, a large increment of resistance was observed, indicating that HSA has adsorbed on NIBC enantiomers modified surfaces and obstructed
Q. Chen et al. / Biochemical Engineering Journal 69 (2012) 155–158
3.2.3. The influence of interaction time between NIBC enantiomers and HSA The influence of interaction time between NIBC enantiomers and HSA from 0 to 100 min on the electrochemical response was shown in Fig. 2A. It can be seen that peak currents of NIBC enantiomers modified surfaces were decreased with the prolongation of interaction time because of a longer interaction time contributed to more adsorption of HSA on a chiral surface and the change of peak current of HSA/l-NIBC-Au (curve a) were larger than HSA/d-NIBCAu (curve b). The amperometric responses leveled off after 60 min for two surfaces. So, 60 min was adopted as incubation time in this work.
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Fig. 2. (A) The time dependence of the adsorption of HSA on (a) l-NIBC and (b) d-NIBC modified surface. (B) The changes of peak current along with the varying concentrations of HSA (a) l-NIBC-Au, (b) d-NIBC-Au, and (c) the difference between l-NIBC-Au and d-NIBC-Au.
the electron transfer of the ferricyanide probe. It was noted that the resistance of HSA/l-NIBC-Au (curve d) was larger than HSA/d-NIBCAu (curve c), implying more HSA molecules adsorbed on l-NIBC modified surfaces and hindered the electron transfer. This result is very consistent with the CV.
3.2.4. Electrochemical response of the modified electrodes with the varying concentrations of HSA The electrochemical response of the l-NIBC and d-NIBC modified electrodes with varying concentrations of HSA were also studied by measuring the change of peak currents. Fig. 2B shows a comparison for the change of peak currents of HSA adsorbed on l-NIBC and d-NIBC surfaces for different HSA concentrations. After NIBC enantiomers modified electrodes inserted in HSA solution for 60 min, the change of peak currents of l-NIBC-Au (Fig. 2Ba) were larger than the d-NIBC-Au (Fig. 2Bb). Fig. 2Bc was the relative difference of response currents between l-NIBC-Au and d-NIBC-Au. It can be seen that the difference of electrochemical signal can be observed even at 0.005 mg mL−1 . It is further evidence that the HSA molecule was selectively adsorbed on NIBC enantiomers modified surfaces. All of those results showed that a larger electrochemical response signal was obtained from l-NIBC-Au surface than the case of d-NIBC-Au under the same conditions. This effect may be caused by a higher amount of HSA molecule adsorbed on the l-NIBC-Au than d-NIBC-Au, which resulted in a greater obstacle for electron transfer between the redox probe and the electrode surfaces. That is to say, HSA could sense the surface chirality and show different adsorption capacities on NIBC enantiomers modified surfaces. The reason for the enantioselective adsorption of HSA molecule on NIBC enantiomers modified surfaces is discussed as following: The existence of COOH, NH and carboxyl groups, which have different spatial arrangements, induce that NIBC enantiomers show different orientations. NIBC enantiomers have different abilities to form effective hydrogen bonding, hydrophobic interactions and specific electrostatic forces with HSA. Namely, the surface chirality may be
Fig. 3. The schematic representation of l-NIBC-Au (a) and d-NIBC-Au (b) interacting with HSA.
