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Highly sensitive impedimetric glycosensor for the determination of a ricin surrogate, Ricinus communis agglutinin I (RCA120)
Journal of Electroanalytical Chemistry 856 (2020) 113735
Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Highly sensitive impedimetric glycosensor for the determination of a ricin surrogate, Ricinus communis agglutinin I (RCA120) Daeho Jeong, Won-Yong Lee ⁎ Department of Chemistry, Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 November 2019 Received in revised form 28 November 2019 Accepted 6 December 2019 Available online 09 December 2019 Keywords: Glycosensor Ricinus communnis agglutinin 120 Electrochemical impedance spectroscopy Security threat toxin Ricin
a b s t r a c t A highly sensitive impedimetric glycosensor has been developed for the label-free detection of Ricinus communis Agglutinin I (RCA120) - a surrogate of potential security threat toxin ricin. In order to accomplish strong binding of RCA120 onto the glycosensor surface, the coverage of self-assembled monolayers of thiolated β-galactose derivative on gold electrode was optimized with varying ratios of thiolated ethylene glycol derivative. The association constant between the RCA120 and the β-galactose derivative with 10% coverage on gold electrode surface has been determined to be ca. 1.54 × 109 M−1, implying that the β-galactose derivative can be used as an effective recognition element for RCA120. The increase in electron transfer resistance through the selective binding of RCA120 onto the glycosensor measured by electron impedance spectroscopy was directly dependent upon the amount of RCA120 in sample solution. The present glycosensor can detect RCA120 from 8.33 × 10−11 M to 4.17 × 10−6 M with a limit of detection (S/N = 3) of 1.23 × 10−11 M, which is much lower than those of the previous reports. Since the proposed glycosensor does not need time-consuming labeling or signal amplification steps commonly carried out in sandwich-type RCA120 immunoassays, it provides the potential for a simple and rapid detection of a security threat toxin ricin. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Ricin is a highly toxic lectin which can be possibly used a biological warfare agent [1]. Ricin can be easily obtained since it is naturally found in the seeds of castor beans. The purified ricin can be made in the form of a powder, a mist, or a pellet, or it can be dissolved in water. The exposure of a small amount of purified ricin to human body via inhalation or injection can cause serious damage or even death [2]. Therefore, it is very important to develop a simple and rapid detection method for a potential security threat toxin ricin in order to prevent the bioterrorism. Up to now, a variety of analytical methods for ricin have been developed. The most common methods use ricin-specific antibodies, including fluorescence-based microarray [3], colloidal gold-based immunochromatographic assay [4], mass change-based magnetoelastic sensor [5], agglutination test of latex particles [2], FRET-based suspension microarray [6], electrochemiluminescence-based immunoassay [7] and carbon nanofiber-based electrochemical impedance measurement [8]. On the other hand, a few methods based on glycosphingolipids GM1 and asialoGM1 coupled with quartz crystal balance [9] and fluorescence-based hydrogel microarrays [10] have been developed. In addition, some methods based on synthesized ⁎ Corresponding author. E-mail address: [email protected] (W.-Y. Lee).
https://doi.org/10.1016/j.jelechem.2019.113735 1572-6657/© 2019 Elsevier B.V. All rights reserved.
carbohydrate derivatives have been developed. For example, a multichannel optical nanosensors based on multiple carbohydrates has been developed for the detection of 5 lectins including a ricin mimic RCA120 [11]. Carbohydrate-stabilized gold nanoparticles have been used for ricin based on surface plasmon resonance detection [12] and also for ricin mimic RCA120 based on microarray fluorescence [13] and colorimetric detections [14]. Although the reported works showed good analytical performance for the detection of ricin and a ricin mimic RCA120, optical transduction methods were used in the majority reports. Therefore, the rapid on-site detection of a potential biological warfare agent ricin with high sensitivity could not be easily realized in the previous reports. Therefore, we have developed a simple and rapid electrochemical detection method for the determination of ricin mimic RCA120. Ricinus communis Agglutinin I (RCA120) is also found in castor bean seeds. RCA120 is a dimeric lectin, and closely similar to the dimeric ricin with the A- and B- chains sharing 93 and 84% identity, respectively [14]. Since RCA120 is much less toxic than ricin, RCA120 is a good model system to develop a bioassay or biosensor for biological warfare agent ricin. Since Ricin and RCA120 are known to specifically bind to galactose and lactose [15], the present glycosensor has utilized the selfassembled monolayers of β-galactose derivatives as a recognition element for target RCA120 containing two galactose binding sites. In the present study, the coverage of self-assembled monolayers (SAMs) of β-galactose derivative on gold electrode was optimized with varying
