Full Paper

Comparative Analysis of Proline Enantiomer in Chiral Recognitionof Biological Macromolecule in Immunoassay

Min Chen,a Yingzi Fu,a* Xin Cui,b Lilan Wang,a Miaosi Lib

a Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and ChemicalEngineering, Southwest University, Chongqing 400715, P. R. China

*e-mail: fyzc@swu.edu.cnb College of Chemistry, Sichuan University, Chengdu 610064, P. R. China

Received: April 6, 2009Accepted: June 26, 2009

AbstractAn interesting phenomenon of the stereoselective interaction between biological macromolecule and amino acidenantiomorphous was described. Firstly, proline enantiomer (L- and D-proline) was assembled on the glassy carbonelectrode surface. Then carcinoembryonic antibody (anti-CEA) was loaded to the enantiomer surface, electro-chemical impedance spectroscopy (EIS) was used to monitor the growth of amino acid film. The assembly process wascharacterized by cyclic voltammetry (CV) and atomic force microscopy (AFM) was applied to image the chiral films.Finally, the developed electrodes had interacted with carcinoembryonic antigen (CEA) in varying concentrationsolutions. The AFM and amperometric results revealed that the proline enantioner had the chiral recognition functionto antibody.

Keywords: Comparative analysis, Proline, Chiral recognition, Electrochemical immunoassay, Immunoassays

DOI: 10.1002/elan.200904688

1. Introduction

The relationship of recognition between amino acids andproteins has received considerable interest in recent yearsbecause of the importance in separation and sensing ofenantiomers, and also influencing the biocompatibility ofmaterials devices and the pathobiologies [1 – 4]. Hofstetter[5 – 9] and Kong groups [10] have used antibodies, bovineserum albumin (BSA) as chiral selectors in electrochemicalchiral discrimination of chiral amino acids based on chiralinteraction respectively. In addition, chiral amino acids asthe major components of proteins have abilities to formchiral membranes, and exhibit stereospecific interactionwith biological macromolecules [11 – 17]. The phenomenanot only give interesting insight for researching a hopefulbiomaterial, but also help to understand the origin ofstereoselectivity in pharmaceutical systems and clinic diag-noses. This prompted us to apply chiral amino acids as onekind material to improve the binding selection to targetbiomolecules in the immunoassays, which is an attractivesubject in clinical diagnosis [18, 19], food industry [20] andenvironmental analysis [21]. Proline is an important unit inliving systems, the L-proline (L) is a neuronal modulator ortransmitter candidate in the central nervous system[22], while the amount of D-proline (D) in the tissue isextremely small and is believed to relate to the renaldisorder [23]. Furthermore, proline is different from theother amino acids for its rigid chiral structure, so as to meetthe geometrical requirements for chiral recognition more

easily than do conformationally unstable structures [24 –27].

Various methods have been developed for researchingchiral amino acids, including chromatography [28], nuclearmagnetic resonance (NMR) [29], molecularly imprintedpolymer (MIP) [30] etc. And immunosensors as the simpletools for monitoring immunochemical substances with highsensitivity and selectivity are shown to be of great interestfor their potential utility in the analysis of chiral recognition[9 – 10]. Electrochemical immunoassay is meet to studychiral recognition for its high sensitivity, low cost, low powerrequirements and high compatibility with advanced micro-machining technologies [31 – 33].

In the present work, electrochemical immunoassay ofCEA based on the stereoselectivity of proline enantiomerwas investigated. Proline enantiomers and carcinoembryon-ic antibody/antigen (a large chiral biopolymer and impor-tant tumor marker [34]) were selected as model systems. Thechiral recognition of L- and D-proline to anti-CEA wasdetected by AFM and CV.

2. Experimental

2.1. Reagents and Apparatus

L-Proline (L), D-proline (D), carcinoembryonic antibodyand antigen (anti-CEA, CEA) were provided by ShanghaiKehua Biotechnol. Co. (Shanghai, China). All other re-

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agents were of analytical grade. Phosphate buffer solution(PBS, 0.1 M, pH 6.97) was prepared with 0.1 M KH2PO4,0.1 M Na2HPO4 which containing 0.1 M KCl. Doubledistilled water was used for all experiments.

