Electrochemical biosensors for monitoring the recognition of glycoprotein–lectin interactions

Analytica Chimica Acta 588 (2007) 292–296

Electrochemical biosensors for monitoring the recognition of glycoprotein–lectin interactions Omowunmi A. Sadik a,∗ , Fei Yan a a

Department of Chemistry, State University of New York at Binghamton, P.O. Box 6000, Binghamton, NY 13902, United States

Received 27 September 2006; received in revised form 2 February 2007; accepted 13 February 2007 Available online 23 February 2007

Abstract Despite the wide applicability and specificity of lectins to carbohydrate moieties, there are few lectin specific biosensors. This is attributed to the difficulty in defining the relevant experimental parameters to measure for sensing. We hereby describe the development of direct and indirect electrochemical sensors to determine the exact trace amounts of probarley lectin (ProBL) and its conversion product wheat germ agglutinin (WGA). In addition to WGA, the antigens (ProBL) employed in this study were over expressed in bacteria, isolated from protein bodies, and purified using immobilized N-acetylglusamine in order to obtain correctly folded active lectins. The amperometric immunosensor uses cell lines producing monoclonal antibody (mAB) to the pro-region of ProBL over expressed from Escherichia coli. The efficacy and sensing characteristics of the lectin were optimized using monoclonal antibody to WGA and the resulting sensor was found to detect only ProBL in the linear range 10−3 –102 ␮g mL−1 and a detection limit of 10−3 ␮g mL−1 . © 2007 Elsevier B.V. All rights reserved. Keywords: Lectins; Probarley lectin; Electrochemical sensors; Affinity

1. Introduction The specificity of lectins to carbohydrate moieties should enable the development of specific sensors for sugars. However, defining the relevant parameters to measure has prevented the development of wide-spread lectin-based biosensors. Lectins are proteins or glycoproteins of plant, animal, or bacterial origin that bind to cell surfaces through specific carbohydratecontaining receptor sites. They have been extensively used to study the nature of specific carbohydrates. Glycomolecules play critical roles in numerous physiological and pathological reactions. Oligosaccharides are major structural components of many cells surface and secreted proteins. Majority of glycoproteins, including major drugs for heart disease, cancer and diabetes rely on correct glycosylation for optimal performance [1,2]. Consequently, a reliable method for assessing glycosylation may assist in rapid identification of therapeutic candidate. Although the determination of the identity of proteins is readily

∗

Corresponding author. Tel.: +1 607 777 4132; fax: +1 607 777 4478. E-mail address: [email protected] (O.A. Sadik).

0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.02.046

assessable using mass spectrometry, there are no consistent methods for characterizing lectins activity. Lectins are polyvalent, i.e. each lectin molecule has at least two carbohydrate binding sites to allow cross linking between cells (by combining with sugars on their surfaces) or between sugar containing macromolecules. Another characteristic property of lectins is that they agglutinate cells or precipitate polysaccharides and glycoproteins, a property that could be used to develop novel assay concepts. Lectins are classified into a small number of specificity groups (d-mannose, d-galactose, d-N-acetylglucosamine, d-N-acetylgalactosamine, l-fructose and d-N-acetylneuraminic acid) according to the monosaccharide, which is the inhibitor of the agglutination of erythrocyte precipitation of carbohydrate polymers, by the lectin [3]. Although lectins have been known for nearly a century, they have recently become the focus of intense interest [3]. There are many reasons for the current interest in lectins. Prominent among these is their usefulness in detecting and studying carbohydrates in solution and cell surfaces. In addition, investigations of the interaction of lectins with carbohydrates are providing information on the precise molecular details of the reactions between