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ratios of ethylene glycol derivative in order to accomplish strong binding of RCA120 onto the glycosensor surface. The association constant between the lectin RCA120 and the β-galactose derivative with 10% coverage on gold electrode surface has been determined to be ca. 1.54 × 109 M−1, implying that the β-galactose derivative can be used as an effective recognition element for RCA120. Upon the selective binding of RCA120 onto the as-prepared glycosensor, the electron transfer resistance at the gold electrode surface of the glycosensor has been increased directly depending upon the amount of RCA120 in sample solution. The electron transfer resistance was directly measured by electrochemical impedance spectroscopy in the presence of 5.0 mM K3Fe (CN)6/K4Fe(CN)6 (1:1, v/v) redox couple. The present glycosensor can detect RCA120 from 8.33 × 10−11 M to 4.17 × 10−6 M with a limit of detection (S/N = 3) down to 1.23 × 10−11 M, which is much lower than those of the previous reports. The present assay was applied to the determination of RCA120 in human serum samples. 2. Material and methods 2.1. Reagents Ricinus communis Agglutinin 1 (RCA120) was purchased form Vetor laboratories. Tri(ethylene glycol)mono-11-mercaptoundecyl ether was purchased from Sigma-Aldrich. Bovine serum albumin, wheat germ agglutinin, and concanavalin A were also purchased from Sigma-Aldrich. RCA120 solutions were prepared in 10 mM PBS buffer at pH 7.7. 2.2. Instrumentation Electrochemical experiments were carried out with EG&G 273A potentiostat (Princeton, NJ, USA) and frequency response detector (Model 5210, Oak Ridge, TN, USA). Three-electrode system was consisted of a platinum wire counter electrode, a gold working electrode (area = 0.070 cm2) and Ag/AgCl (3 M NaCl) reference electrode in a 10 mL cell. Electrochemical impedance spectroscopy and cyclic voltammograms were recorded in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1, v/v) solution dissolved in 50 mM phosphate buffer at pH 7.0 as reported previously [16]. 2.3. Preparation of glycosensor The β-galactoside containing a thiol functional group at the terminal position as shown in Fig. 1 has been prepared in a multistep sequence from galactosyl trichoroacetimidate and alcohol according to our previous report [17]. The bare gold electrode was precleaned according to the previous work [18]. In order to form the mixed SAMs of β-galactose and ethylene glycol derivatives on gold electrode, pre-cleaned gold electrode was soaked in 10 μM as-prepared thiolated β-galactoside derivative ethanol solution and thiolated ethylene glycol derivative solution for 12 h.
2.4. Electrochemical experiments The gold electrode self-assembled with β-galactose and ethylene glycol was immersed in RCA120 solution for 1 h. The EIS and CV experiments have been carried out according to our previous report [18]. ZSimpWin program (Princeton Applied Research, Oak Ridge, TN, USA) was used to fit the experimental results into equivalent circuit and estimate the diameter of the semicircles [19]. 3. Results and discussion 3.1. Fabrication of glycosensor The mixed SAMs of the β-galactoside and ethylene glycol derivatives have been well formed on a gold electrode surface by placing a bare gold electrode in the 10 μM β-galactoside and tri(ethylene glycol)mono-11mercaptoundecyl ether solution (1:10, v/v) prepared in ethanol for 12 h as shown in Fig. 2. The polyethyleneglycol (PEG) spacers were introduced both in the thiolated β-galactoside derivative and in the diluting molecules of thiolated ethylene glycol derivative in order to minimize the nonspecific adsorption of interfering proteins onto gold electrode surface as we previously reported [16]. The formation of mixed SAMs of β-galactoside and ethylene glycol derivatives has been verified by EIS and CV in the presence of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1, v/ v) prepared in 0.05 M phosphate buffer solution (pH 7.0) as a redox probe. Fig. 3A shows the Nyquist plots of EIS obtained at a bare gold electrode and the fabricated glycosensors in the absence and presence of RCA120. An equivalent circuit model (inset of Fig. 3) has been used in order to fit the EIS experimental results, in which Rct is charge transfer resistance, Rs is solution resistance, Qdl is double-layer capacitance, and W is Warberg impedance. The formation of mixed SAMs on a gold electrode significantly increased the diameter of the semicircle in Nyquist plot, corresponding to Rct, from 3.43 × 102 Ω (a) to 8.90 × 104 Ω (b). In addition, Qdl and W double- have been changed from 3.78 μS•s to 0.910 μS•s and from 819.5 μS•s1/2 to 142.6 μS•s1/2, respectively. Fig. 3B exhibits the cyclic voltammograms obtained at a bare gold electrode and the as-prepared glycosensors in the absence and presence of RCA120. The formation of the mixed SAMs on a gold electrode dramatically decreased the reduction peak current from 7.26 × 10−5 A (a) to an almost negligible value (b). The results obtained from the EIS and CV measurements strongly indicate that the mixed SAMs have been well formed on a bare gold electrode and the tightly organized mixed SAMs on the gold electrode surface inhibit the electron transfer between the electrode surface and the redox probe in the electrolyte solution. In order to examine the binding capability of the fabricated glycosensor towards RCA120, the glycosensor was placed in 4.17 × 10−6 M RCA120 solution for 1 h, As shown in Fig. 3A, the Rct in EIS was significantly increased from 8.90 × 104 Ω (b) 3.78 × 105 Ω (c). In addition, Qdl and W double- have been changed from 0.910 μS•s to 0.840 μS•s and from 142.6 μS•s1/2 to 153.4 μS•s1/2, respectively. The reduction peak current in CV was also almost negligible (Fig. 3B, c) because the RCA120, which was selectively bound on the glycosensor, works as an insulating layer that blocks the redox couple to reach the glycosensor surface. The results obtained from the EIS and CV measurements confirm the successful fabrication of the RCA120 glycosensor. 3.2. Optimization of β-galactose coverage on gold electrode
Fig. 1. Chemical structures of thiol-modified β-galactose derivative (a) and triethylene glycol mono-11-mercaptoundecyl ether (b).