Eelectrochemical impedance spectroscopy (EIS) andcyclic voltammetry (CV) were carried out on a PAR-STAT2273 electrochemical workstation (Advanced Mea-surement Tech., Inc., USA) in a three-electrode electro-chemical cell consisting of bare or modified glassy carbonelectrode (GCE, ˘¼ 3 mm) as a working electrode, aplatinum wire and a saturated calomel electrode (SCE) asthe counter and the reference electrode. Atomic forcemicroscopy (AFM) images were obtained from a scanningprobe microscope using the gold quartz slides (Vecco,Alpharetta, GA).

2.2. Preparation of Working Electrodes

The GCEs were polished successively with 1.0, 0.3 and0.05 mm alumina slurry, sonicated in ethanol and water for3 min each, then were immersed in a solution containing2.5% K2Cr2O7 and 20% H2SO4 and held atþ1.5 V for 1 min.Following that, the electrodes were immersed into 10 mM Lor D solutions containing 0.01 M HCl for 36 h (D(L)/GCE).After thorough rinsing, the electrodes were incubated in40 mL anti-CEA solution overnight at 4 8C (anti-CEA/D(L)/

GCE). At last, the modified electrodes were incubated in0.25 wt% BSA for 1 h at 37 8C to block the remaining activegroups and eliminate non-specific adsorption (BSA/anti-CEA/D(L)/GCE). The obtained modified electrodes wereused for the following experiments and stored at 4 8C whennot used. The stepwise fabrication process of modifiedelectrode was displayed in Scheme 1.

2.3. Experimental Measurements

The swept potential of CV was from �0.2 to þ0.6 V at50 mV/s scan rate. EIS was measured in the frequency rangefrom 0.01 Hz to 100 kHz at 220 mV. CV and EIS were in a5 mL unstirred electrochemical cell containing 2.5 mM[Fe(CN)6]

4�/3� mixture solution (0.1 M PBS, pH 6.97) atroom temperature.

3. Results and Discussion

3.1. Electrochemical Characteristics of Chiral Films

The cyclic voltammograms (CVs) of the developed enan-tiomer electrodes were similar (Fig. 1 A and B). Well-

Scheme 1. The stepwise fabrication processes of the developedelectrodes.

Fig. 1. CVs of the modified electrodes in 2.5 mM [Fe(CN)6]4�/3�

(0.1 M PBS, pH 6.97): (a) bare GCE; (b) L/GCE (A), D/GCE (B);(c) anti-CEA/L/GCE (A), anti-CEA/D/GCE (B); (d) BSA/ anti-CEA/L/GCE (A), BSA/anti-CEA/D/GCE (B) at 50 mV/s scanrate.

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defined and stable redox peaks of the bare GCE, indicatingthe surfaces were clean and smooth (Fig. 1a). It wasobserved that the redox peaks turned low and the potentialbecame broad (Fig. 1b) at the proline-modified electrodes,suggesting that D or L could be easily adsorbed onto the

surface of GCE, which was rich in hydroxyl and carboxylgroups after the physical and chemical pretreatment [35],via H-bond in the acidic condition to form a chiral film [13].After dipping D(L)/GCE into anti-CEA solution, theobvious decrease in the peak current and increase in the

Fig. 2. AFM images of (a) bare, (b) D, (c) L, (d) anti-CEA/D, (e) anti-CEA/L gold quartz slide substrates surface.

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peak-to-peak separation were observed (Fig. 1c). It showedthat anti-CEA acted as an inert electron layer and hinderedthe diffusion of ferricyanide toward the electrode surface.At last, BSA was employed to block the possible remainingactivity sites (Fig. 1d), the peak currents decreased a little.

3.2. AFM Characterization of Chiral Films

AFM is a versatile technique to image the biomoleculessurfaces, that has been extensively utilized to study theproperties of biomolecules under different circumstances orthe interaction between biomolecules under the differentcircumstances [37, 38]. In this experiment, AFM was used toobtain the dynamic images of different chiral films.

The AFM image of the bare gold substrate (Fig. 2a) wassmooth, while covered by proline the image became rough.Compared the AFM image of the enantiomer, a three-dimensional �network� topography was observed clearly onD (Fig. 2b), while the �roots� shape was seen definitely on L(Fig. 2c), implying that D and L had different self-assemblymode due to their different configurations under the sameconditions. When anti-CEA compactly loaded on D or L,the surface topography varied dense and uniform (Fig. 2d)or only became �fat�, that suggested D bind better with anti-CEA than L.