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proteins and carbohydrates in general, as well as between cells [4]. Probarley lectin is a protein found in plants that differs from its conversion product, Barley lectin (BL) through a carboxyterminal propeptide (CTPP) of 15-amino acid sequence [5]. It has been shown that only the peptide portion of the CTPP is needed for correct and efficient targeting of ProBL in plants [6]. BL exhibits specificity for N-acetyl-d-glucosamine like its analogue wheat germ agglutinin (WGA). BL and WGA share strong sequence identity (about 95%), for which the biochemical properties and crystal structure have been extensively characterized [5,6]. Despite their wide applicability and specificity to carbohydrate moieties, there are few lectin specific sensors [7,8]. This is attributed to the difficulty in defining the relevant experimental parameters to measure for sensing. We propose the use of electrochemistry as a tool for studying the affinity of glycoproteins with lectins in real time. Signal generation for bioaffinity recognition must provide reversible and continuous monitoring without the use of chemical regeneration steps. In this context, we have studied the theoretical and experimental approaches for monitoring the interfacial biomolecular reaction, specifically antibody–antigen (Ab–Ag) recognition using electrochemical techniques. These studies were presented in a series of systematic reports focusing on the mechanistic, analytical and detection aspects [9–13]. Since these interactions are based on the affinity of the antigen for the corresponding antigen, they may be applied to monitor the interactions of other bioaffinity surfaces such as that of lectins and carbohydrates. We hereby describe the development of direct and indirect strategies to determine the exact trace amounts of ProBL. The efficacy and sensing characteristics of such a sensor using monoclonal antibody to WGA or CTPP was optimized and the resulting sensor was found to detect only ProBL with a detection limit of 10−3 ␮g mL−1 .

2. Materials 2.1. Chemicals Lectin from Triticum vulgaris (wheat germ, WGA, Product No. L-9640), its corresponding antibody (Product No. T-4144. Developed in Rabbit, fractionated Antiserum), Horseradish peroxidase (HRP) (No. P-6782) and Protein G were obtained from Sigma. 3-Aminopropyltriethoxysilane (APTS) was from Aldrich Chemicals, Milwaukee, WI. All other chemicals were of the highest purity and were obtained from Pierce.

2.2. Stock solutions All solutions were prepared from deionized water (Nanopure). Phosphate buffered saline (PBS buffer, pH 7.0) was made by dissolving 8.0 g NaCl, 0.2 g KH2 PO4 , 1.15 g Na2 HPO4 ·H2 O, 0.2 g KCl and 0.2 g NaN3 in 1 L deionized water.

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2.3. Monoclonal antibody: isolation and characterization Besides WGA, the antigen used in this study was a bacterially-produced probarley lectin (ProBL) as described by Schroeder and co-workers [5,6,14]. The protein was over expressed in bacteria, isolated from protein bodies, denatured and refolded as described by Tsuji et al. [15] and then purified using immobilized N-acetylglucosamine (Sigma Chemicals) in order to obtain correctly folded active lectin. The cell line producing a monoclonal antibody to the pro-region of ProBL was isolated by Schroeder, and its characterization was described by Runeberg-Roos and co-workers [5,16]. The antibody was purified from the culture supernate of these cells using an immobilized Protein G disc following the manufacturer’s (Sigma Chemical) instructions. The proteins were quantified using the typical bicinchoninic protein assay [17] (Sigma® Chemical) and the identity and antigenicity were confirmed using western blots [18]. The monoclonal antibody horseradish peroxidase (mAb–HRP) conjugate was synthesized using the common periodate procedure as described previously [19,20] and was stored frozen as solution in 50 mM phosphate, pH 7.0. 2.4. Design of immunosensor for lectin Platinum metal surface was activated by silanization with APTS according to a previous report [21]. We followed a different approach, briefly, the electrodes were first cleansed in acetone and incubated for 1.5 h in 5% APTS solution. After drying for 1 h at 100 ◦ C, the remaining free APTS was removed by careful washing with acetone. Lectin immobilization was obtained directly from the APTS molecule. 2.5. Measurement of antibody–antigen binding using amperometric measurements A Princeton Applied Research potentiostat (EG&G PAR) equipped with EG&G research electrochemistry software Model 270/250 was used for data collection. All measurements were carried out in a one-compartment electrochemical cell comprised of the platinum electrode as a working electrode, a Ag/AgCl (3 M) electrode as reference electrode and a platinum coil as the auxiliary electrode. All potentials are reported with respect to Ag/AgCl (3 M) reference electrode. After incubating in HRP–Ab for 30 min and substrate solutions (6 mM HQ, 1.62 ␮M H2 O2, PBS buffer with pH 7.0)] for at least 45 min, the working potential (−300 mV versus Ag/AgCl reference electrode) was applied to the sensor and the pseudo-steady current at 1 min was read and used for evaluations [22–24]. In a typical measurement, the electrodes were placed in a BAS cell (5 mL), which were connected to the electrochemical analyzer. Before each test, the Pt working electrode was polished with alumina, sonicated for 30 min in a 0.5 mol L−1 H2 SO4 solution and washed with distilled water. The state of the electrode surface was checked before each experiment by recording a cyclic voltammogram in 0.1 mol L−1 phosphate buffer solution at pH 7.0. Cyclic voltammetric studies for ProBL and other lectin derivatives were registered in 0.1 M phosphate buffer solution