It is reported that the interaction between carbohydrate and lectin is strongly influenced by the amount of immobilized carbohydrate on surface because the extent of multivalent binding is dependent upon the surface density of active carbohydrates on surface [20,21]. Therefore, the surface coverage of β-galactose derivative self-assembled on gold electrode has been optimized with varying ratios of the diluting molecules of thiolated ethylene glycol derivative in order to accomplish strong binding of RCA120 onto the glycosensor surface. The association
D. Jeong, W.-Y. Lee / Journal of Electroanalytical Chemistry 856 (2020) 113735
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Fig. 2. Schematic illustration of the diffusion of K3Fe(CN)6/K4Fe(CN)6 at a bare gold electrode (a) and the glycosensor based on the mixed SAMs of β-galactose and ethylene glycol derivatives on gold electrode before (b) and after the binding of 4.17 × 10−6 M RCA120 (c).
constant, Ka, for the binding interaction between the as-prepared glycosensor and RCA120 has been determined upon varying ratios of ethylene glycol derivative to β-galactose derivative on gold by using Langmuir adsorption isotherm as a function of RCA120 concentration with the following equation [18]. ½RCA120 ½RCA120 1 ¼ þ ΔS ΔSmax K a ΔSmax where ΔS is a change of Ret value induced by the RCA120 binding to the glycosensor and [RCA120] is in molar concentration. Since the binding of the RCA120 to the β-galactose SAM-modified gold electrode prevents the diffusion of redox couple towards the electrode surface, the electron transfer resistance measured from EIS should increase linearly as the concentration of the RCA120 increases. In the present EIS measurements, normalized electron-transfer resistance change, ΔRet = [Ret(i) - Ret(0)]/Ret(0), was used, in which Ret(0) and Ret(i) represent the electron-transfer resistance before and after the binding of RCA120 to the glycosensor as shown in Figs. 4, 5, 6 and 7. First, self-assembly process has been carried out by soaking a gold electrode in thiolated β-galactose derivative solution without the addition of thiolated ethylene glycol derivatives. As shown in Fig. 4A, the asprepared glycosensor based on the β-galactose derivative SAMs with 100% full coverage has followed the linear eq. (1.49 × 10−1 [CT] + 3.92 × 10−8 (r2 = 0.999, n = 3) in the reciprocal plot of [RCA120]/ΔS vs. [RCA120]. From fitting the equation, the association constant has been calculated to be 3.81 × 106 M−1. In order to determine the optimal surface coverage of galactose derivative on gold electrode surface, the gold electrode was selfassembled with thiolated galactose derivative in varying ratios of thiolated ethylene glycol derivatives as the diluting molecules. The surface coverage of β-galactose derivatives on gold electrode was assumed to be the same as that of the solution, i.e. a solution of 10% thiolated βgalactose derivatives and 90% thiolated ethylene glycol derivatives is assumed to give the self-assembled monolayers containing 10% β-
galactose derivatives on gold electrode. In the surface coverage tested from 5% to 100%, the glycosensor based on the β-galactose derivative SAMs with 10% coverage on gold electrode surface has shown the largest electron-transfer resistance change (data not shown) for 4.17 × 10−8 M RCA120. The as-prepared glycosensor based on the βgalactose derivative SAMs with 10% coverage has followed the linear eq. (5.24 × 10−1[CT] + 3.40 × 10−10, r2 = 0.999, n = 3) in the reciprocal plot of [RCA120]/ΔS vs. [RCA120] as shown in Fig. 4B. From fitting the equation, the association constant has been calculated to be 1.54 × 109 M−1, which is much larger than that obtained at the glycosensor based on the β-galactose derivative SAMs with 100% full coverage (3.81 × 106 M−1). This result indicates that stronger interaction of β-galactose-RCA120 could be obtained at less densely selfassembled β-galactose on surface rather than at more densely selfassembled β-galactose because the less densely self-assembled βgalactose derivative on gold might be more accessible for the binding of large RCA120 to that of more densely self-assembled β-galactose as similar to the previously reported carbohydrate-lectin interactions [18,21]. 3.3. Impedimetric detection of RCA120 The calibration curve was constructed by plotting the impedance change as a function of the RCA120 concentration. As shown in Fig. 5, a linear regression equation was obtained as ΔRet = 0.572 (±0.0251) [RCA120] + 6.270 (±0.204) (r2 = 0.989, n = 3). The present RCA120 glycosensor based on the β-galactose derivative SAMs with 10% coverage gave a linear response for RCA120 from 8.33 × 10−11 M to 4.17 × 10−6 M with a detection limit (S/N = 3) of 1.23 × 10−11 M. As summarized in Table 1, the detection limit of the present impedimetric glycosensor is clearly lower than those obtained with β-galactose-based optical detection methods such as fluorescence [13], fluorescence resonance energy transfer [11] and colorimetry [14]. As mentioned earlier, RCA120 is a good model system to develop a bioassay or biosensor for biological warfare agent ricin because it is much less toxic than ricin. The
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Fig. 3. (A) Nyquist plots for the faradaic impedance measurements in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 in 50 mM phosphate buffer (pH 7.0) at bare gold electrode (a) mixed SAM-modified gold electrode (b) and RCA120 treated gold electrode (c). Inset: four-component equivalent circuit R(QR)W, Rct: charge transfer resistance, Rs: solution resistance, Q: double-layer capacitance, W: Warburg impedance. (B) Cyclic voltammograms in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 in 50 mM phosphate buffer (pH 7.0) of bare gold electrode (a) mixed SAM-modified gold electrode (b) and RCA120 treated gold electrode (c).