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recognized through special chemical interaction between the chiral moieties of the surface and HSA, which may release different signals. From the results of above experiments, it could be reasonably speculated that the molecular configuration of l-NIBC may match well with the HSA and can bind with the major HSA through hydrogen bonding, hydrophobic interactions and the specific electrostatic forces interaction, which results in a higher adsorption of HSA (Fig. 3a), while a weak adsorption on the d-NIBC surface for the steric hindrance (Fig. 3b). 4. Conclusions In conclusion, the adsorption of HSA on the NIBC enantiomers modified surfaces was investigated by electrochemical methods. All of the results showed that a larger electrochemical response signal was obtained from l-NIBC-Au than d-NIBC-Au, and the surface chirality has great influence on the adsorption of HSA. The molecular configurations of l-NIBC and d-NIBC greatly influence the formation of hydrogen bonding, hydrophobic interactions and the specific electrostatic forces interactions between NIBC enantiomers and HSA molecules. The detailed analysis of the interaction between HSA and NIBC enantiomers is helpful for knowing the properties of protein. This work gives us the great opportunity to get to know the reaction between protein and chiral medicine, and provides us a reference for studying chiral drugs. Acknowledgment This work was supported by the National Natural Science Foundation of China No. 20972128 References [1] X.X. Zhang, J.S. Bradshaw, R.M. Izatt, Enantiomeric recognition of amine compounds by chiral macrocyclic receptors, Chem. Rev. 97 (1997) 3313–3362. [2] Z.B. Li, J. Lin, Y.C. Qin, L. Pu, Enantioselective fluorescent recognition of a soluble “supported” chiral acid: toward a new method for chiral catalyst screening, Org. Lett. 7 (2005) 3441–3444. [3] N.M. Maier, P. Franco, W. Lindner, Separation of enantiomers: needs, challenges, perspectives, J. Chromatogr. A 906 (2001) 30–33. [4] K.J. Tang, H. Gan, Y. Li, L.F. Chi, T.L. Sun, H. Fuchs, Stereoselective interaction between DNA and chiral surfaces, J. Am. Chem. Soc. 130 (2008) 11284–11285. [5] H. Gan, K.J. Tang, T.L. Sun, M. Hirtz, Y. Li, L.F. Chi, Selective adsorption of DNA on chiral surfaces: supercoiled or relaxed conformation, Angew. Chem. 121 (2009) 5386–5390. [6] T.L. Sun, D. Sun, K. Rhemann, L.F. Chi, H. Fuchs, Stereospecific interaction between immune cells and chiral surfaces, J. Am. Chem. Soc. 129 (2007) 1496–1497.
[7] M. Matsunaga, T. Ueno, T. Nakanishi, T. Osaka, Enantioselective potential response of a human serum albumin-modified ITO electrode for tryptophan, Electrochem. Commun. 10 (2008) 1844–1846. [8] Y.Z. Fu, Q. Chen, J. Zhou, Q. Han, Y.H. Wang, Enantioselective recognition of man delic acid based on ␥-globulin modified glassy carbon electrode, Anal. Biochem. 421 (2011) 103–107. [9] G. Contini, P. Gorit, F. Roncit, N. Zema, S. Colonnat, M. Aschi, A. Palma, S. Turchinit, D. Catonet, A. Cricentit, T. Prosperit, Chirality transfer from a single chiral molecule to 2D superstructures in alaninol on the Cu (1 0 0) surface, Langmuir 27 (2011) 7410–7418. [10] W. Su, W. Zhang, S. Zhang, J. Fan, X. Yin, M. Luo, S. Choon Ng, A novel strategy for rapid real-time chiral discrimination of enantiomers using serum albumin functionalized QCM biosensor, Biosens. Bioelectron. 25 (2009) 488–492. [11] H. Xu, X.D. Yu, H.Y. Chen, Analysis of conformational change of human serum albumin using chiral capillary electrophoresis, J. Chromatogr. A 1055 (2004) 209–214. [12] D.M. Kanani, E. Komkova, T. Wong, A. Mika, R.F. Childs, R. Ghosh, Separation of human plasma proteins HSA and HIgG using high-capacity macroporous gel-filled membranes, Biochem. Eng. J. 35 (2007) 295–300. [13] M.R. Alexander, B. Alexander, A.L. Mustion, R. Spector, C.B. Wright, Therapeutic use of albumin: 2, J. Am. Med. Assoc. 247 (1982) 831–833. [14] C. Bertucci, E. Domenici, Reversible and covalent binding of drugs to human serum albumin: methodological approaches and physiological relevance, Curr. Med. Chem. 9 (2002) 1463–1481. [15] N.A. Pchelintsev, A. Vakurov, H.H. Hays, P.A. Millner, Thiols deposition onto the surface of glassy carbon electrodes mediated by electrical potential, Electrochim. Acta 56 (2011) 2696–2702. [16] J.C. Love, L.A. Estroff, J.K. Kriebel, R.G. Nuzzo, G.M. Whitesides, Self-assembled monolayers of thiolates on metals as a form of nanotechnology, Chem. Rev. 105 (2005) 1103–1170. [17] M. Matsunaga, T. Nakanishi, T. Asahi, T. Osaka, Effect of surface coverage of gold (1 1 1) electrode with cysteine on the chiral discrimination of dopa, Chirality 19 (2007) 295–299. [18] M. Matsunaga, T. Nakanishi, T. Asahi, T. Osaka, Highly enantioselective discrimination of amino acids using copper deposition on a gold electrode modified with homocysteine monolayer, Electrochem. Commun. 