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at pH 7.0 (PBS). Cyclic voltammetric experiments were carried out between −500 and 100 mV versus Ag/AgCl reference electrode. −300 mV was selected as the optimized potential and to prevent interferences from ambient oxygen, antibody–antigen binding reaction was carried out in saturated oxygen solutions. 3. Results and discussion Two functions of lectins have attracted most attention [2]: (a) as mediators of symbiosis between nitrogen fixing microorganisms, primarily rhizobia, and plants; and (b) in protection of plants against phytopathogens. However, experimental evidence in support of such roles is still scanty at best. Whatever role(s) lectin may play, it is important to determine the localization and distribution in various parts of the plant, as temporal changes in their distribution may help to formulate ideas about the functions of lectins. Hence, the goal of this work is to develop a screening method for ProBL using electrochemical technique. 3.1. Immunoreaction The main principle of the immunosensor follows the reaction of peroxide with hydroquinone in the presence of the enzyme peroxidase: Peroxidase (POD) is widely used as an enzyme label in electrochemical biosensors for selective detection of hydrogen peroxide (H2 O2 ) [21–24]. The hydroquinone/quinoline (HQ/Q) redox pair serves as a mediator for transferring electrons from the electrode to peroxidase. In the presence of H2 O2 and HQ, POD activity can be then determined amperometrically, wherever POD exists as a free form or in the form of an antigen/mAb–POD conjugate complex (Scheme 1). Hydroquinone exhibits a fast reaction rate with POD characterized by kinetic rate constant of 3 × 106 [25]. The concentration of the quinone is proportional to the amount of peroxide and can be detected amperometrically at −300 mV versus Ag/AgCl reference electrode.

Fig. 1. Cyclic voltammogram of hydroquinone at different concentrations of H2 O2 : (a) 0.096 ␮M; (b) 0.19 ␮M; (c) 0.28 ␮M; (d) 0.37 ␮M; potential sweeping from −350 mV to 100 mV, scan rate = 50 mV s−1 .

provide good responses (Fig. 2(a)). All subsequent amperometric measurements were thus conducted using the optimized potential. The optimal concentration of H2 O2 was selected by comparing the magnitude of signals using the same amount of HQ and applied potential (Fig. 2(b)). 1.62 ␮M was chosen for further experiments. 3.3. Amperometric antibody-monolayer immunosensors The concept of an amperometric antibody sensor is outlined in Fig. 3(a). Similar to antigen-monolayer immunosensor [26,27], interaction of the antibody-functionalized electrode with the respective antigen results in the association of the antigen to the monolayer and the insulation of the electrode

3.2. Optimization Fig. 1 shows the cyclic voltammograms of hydroquinone in the presence of variable concentrations of hydrogen peroxide. It was demonstrated that well-defined redox peaks resulting from the electron transfer of HQ is proportional to the concentrations of H2 O2 in the solution. Thus, it was possible to determine the POD activity by using HQ and H2 O2 as the substrate solution. The dependence of signal to applied potential in the presence of H2 O2 shows that a potential of −300 mV was sufficient to

Scheme 1. Schematic representation of the electrochemical determination of glycoprotein–lectin interactions via measurement of mAb–POD activity.

Fig. 2. Optimization of operating potential for amperometric measurement. WGA modified Pt incubated in HRP–mAb solution (10 ␮g mL−1 ) for 30 min; substrate solution [6 ␮M HQ, 1.62 mM H2 O2, PBS buffer (pH 7.0)] 30 min; (b) Optimization of H2 O2 concentration.

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Fig. 5. Cyclic voltammograms of antibody-functionalized electrode (a) in the presence of [K3 Fe(CN)6 ] = 1 mM solution without lectin; (b) upon addition of 0.225 ␮g ␮L−1 extract; (c) upon addition of 1.02 ␮g ␮L−1 extract and 0.034 ␮g ␮L−1 ProBL.