lethal dose (LD50) of ricin is known to be 3–5 μg/kg body weight in case of inhalation and 20 mg/kg in case of oral injection [7]. In case of acute intoxication, the concentration of ricin in human plasma is known to be around 25 pM concentration ranges [22]. Therefore, the present glycosensor with extremely low detection limit of 12.3 pM could be employed to detect biological warfare agent ricin because both RCA120 and ricin can specifically bind to β-galactose. In the present study, the present RCA120 glycosensor have been used only once for the measurement of a specific concentration and then discarded. The present glycosensor showed good sensor-sensor reproducibility for the measurement of a specific concentration. For example, the relative of standard deviation in electron transfer resistance values were 13.01%, 7.48%, 5.52%, 5.73%, 3.52%, 5.59% and 1.55% (n = 3) at 8.33 × 10−11 M, 4.17 × 10−10 M, 8.33 × 10−10 M, 4.17 × 10−9 M, 4.17 × 10−8 M, 4.17 × 10−7 M and 4.17 × 10−6 M, respectively as shown in Fig. 5. The binding kinetics between RCA120 and the present glycosensor has been studied with an exposure to 4.17 × 10−8 M RCA120 solution at room temperature over 50 min period. As shown in Fig. 6, the ΔRet induced by the RCA120 binding has been rapidly increased up to around 20 min and then very slowly increased up to 30 min. Further incubation beyond 30 min did not lead to further increase in the ΔRet, indicating that the β-galactoside SAMs on gold electrode are fully bound with
Fig. 4. (A) Reciprocal plot of [RCA120]/ΔRet vs. [RCA120] in EIS experiments obtained at the glycosensor with β-galactose SAM with full coverage. (B) Reciprocal plot of [RCA120]/ΔRet vs. [RCA120] in EIS experiments obtained at the glycosensor with β-galactose SAM with 10% coverage.
RCA120. In order to secure high sensitivity and good reproducibility, the incubation time was set to 60 min before EIS measurement in all subsequent experiments. In case the rapid analysis is required, the shorter incubation time could be employed although slightly smaller EIS response is obtained (e.g. 80% response if the incubation time is 10 min).
3.4. Selectivity and recovery test The selectivity of the present glycosensor based on the β-galactose modified gold electrode has been examined against possible interfering proteins and lectins such as bovine serum albumin (BSA), wheat germ agglutinin (WGA) and concanavalin A (Con A). As shown in Fig. 7, the relative signals of the present glycosensor to BSA, WGA, and Con A were 20.1%, 2.95%, and 9.07%, respectively, in comparison to that measured for the same concentration of RCA120 (4.17 × 10−8 M). There are two types of RCA including RCA60 and RCA120. Since RCA60, also called ricin, is highly toxic, it has not been included in the present selectivity test. In addition, the recovery test of the spiked RCA120 of 8.33 × 10−10 M in human plasma sample has been performed. The test results were satisfactory with the recovery of 76.8 ± 0.38% (n = 3) for spiked RCA120 solutions. This result strongly suggests that the present glycosensor based on the β-galactose modified gold electrode
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Fig. 7. Selectivity studies of the present glycosensor in the same concentration (4.17 × 10−6 M) of various protein samples. Each sample: BSA, WGA and Con A. Error bar represent the standard deviation of each sample, n = 3.
has good selectivity towards RCA120, and shows a potential to be applied for the determination of RCA120 in real samples. The long-term storage stability of the present glycosensor was examined during 2 weeks by measuring EIS response for 4.17 × 10−10 M RCA120. The present glycosensor has shown good stability, in which 93% of its initial activity was retained after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0. 4. Conclusions
Fig. 5. (A) Nyquist plots for the faradaic impedance measurements in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 at the glycosensor before (a) and after the binding of 8.33 × 10−11 M (b), 4.17 × 10−10 M (c), 8.33 × 10−10 M (d), 4.17 × 10−9 M (e), 4.17 × 10−8 M (f), 4.17 × 10−7 M (g), 4.17 × 10−6 M (h) RCA120. (B) Calibration curve for RCA120 obtained at the present glycosensor.
This work has demonstrated that the impedimetric glycosensor based on the mixed SAMs of β-galactose and ethylene glycol derivatives can be effectively used for the label-free detection of RCA120 - a surrogate of potential security threat toxin ricin. The surface coverage of SAMs of β-galactose derivative on gold electrode has strongly affected the association constant between the target RCA120 and the βgalactose derivative. The association constant between the RCA120 and the β-galactose derivative with 10% coverage on gold electrode surface has been determined to be ca. 1.54 × 109 M−1, implying that the βgalactose derivative can be used as an effective recognition element for RCA120. The present glycosensor can selectively detect the RCA120 Table 1 The comparison of the present glycosensor with other reported sensors based on different recognition element and detection methods for ricin or RCA120.