On the basis of AFM results, we noticed that the D or Lcould absorb onto the substrate surface by differentassembly means, while assembling with constant anti-CEAon the enantiomer surfaces, the morphologies had remark-able differences. Since anti-CEA is a biomolecule withdistinct three-dimensional structure and possible to assignspecific electrostatic or hydrogen bonding sites [39], it couldcontact with the chiral proline films to form H-bondaccording to the spatial arrangements of the existence ofthe �OH, �NH- and carboxyl groups in different mode(Scheme 1b and b’), that is to say, the proline enantiomerhad chiral recognition function to antibody.

3.3. Chiral Recognition via Immunoassay

3.3.1. Selection of Experimental Condition

Firstly, the pullulation time of amino acid films coated onGCEs was studied. The GCEs were immersed in 10 mM Dor L solution 4 – 72 h, respectively. It was found that thesemicircles of the Nyquist lines had grown with time at first36 h, then increased slowly and overlapped (Fig. 3). By acombination of the experiment period, we selected 36 h asthe pullulation time for preparing chiral amino acid film.

Secondly, the incubation time was examined from 5 to25 min at 37 8C. The peak current of the immunoelectrodesto 20 ng/mL CEA decreased with the incubation time andleveled off after 20 min (Fig. 4). Thus, 20 min was chosen asthe incubation time in the following experiments.

Finally, to simplify the analytical process and keep activityof biological molecules, 37 8C and pH 6.97 which were near

physiological condition were used as the experimentalconditions of immunochemical incubation.

Fig. 3. EIS of D/GCE (A) and L/GCE (B) immersing in prolinesolutions at different pullulation time: (a) 0, (b) 4, (c) 12, (d) 24,(e) 36, (f) 48, (g) 60, (f) 72 h.

Fig. 4. Effect of the incubation time on the response of D (a) andL (b) modified immunoelectrodes at 37 8C.

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3.3.2. The Comparison of Amperometric Response

To further compare the chiral recognition of the developedimmunoelectrodes, the antigen-antibody reaction had beenperformed. Under the same experimental treatment proce-dure, the peak currents of D (Fig. 5a) and L (Fig. 5b)modified immunoelectrodes decreased with increasingCEA concentration. The percentage of the peak currentdecrease was proportional to the CEA concentration in tworanges with different slope rate (S) and correlation coef-ficient (R): one from 0.5 to 10.0 ng/mL (SD¼ 0.1672,SL¼0.0881; RD¼ 0.9763, RL¼0.9921), the other from 10.0to 80.0 ng/mL (SD¼ 0.0188, SL¼0.0127; RD¼ 0.9932,RL¼0.9901). The detection limits were 0.17 ng/mL (curvea) and 0.29 ng/mL (curve b) at 3s (where s was the standarddeviation of a blank solution, n¼ 10). The inter-assayprecision of CEA was estimated using three immunoelectr-odes for every CEA concentration. The coefficient ofvariation of CV of L modified immunoelectrodes for inter-assay on this method was 3.8% at 5 ng mL�1, 2.9% at 60 ngmL�1, and it of D modified immunoelectrodes was 3.3% at5 ng mL�1, 3.0% at 60 ng mL�1, indicating good detectionprecision and reproducibility of the immunoelectrodes.

The experimental results revealed that the D modifiedimmunoelectrodes had stronger amperometric responsethan L, further demonstrated that D had a better recog-nition function to anti-CEA than L, that was consistent withthe AFM observation. The behaviors of anti-CEA weregreatly influenced by L or D, the surface on D mightstrengthen and promote the antibody adhesion. It impliedthat designing chiral surfaces of amino acids might bring anew direction for biomaterials, which was potentiallycomplementary to improve the biocompatibility. Mean-while the interesting phenomenon that D combined wellwith anti-CEA rather L might intimate the origin ofstereoselectivity in pharmaceutical systems and clinic diag-noses.

4. Conclusions

In this paper, the preliminary study based on comparativeanalysis of L-proline and D-proline forming chiral mem-branes in recognition of anti-CEA in immunoassay, wasproposed for bringing novel application insights for theproline and other chiral molecules to improve the biocom-patibility. The AFM images exhibited that the L and D haddifferent interaction mode with anti-CEA, and the chiralrecognition was further investigated by the different am-perometric response. Hence, the amino acid enantiomerused as a kind of modified material in immunoassay mayassist or regulate chiral function in biomaterial and phar-maceutical system, help to understand the origin of stereo-selectivity in pharmaceutical systems and clinic diagnoses.

5. Acknowledgements

This project was supported by The National Natural ScienceFoundation of China (20732003) and The Foundation forDoctoral Research (SWUB 2006008) of Southwest Univer-sity of China.

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