Fig. 3. Affinity interface and (a) direct and (b) competitive immunosensor reactions for lectins.

toward the redox probe [28,29]. The extent of electrode insulation depends on the antigen concentration in the analyzed solution and time of incubation of the monolayer with the antigen solution. Thus, the decrease in amperometric response of the antibody monolayer electrode provides a quantitative measure to the antigen concentration upon interaction with the respective antigen solution at a fixed time. Fig. 4 shows the decrease of the amperometric responses upon treatment with ProBL standards at variable concentrations (0.1–20 ␮g mL−1 ). The response saturated after a higher concentration than 10 ␮g mL−1 , given the same amount of antibody immobilized on the platinum surface. The statistical data obtained for ProBL calibration using this

Fig. 4. Calibration curve for the amperometric responses of the anti-ProBLfunctionalized electrode after treatment with different concentrations of analytes including ProBL.

approach is y = 0.0193X2 − 0.7718X + 13.96 with a correlation coefficient of 0.9922. Fig. 5 shows the cyclic voltammogram of the antibody monolayer in the presence of K4 Fe(CN)6 after treatment with plant extract or extract ProBL mixture. The electrical communication between the redox probe and the electrode decreases and becomes irreversible. This is attributed to the association of the bulky ProBL to the antibody-functionalized electrode, which perturbs the electrical contact and electron transfer rate between the K4 Fe(CN)6 and the electrode surface. The difference between the amperometric signals at the antibody electrode (A), and that of the antibody electrode treated with variable amounts of extract (including ProBL), indicates the extent of the electrode insulation. The current value decreases as the amount of ProBL present in the analyte samples. Using these optimized conditions, ProBL concentrations as low as 0.002 ␮g mL−1 was detected. 3.4. Amperometric competitive immunosensors The concept of an amperometric competitive sensor is shown in Fig. 3(b). The analyte from the sample competes with immobilized analyte for binding to labeled antibody molecules. A certain amount of Lectin (i.e. WGA) was immobilized directly onto the platinum electrode. Free WGA in the bulk solution competes with the immobilized WGA for a limited amount of POD–mAb conjugates inside the cell. After a preset incubation period, the electrode was washed, and the substrate solution is added and POD activity is quantified. The measured signal is directly proportional to the amount of immunocomplexes WGA–(mAb–POD) remaining in the cell, and is indirectly proportional to the concentration of WGA in the sample. Typically, a bifunctional crosslinking reagent such as glutaraldehyde is used to achieve a better stability of adsorbed proteins [19,20]. It was reported that the best response could be achieved without adding any spacer molecules, probably because the spacer molecules might block the electrochemically active surface area of the electrode. Although it was previ-

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more preferable to ensure a good reproducibility of different trials. Acknowledgments The authors acknowledge the following agencies for financial supports: NYS Great Lakes Protection Fund, NYS-Center for Advanced Technology through the IEEC; National Science Foundation, US-Environmental Protection Agency. The authors also thank Dr. Susannah Gal from the Department of Biological Science, SUNY-Binghamton for supplying the WGA antigen used in this work. Fig. 6. Calibration curve for WGA determination. Conditions see Materials Section. After 30 min incubation with substrate, the potential of −300 mV was applied and current was recorded for 1 min.

ously shown that measurements using platinum electrode on the screen-printed strip were complicated by the time required to stabilize the background currents, we found that good responses could be obtained by using normal platinum electrodes. Variation is attributed to the mechanical and electrical differences between the screen-printed strip and pure metal electrodes. 3.5. Characterization of competitive immunosensor for lectin The calibration curve (Fig. 6) for free WGA was constructed by first immobilizing WGA (0.50 mg mL−1 ) on the platinum electrode at incubation time of 30 min. As the amount of free WGA in the solution increases, the antibody–POD conjugate available for binding to immobilized WGA decreases, thus the resulting current decreases. Linearity was observed in the range 10−3 –102 ␮g mL−1 for WGA. The statistical data recorded for WGA using this biosensor approach is y = 2.0225e − 0.1341x with a correlation coefficient of 0.9951. 4. Conclusion This work has shown that electrochemical immunosensor is a suitable approach for the determination of lectins such as WGA and ProBL. The methodology may provide a generic protein screening method for any lectins if specific antibodies to those proteins are available. We demonstrated two different immunosensing strategies. In both cases, the obtained signal was inversely related to the analyte concentration. By combining the specificity of antibodies with the degree of signal amplification afforded by the catalytic effects of an enzyme, a high level selectivity and sensitivity can be obtained. The amperometric immunosensor could therefore provide a rapid and convenient analytical method over traditional technique such as western blotting. The ideal situation would be to integrate a reversible antibody–antigen reaction in a sensor that is able to maintain the same activity through a high number of assays. However, most immunosensors can be used only for a limited number of assays. Thus, disposable amperometric immunosensor is

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