Fig. 6. Plot of ΔRet versus binding time of the glycosensor in 4.17 × 10−8 M RCA120.
Recognition element
Detection method
Target
Limit of detection (M)
Reference
Ab Ab Ab Ab Ab Ab Ab/Aptamer GM1 Lactose β-Galactose β-Lactoside β-Galactose β-Galactose β-Galactose
Fluorescence Immunochromatographic Magnetoelastic Radioimmunoassay FRET ECL Impedance QCM Fluorescence FRET SPR Colorimetric Fluorescence Impedance
Ricin Ricin Ricin Ricin Ricin Ricin Ricin Ricin Ricin RCA120 Ricin RCA120 RCA120 RCA120
3.3 × 10−9 8.3 × 10−10 8.3 × 10−11 7.5 × 10−8 1.7 × 10−11 1.7 × 10−10 N/A 8.3 × 10−8 5.0 × 10−10 5.7 × 10−9 5.0 × 10−10 9.0 × 10−9 2.0 × 10−8 1.23 × 10−11
3 4 5 2 6 7 8 9 10 11 12 13 14 This study
Ab: antibody, FL: fluorescence, FRET: fluorescence resonance energy transfer, SPR: surface plasmon resonance, QCM: quartz crystal microbalance, ECL: electrochemiluminescence.
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from 8.33 × 10−11 M to 4.17 × 10−6 M with a detection limit (S/N = 3) of 1.23 × 10−11 M, which is much lower than those of previous reports. Since the proposed glycosensor does not need time-consuming labeling or signal amplification steps commonly carried out in antibody-based immunosensor or RCA120 immunoassays, it provides the potential for a simple and rapid (less than 1 h) on-site detection of a security threat toxin ricin. Author contributions Daeho Jeong: Conceived and designed the analysis (first author designed the RCA detection using glycosensor), collected the data (he collected all data), contributed data or analysis tools (he dealt with EIS data analysis), performed the analysis (he performed electrochemical analysis), wrote the paper (he wrote the first draft), other contribution (he checked and approved the final version). Won-Yong Lee: Conceived and designed the analysis (he developed the idea of RCA detection using glycosensor), performed the analysis (he interpreted all data), wrote the paper (he wrote the final manuscript), other contribution (he worked as a principle investigator in the present work and shared all information with the first author regarding the paper work). Acknowledgements Financial support for this work has been provided by Basic Science Research Program through the National Research Foundation of Korea (NRF-2018R1D1A1A09082076).
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Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Highly sensitive impedimetric glycosensor for the determination of a ricin surrogate, Ricinus communis agglutinin I (RCA120) Daeho Jeong, Won-Yong Lee ⁎ Department of Chemistry, Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 November 2019 Received in revised form 28 November 2019 Accepted 6 December 2019 Available online 09 December 2019 Keywords: Glycosensor Ricinus communnis agglutinin 120 Electrochemical impedance spectroscopy Security threat toxin Ricin
a b s t r a c t A highly sensitive impedimetric glycosensor has been developed for the label-free detection of Ricinus communis Agglutinin I (RCA120) - a surrogate of potential security threat toxin ricin. In order to accomplish strong binding of RCA120 onto the glycosensor surface, the coverage of self-assembled monolayers of thiolated β-galactose derivative on gold electrode was optimized with varying ratios of thiolated ethylene glycol derivative. The association constant between the RCA120 and the β-galactose derivative with 10% coverage on gold electrode surface has been determined to be ca. 1.54 × 109 M−1, implying that the β-galactose derivative can be used as an effective recognition element for RCA120. The increase in electron transfer resistance through the selective binding of RCA120 onto the glycosensor measured by electron impedance spectroscopy was directly dependent upon the amount of RCA120 in sample solution. The present glycosensor can detect RCA120 from 8.33 × 10−11 M to 4.17 × 10−6 M with a limit of detection (S/N = 3) of 1.23 × 10−11 M, which is much lower than those of the previous reports. Since the proposed glycosensor does not need time-consuming labeling or signal amplification steps commonly carried out in sandwich-type RCA120 immunoassays, it provides the potential for a simple and rapid detection of a security threat toxin ricin. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Ricin is a highly toxic lectin which can be possibly used a biological warfare agent [1]. Ricin can be easily obtained since it is naturally found in the seeds of castor beans. The purified ricin can be made in the form of a powder, a mist, or a pellet, or it can be dissolved in water. The exposure of a small amount of purified ricin to human body via inhalation or injection can cause serious damage or even death [2]. Therefore, it is very important to develop a simple and rapid detection method for a potential security threat toxin ricin in order to prevent the bioterrorism. Up to now, a variety of analytical methods for ricin have been developed. The most common methods use ricin-specific antibodies, including fluorescence-based microarray [3], colloidal gold-based immunochromatographic assay [4], mass change-based magnetoelastic sensor [5], agglutination test of latex particles [2], FRET-based suspension microarray [6], electrochemiluminescence-based immunoassay [7] and carbon nanofiber-based electrochemical impedance measurement [8]. On the other hand, a few methods based on glycosphingolipids GM1 and asialoGM1 coupled with quartz crystal balance [9] and fluorescence-based hydrogel microarrays [10] have been developed. In addition, some methods based on synthesized ⁎ Corresponding author. E-mail address: [email protected] (W.-Y. Lee).