9 (2007) 725–728. [19] Y.J. Kang, J.W. Oh, Y.R. Kim, J.S. Kim, H. Kim, Chiral gold nanoparticlebased electrochemical sensor for enantioselective recognition of 3,4dihydroxyphenylalanine, Chem. Commun. 46 (2010) 5665–5667. [20] K. Yun, E. Kobatake, T. Haruyama, M.L. Laukkanen, K. Keinanen, M. Aizawa, Use of a quartz crystal microbalance to monitor immunoliposome–antigen interaction, Anal. Chem. 70 (1998) 260–264. [21] H. Bückner, S. Haasmann, M. Langer, T. Westhauser, R. Wittner, Liquid chromatographic determination of d- and l-amino acids by derivatization with o-phthaldialdehyde and chiral thiols: applications with reference to biosciences, J. Chromatogr. A 666 (1994) 259–273. [22] Z.D. Zhivkova, V.N. Russeva, Stereoselective binding of ketoprofen enantiomers to human serum albumin studied by high-performance liquid affinity chromatography, J. Chromatogr. B 714 (1998) 277–283. [23] R. Kaliszan, T.A. Noctor, I.W. Wainer, Stereochemical aspects of benzodiazepine binding to human serum albumin. II. Quantitative relationships between structure and enantioselective retention in high performance liquid affinity chromatography, Mol. Pharmacol. 42 (1992) 512–517. [24] J. Hermansson, Enantiomeric separation of drugs and related compounds based on their interaction with ␣-acid glycoprotein, Trends Anal. Chem. 8 (1989) 251–259.
Contents lists available at SciVerse ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
The selective adsorption of human serum albumin on N-isobutyryl-cysteine enantiomers modified chiral surfaces Qiao Chen, Juan Zhou, Qian Han, Yonghua Wang, Yingzi Fu ∗ Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
a r t i c l e
i n f o
Article history: Received 2 May 2012 Received in revised form 18 August 2012 Accepted 8 September 2012 Available online 13 September 2012 Keywords: N-isobutyryl-cysteine Human serum albumin Adsorption Amino acids Protein Sensors
a b s t r a c t The stereoselective interaction between human serum albumin (HSA) and N-isobutyryl-cysteine (NIBC) enantiomers was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical responses of the NIBC enantiomers modified surfaces were varied along with the inserted time and the concentration of HSA. The results showed that HSA molecules displayed different adsorption behaviors on NIBC enantiomers modified surfaces and preferred to be adsorbed on the l-NIBC modified surface. That is to say, l-NIBC modified surface had stronger interaction with HSA molecules than d-NIBC modified surface, suggesting that the molecular configuration of l-NIBC had matched better with HSA than the case of d-NIBC. The investigation of the interaction between protein and chiral molecules may not only help to understand the high selectivity of chirality in biosystems, but also provide a reference for studying chiral drugs. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Chiral recognition is one of the most fundamental and crucial properties of various natural systems, which plays a very important role in understanding the interactions of biological molecules [1,2]. Living systems contain large numbers of biological macromolecules, and most of them show natural enantioselectivity [3]. For example, cells and DNA can sense the surface chirality and exhibit different adhesion and activation behaviors on enantiomorphous surfaces [4–6], human serum albumin (HSA) modified indium tin oxide (ITO) electrodes can selectively adsorb tryptophan enantiomers [7] and ␥-gluoblin can selectively recognize mandelic acid enantiomers [8]. Therefore, the study on the interaction between biomacromolecules and chiral molecules is valuable and fascinating in understanding the properties of biomacromolecules and the high selectivity of chiral molecules in biosystems [9]. Serum albumin is the most abundant proteins in the circulatory system and maintains the normal plasma osmotic pressure [10]. HSA which contains 585 amino acid residues is 67,000 Da molecular mass and it is a valuable biomarker of many diseases and widely used in clinical treating of diseases [11–13]. At the same time, HSA plays an important role in the transport of drugs, metabolites, and endogenous ligands [14].