https://doi.org/10.1016/j.jelechem.2019.113735 1572-6657/© 2019 Elsevier B.V. All rights reserved.
carbohydrate derivatives have been developed. For example, a multichannel optical nanosensors based on multiple carbohydrates has been developed for the detection of 5 lectins including a ricin mimic RCA120 [11]. Carbohydrate-stabilized gold nanoparticles have been used for ricin based on surface plasmon resonance detection [12] and also for ricin mimic RCA120 based on microarray fluorescence [13] and colorimetric detections [14]. Although the reported works showed good analytical performance for the detection of ricin and a ricin mimic RCA120, optical transduction methods were used in the majority reports. Therefore, the rapid on-site detection of a potential biological warfare agent ricin with high sensitivity could not be easily realized in the previous reports. Therefore, we have developed a simple and rapid electrochemical detection method for the determination of ricin mimic RCA120. Ricinus communis Agglutinin I (RCA120) is also found in castor bean seeds. RCA120 is a dimeric lectin, and closely similar to the dimeric ricin with the A- and B- chains sharing 93 and 84% identity, respectively [14]. Since RCA120 is much less toxic than ricin, RCA120 is a good model system to develop a bioassay or biosensor for biological warfare agent ricin. Since Ricin and RCA120 are known to specifically bind to galactose and lactose [15], the present glycosensor has utilized the selfassembled monolayers of β-galactose derivatives as a recognition element for target RCA120 containing two galactose binding sites. In the present study, the coverage of self-assembled monolayers (SAMs) of β-galactose derivative on gold electrode was optimized with varying
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ratios of ethylene glycol derivative in order to accomplish strong binding of RCA120 onto the glycosensor surface. The association constant between the lectin RCA120 and the β-galactose derivative with 10% coverage on gold electrode surface has been determined to be ca. 1.54 × 109 M−1, implying that the β-galactose derivative can be used as an effective recognition element for RCA120. Upon the selective binding of RCA120 onto the as-prepared glycosensor, the electron transfer resistance at the gold electrode surface of the glycosensor has been increased directly depending upon the amount of RCA120 in sample solution. The electron transfer resistance was directly measured by electrochemical impedance spectroscopy in the presence of 5.0 mM K3Fe (CN)6/K4Fe(CN)6 (1:1, v/v) redox couple. The present glycosensor can detect RCA120 from 8.33 × 10−11 M to 4.17 × 10−6 M with a limit of detection (S/N = 3) down to 1.23 × 10−11 M, which is much lower than those of the previous reports. The present assay was applied to the determination of RCA120 in human serum samples. 2. Material and methods 2.1. Reagents Ricinus communis Agglutinin 1 (RCA120) was purchased form Vetor laboratories. Tri(ethylene glycol)mono-11-mercaptoundecyl ether was purchased from Sigma-Aldrich. Bovine serum albumin, wheat germ agglutinin, and concanavalin A were also purchased from Sigma-Aldrich. RCA120 solutions were prepared in 10 mM PBS buffer at pH 7.7. 2.2. Instrumentation Electrochemical experiments were carried out with EG&G 273A potentiostat (Princeton, NJ, USA) and frequency response detector (Model 5210, Oak Ridge, TN, USA). Three-electrode system was consisted of a platinum wire counter electrode, a gold working electrode (area = 0.070 cm2) and Ag/AgCl (3 M NaCl) reference electrode in a 10 mL cell. Electrochemical impedance spectroscopy and cyclic voltammograms were recorded in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1, v/v) solution dissolved in 50 mM phosphate buffer at pH 7.0 as reported previously [16]. 2.3. Preparation of glycosensor The β-galactoside containing a thiol functional group at the terminal position as shown in Fig. 1 has been prepared in a multistep sequence from galactosyl trichoroacetimidate and alcohol according to our previous report [17]. The bare gold electrode was precleaned according to the previous work [18]. In order to form the mixed SAMs of β-galactose and ethylene glycol derivatives on gold electrode, pre-cleaned gold electrode was soaked in 10 μM as-prepared thiolated β-galactoside derivative ethanol solution and thiolated ethylene glycol derivative solution for 12 h.
2.4. Electrochemical experiments The gold electrode self-assembled with β-galactose and ethylene glycol was immersed in RCA120 solution for 1 h. The EIS and CV experiments have been carried out according to our previous report [18]. ZSimpWin program (Princeton Applied Research, Oak Ridge, TN, USA) was used to fit the experimental results into equivalent circuit and estimate the diameter of the semicircles [19]. 3. Results and discussion 3.1. Fabrication of glycosensor The mixed SAMs of the β-galactoside and ethylene glycol derivatives have been well formed on a gold electrode surface by placing a bare gold electrode in the 10 μM β-galactoside and tri(ethylene glycol)mono-11mercaptoundecyl ether solution (1:10, v/v) prepared in ethanol for 12 h as shown in Fig. 2. The polyethyleneglycol (PEG) spacers were introduced both in the thiolated β-galactoside derivative and in the diluting molecules of thiolated ethylene glycol derivative in order to minimize the nonspecific adsorption of interfering proteins onto gold electrode surface as we previously reported [16]. The formation of mixed SAMs of β-galactoside and ethylene glycol derivatives has been verified by EIS and CV in the presence of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1, v/ v) prepared in 0.05 M phosphate buffer solution (pH 7.0) as a redox probe. Fig. 3A shows the Nyquist plots of EIS obtained at a bare gold electrode and the fabricated glycosensors in the absence and presence of RCA120. An equivalent circuit model (inset of Fig. 3) has been used in order to fit the EIS experimental results, in which Rct is charge transfer resistance, Rs is solution resistance, Qdl is double-layer capacitance, and W is Warberg impedance. The formation of mixed SAMs on a gold electrode significantly increased the diameter of the semicircle in Nyquist plot, corresponding to Rct, from 3.43 × 102 Ω (a) to 8.90 × 104 Ω (b). In addition, Qdl and W double- have been changed from 3.78 μS•s to 0.910 μS•s and from 819.5 μS•s1/2 to 142.6 μS•s1/2, respectively. Fig. 3B exhibits the cyclic voltammograms obtained at a bare gold electrode and the as-prepared glycosensors in the absence and presence of RCA120. The formation of the mixed SAMs on a gold electrode dramatically decreased the reduction peak current from 7.26 × 10−5 A (a) to an almost negligible value (b). The results obtained from the EIS and CV measurements strongly indicate that the mixed SAMs have been well formed on a bare gold electrode and the tightly organized mixed SAMs on the gold electrode surface inhibit the electron transfer between the electrode surface and the redox probe in the electrolyte solution. In order to examine the binding capability of the fabricated glycosensor towards RCA120, the glycosensor was placed in 4.17 × 10−6 M RCA120 solution for 1 h, As shown in Fig. 3A, the Rct in EIS was significantly increased from 8.90 × 104 Ω (b) 3.78 × 105 Ω (c). In addition, Qdl and W double- have been changed from 0.910 μS•s to 0.840 μS•s and from 142.6 μS•s1/2 to 153.4 μS•s1/2, respectively. The reduction peak current in CV was also almost negligible (Fig. 3B, c) because the RCA120, which was selectively bound on the glycosensor, works as an insulating layer that blocks the redox couple to reach the glycosensor surface. The results obtained from the EIS and CV measurements confirm the successful fabrication of the RCA120 glycosensor. 3.2. Optimization of β-galactose coverage on gold electrode
Fig. 1. Chemical structures of thiol-modified β-galactose derivative (a) and triethylene glycol mono-11-mercaptoundecyl ether (b).
It is reported that the interaction between carbohydrate and lectin is strongly influenced by the amount of immobilized carbohydrate on surface because the extent of multivalent binding is dependent upon the surface density of active carbohydrates on surface [20,21]. Therefore, the surface coverage of β-galactose derivative self-assembled on gold electrode has been optimized with varying ratios of the diluting molecules of thiolated ethylene glycol derivative in order to accomplish strong binding of RCA120 onto the glycosensor surface. The association
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Fig. 2. Schematic illustration of the diffusion of K3Fe(CN)6/K4Fe(CN)6 at a bare gold electrode (a) and the glycosensor based on the mixed SAMs of β-galactose and ethylene glycol derivatives on gold electrode before (b) and after the binding of 4.17 × 10−6 M RCA120 (c).
constant, Ka, for the binding interaction between the as-prepared glycosensor and RCA120 has been determined upon varying ratios of ethylene glycol derivative to β-galactose derivative on gold by using Langmuir adsorption isotherm as a function of RCA120 concentration with the following equation [18]. ½RCA120 ½RCA120 1 ¼ þ ΔS ΔSmax K a ΔSmax where ΔS is a change of Ret value induced by the RCA120 binding to the glycosensor and [RCA120] is in molar concentration. Since the binding of the RCA120 to the β-galactose SAM-modified gold electrode prevents the diffusion of redox couple towards the electrode surface, the electron transfer resistance measured from EIS should increase linearly as the concentration of the RCA120 increases. In the present EIS measurements, normalized electron-transfer resistance change, ΔRet = [Ret(i) - Ret(0)]/Ret(0), was used, in which Ret(0) and Ret(i) represent the electron-transfer resistance before and after the binding of RCA120 to the glycosensor as shown in Figs. 4, 5, 6 and 7. First, self-assembly process has been carried out by soaking a gold electrode in thiolated β-galactose derivative solution without the addition of thiolated ethylene glycol derivatives. As shown in Fig. 4A, the asprepared glycosensor based on the β-galactose derivative SAMs with 100% full coverage has followed the linear eq. (1.49 × 10−1 [CT] + 3.92 × 10−8 (r2 = 0.999, n = 3) in the reciprocal plot of [RCA120]/ΔS vs. [RCA120]. From fitting the equation, the association constant has been calculated to be 3.81 × 106 M−1. In order to determine the optimal surface coverage of galactose derivative on gold electrode surface, the gold electrode was selfassembled with thiolated galactose derivative in varying ratios of thiolated ethylene glycol derivatives as the diluting molecules. The surface coverage of β-galactose derivatives on gold electrode was assumed to be the same as that of the solution, i.e. a solution of 10% thiolated βgalactose derivatives and 90% thiolated ethylene glycol derivatives is assumed to give the self-assembled monolayers containing 10% β-