Thiol molecules modified surfaces have received steadily increasing attention [15,16]. Chiral thiol molecules have been widely studied and applied in chiral recognition [17]. For example, l-homocysteine modified electrode has been used to selectively recognize amino acids enantiomers in the presence of copper ions [18], and penicillamine enantiomers functionalized gold nanoparticles can enantioselectively recognize 3,4-dihydroxyphenylalanine [19]. Protein-based biosensor, prepared by the interaction between a self-assembled functional monolayer film and protein molecules, has wide applications [20]. N-isobutyryl-cysteine (NIBC) enantiomers, one of the derivatives of cysteine, have been reported to selectively recognize amino acids [21]. Here, the adsorption behaviors of HSA on NIBC enantiomers modified gold surfaces have been studied. To date, various methods have been developed to study the interaction between protein and chiral molecules [22–24]. In this work, the stereoselective interaction between HSA and N-isobutyryl-cysteine enantiomer modified chiral surfaces was investigated via electrochemical methods. The results shown that larger response signal was observed from the interaction between the l-NIBC modified gold surface and HSA molecules than the case of d-NIBC modified gold surface. 2. Experimental 2.1. Materials and methods
∗ Corresponding author. Tel.: +86 023 68252360; fax: +86 023 68253195. E-mail address: [email protected] (Y. Fu). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.09.008
Human serum albumin, N-isobutyryl-l(d)-cysteine (l- or dNIBC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA)
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Fig. 1. (A) Cyclic voltammograms and (B) electrochemical impedance spectroscopy of different electrodes in 5 mM [Fe(CN)6 ]4−/3− solution (pH 6.7) (a) l-NIBC-Au, (b) d-NIBC-Au, after (c) d-NIBC-Au and (d) l-NIBC-Au interacted with 0.1 mM HSA for 1 h.
and stored in refrigerator. Other chemical reagents were analytical grade and used without further purification. All aqueous solutions were prepared with double distilled water. All experiments were completed at room temperature (25 ± 0.5 ◦ C). 2.2. Apparatus Electrochemical impedance spectroscopy (EIS) measurements were performed on Model IM6e (ZAHNER Elektrick Co., Germany). Cyclic voltammetry (CV) measurements were performed on a CHI 660D electrochemistry workstation (Shanghai Chenhua Instruments Co., China). Electrochemical experiments were performed in an unstirred electrochemical cell. A working electrode (the modified electrode or bare electrode), a platinum wire auxiliary electrode and a saturated calomel electrode (SCE) constituted the standard three-electrode system. 2.3. The formation of chiral surface The gold electrodes (Ø = 4 mm) were polished on microcloth carefully with 1.0, 0.3 and 0.05 m alumina slurries, respectively. Following that, the electrodes were sonicated in double distilled water, ethanol and double distilled water each for 5 min. Then, the cleaned electrodes were immediately immersed in 5 mM l- or dNIBC ethanol solution overnight at 4 ◦ C. The l- or d-NIBC modified chiral surfaces (l-/d-NIBC-Au) were obtained. 2.4. Experimental measurements The scan of CV was taken from −0.2 to 0.6 V (vs. SCE) at 100 mV s−1 in 5 mM [Fe(CN)6 ]4−/3− (0.1 M PBS, pH 6.73). The frequency range of EIS measurements from 0.1 to 105 Hz in a given open circuit voltage, amplitude was 0.22 V. The enantioselective detection was based on the difference of current response (I = I0 − I1 ) before and after the NIBC enantiomers modified surfaces immersed in HSA solution. The peak currents of the NIBC enantiomers modified electrodes were regarded as I0 , after it immersed in HSA solution, the peak current was regarded as I1.
3. Results and discussion 3.1. Characterization of the NIBC enantiomers modified surfaces As an effective and convenient method for investigating the feature of the electrode surface, CV was used to study the characterization of NIBC enantiomers modified surfaces. The bare gold electrode showed well redox peaks. A large decrease of peak current was observed after the gold electrode immersed in ethanol solutions of d- or l-NIBC overnight, suggesting d- or l-NIBC has successfully self-assembled on gold electrodes. 3.2. The selective adsorption of HSA on NIBC enantiomers modified surfaces 3.2.1. The adsorption characterization of HSA As shown in Fig. 1A, the electrochemical responses of lNIBC-Au (curve a) and d-NIBC-Au (curve b) were almost the same. The peak currents were decreased after l-NIBC and dNIBC modified surfaces interacted with 0.1 mM HSA about 60 min, hinting the adsorbed HSA insulated the interfacial electron transfer. However, the decreasing of peak current of l-NIBCAu (curve d) was larger than d-NIBC-Au (curve c), implying the adsorbed HSA molecules on l-NIBC-Au surface was higher. The adsorption of HSA has been greatly influenced by the surface chirality, and the interaction between HSA and l-NIBC-Au was larger, suggesting HSA prefers to adsorb on l-NIBC-Au surface. 3.2.2. EIS characterization of the adsorption of HSA As shown in Fig. 1B, the impedance spectra include a semicircle portion and a linear portion. The semicircle diameter equals the interfacial electron transfer resistance (Ret ). The l-NIBC-Au (curve a) and d-NIBC-Au (curve b) surfaces had almost the same Ret . After the modified surfaces inserted in HSA solution for 60 min, a large increment of resistance was observed, indicating that HSA has adsorbed on NIBC enantiomers modified surfaces and obstructed
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3.2.3. The influence of interaction time between NIBC enantiomers and HSA The influence of interaction time between NIBC enantiomers and HSA from 0 to 100 min on the electrochemical response was shown in Fig. 2A. It can be seen that peak currents of NIBC enantiomers modified surfaces were decreased with the prolongation of interaction time because of a longer interaction time contributed to more adsorption of HSA on a chiral surface and the change of peak current of HSA/l-NIBC-Au (curve a) were larger than HSA/d-NIBCAu (curve b). The amperometric responses leveled off after 60 min for two surfaces. So, 60 min was adopted as incubation time in this work.
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Fig. 2. (A) The time dependence of the adsorption of HSA on (a) l-NIBC and (b) d-NIBC modified surface. (B) The changes of peak current along with the varying concentrations of HSA (a) l-NIBC-Au, (b) d-NIBC-Au, and (c) the difference between l-NIBC-Au and d-NIBC-Au.
the electron transfer of the ferricyanide probe. It was noted that the resistance of HSA/l-NIBC-Au (curve d) was larger than HSA/d-NIBCAu (curve c), implying more HSA molecules adsorbed on l-NIBC modified surfaces and hindered the electron transfer. This result is very consistent with the CV.
3.2.4. Electrochemical response of the modified electrodes with the varying concentrations of HSA The electrochemical response of the l-NIBC and d-NIBC modified electrodes with varying concentrations of HSA were also studied by measuring the change of peak currents. Fig. 2B shows a comparison for the change of peak currents of HSA adsorbed on l-NIBC and d-NIBC surfaces for different HSA concentrations. After NIBC enantiomers modified electrodes inserted in HSA solution for 60 min, the change of peak currents of l-NIBC-Au (Fig. 2Ba) were larger than the d-NIBC-Au (Fig. 2Bb). Fig. 2Bc was the relative difference of response currents between l-NIBC-Au and d-NIBC-Au. It can be seen that the difference of electrochemical signal can be observed even at 0.005 mg mL−1 . It is further evidence that the HSA molecule was selectively adsorbed on NIBC enantiomers modified surfaces. All of those results showed that a larger electrochemical response signal was obtained from l-NIBC-Au surface than the case of d-NIBC-Au under the same conditions. This effect may be caused by a higher amount of HSA molecule adsorbed on the l-NIBC-Au than d-NIBC-Au, which resulted in a greater obstacle for electron transfer between the redox probe and the electrode surfaces. That is to say, HSA could sense the surface chirality and show different adsorption capacities on NIBC enantiomers modified surfaces. The reason for the enantioselective adsorption of HSA molecule on NIBC enantiomers modified surfaces is discussed as following: The existence of COOH, NH and carboxyl groups, which have different spatial arrangements, induce that NIBC enantiomers show different orientations. NIBC enantiomers have different abilities to form effective hydrogen bonding, hydrophobic interactions and specific electrostatic forces with HSA. Namely, the surface chirality may be
Fig. 3. The schematic representation of l-NIBC-Au (a) and d-NIBC-Au (b) interacting with HSA.
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recognized through special chemical interaction between the chiral moieties of the surface and HSA, which may release different signals. From the results of above experiments, it could be reasonably speculated that the molecular configuration of l-NIBC may match well with the HSA and can bind with the major HSA through hydrogen bonding, hydrophobic interactions and the specific electrostatic forces interaction, which results in a higher adsorption of HSA (Fig. 3a), while a weak adsorption on the d-NIBC surface for the steric hindrance (Fig. 3b). 4. Conclusions In conclusion, the adsorption of HSA on the NIBC enantiomers modified surfaces was investigated by electrochemical methods. All of the results showed that a larger electrochemical response signal was obtained from l-NIBC-Au than d-NIBC-Au, and the surface chirality has great influence on the adsorption of HSA. The molecular configurations of l-NIBC and d-NIBC greatly influence the formation of hydrogen bonding, hydrophobic interactions and the specific electrostatic forces interactions between NIBC enantiomers and HSA molecules. The detailed analysis of the interaction between HSA and NIBC enantiomers is helpful for knowing the properties of protein. This work gives us the great opportunity to get to know the reaction between protein and chiral medicine, and provides us a reference for studying chiral drugs. Acknowledgment This work was supported by the National Natural Science Foundation of China No. 20972128 References [1] X.X. Zhang, J.S. Bradshaw, R.M. Izatt, Enantiomeric recognition of amine compounds by chiral macrocyclic receptors, Chem. Rev. 97 (1997) 3313–3362. [2] Z.B. Li, J. Lin, Y.C. Qin, L. Pu, Enantioselective fluorescent recognition of a soluble “supported” chiral acid: toward a new method for chiral catalyst screening, Org. Lett. 7 (2005) 3441–3444. [3] N.M. Maier, P. Franco, W. Lindner, Separation of enantiomers: needs, challenges, perspectives, J. Chromatogr. A 906 (2001) 30–33. [4] K.J. Tang, H. Gan, Y. Li, L.F. Chi, T.L. Sun, H. Fuchs, Stereoselective interaction between DNA and chiral surfaces, J. Am. Chem. Soc. 130 (2008) 11284–11285. [5] H. Gan, K.J. Tang, T.L. Sun, M. Hirtz, Y. Li, L.F. Chi, Selective adsorption of DNA on chiral surfaces: supercoiled or relaxed conformation, Angew. Chem. 121 (2009) 5386–5390. [6] T.L. Sun, D. Sun, K. Rhemann, L.F. Chi, H. Fuchs, Stereospecific interaction between immune cells and chiral surfaces, J. Am. Chem. Soc. 129 (2007) 1496–1497.
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