galactose derivatives on gold electrode. In the surface coverage tested from 5% to 100%, the glycosensor based on the β-galactose derivative SAMs with 10% coverage on gold electrode surface has shown the largest electron-transfer resistance change (data not shown) for 4.17 × 10−8 M RCA120. The as-prepared glycosensor based on the βgalactose derivative SAMs with 10% coverage has followed the linear eq. (5.24 × 10−1[CT] + 3.40 × 10−10, r2 = 0.999, n = 3) in the reciprocal plot of [RCA120]/ΔS vs. [RCA120] as shown in Fig. 4B. From fitting the equation, the association constant has been calculated to be 1.54 × 109 M−1, which is much larger than that obtained at the glycosensor based on the β-galactose derivative SAMs with 100% full coverage (3.81 × 106 M−1). This result indicates that stronger interaction of β-galactose-RCA120 could be obtained at less densely selfassembled β-galactose on surface rather than at more densely selfassembled β-galactose because the less densely self-assembled βgalactose derivative on gold might be more accessible for the binding of large RCA120 to that of more densely self-assembled β-galactose as similar to the previously reported carbohydrate-lectin interactions [18,21]. 3.3. Impedimetric detection of RCA120 The calibration curve was constructed by plotting the impedance change as a function of the RCA120 concentration. As shown in Fig. 5, a linear regression equation was obtained as ΔRet = 0.572 (±0.0251) [RCA120] + 6.270 (±0.204) (r2 = 0.989, n = 3). The present RCA120 glycosensor based on the β-galactose derivative SAMs with 10% coverage gave a linear response for RCA120 from 8.33 × 10−11 M to 4.17 × 10−6 M with a detection limit (S/N = 3) of 1.23 × 10−11 M. As summarized in Table 1, the detection limit of the present impedimetric glycosensor is clearly lower than those obtained with β-galactose-based optical detection methods such as fluorescence [13], fluorescence resonance energy transfer [11] and colorimetry [14]. As mentioned earlier, RCA120 is a good model system to develop a bioassay or biosensor for biological warfare agent ricin because it is much less toxic than ricin. The
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Fig. 3. (A) Nyquist plots for the faradaic impedance measurements in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 in 50 mM phosphate buffer (pH 7.0) at bare gold electrode (a) mixed SAM-modified gold electrode (b) and RCA120 treated gold electrode (c). Inset: four-component equivalent circuit R(QR)W, Rct: charge transfer resistance, Rs: solution resistance, Q: double-layer capacitance, W: Warburg impedance. (B) Cyclic voltammograms in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 in 50 mM phosphate buffer (pH 7.0) of bare gold electrode (a) mixed SAM-modified gold electrode (b) and RCA120 treated gold electrode (c).
lethal dose (LD50) of ricin is known to be 3–5 μg/kg body weight in case of inhalation and 20 mg/kg in case of oral injection [7]. In case of acute intoxication, the concentration of ricin in human plasma is known to be around 25 pM concentration ranges [22]. Therefore, the present glycosensor with extremely low detection limit of 12.3 pM could be employed to detect biological warfare agent ricin because both RCA120 and ricin can specifically bind to β-galactose. In the present study, the present RCA120 glycosensor have been used only once for the measurement of a specific concentration and then discarded. The present glycosensor showed good sensor-sensor reproducibility for the measurement of a specific concentration. For example, the relative of standard deviation in electron transfer resistance values were 13.01%, 7.48%, 5.52%, 5.73%, 3.52%, 5.59% and 1.55% (n = 3) at 8.33 × 10−11 M, 4.17 × 10−10 M, 8.33 × 10−10 M, 4.17 × 10−9 M, 4.17 × 10−8 M, 4.17 × 10−7 M and 4.17 × 10−6 M, respectively as shown in Fig. 5. The binding kinetics between RCA120 and the present glycosensor has been studied with an exposure to 4.17 × 10−8 M RCA120 solution at room temperature over 50 min period. As shown in Fig. 6, the ΔRet induced by the RCA120 binding has been rapidly increased up to around 20 min and then very slowly increased up to 30 min. Further incubation beyond 30 min did not lead to further increase in the ΔRet, indicating that the β-galactoside SAMs on gold electrode are fully bound with
Fig. 4. (A) Reciprocal plot of [RCA120]/ΔRet vs. [RCA120] in EIS experiments obtained at the glycosensor with β-galactose SAM with full coverage. (B) Reciprocal plot of [RCA120]/ΔRet vs. [RCA120] in EIS experiments obtained at the glycosensor with β-galactose SAM with 10% coverage.
RCA120. In order to secure high sensitivity and good reproducibility, the incubation time was set to 60 min before EIS measurement in all subsequent experiments. In case the rapid analysis is required, the shorter incubation time could be employed although slightly smaller EIS response is obtained (e.g. 80% response if the incubation time is 10 min).
3.4. Selectivity and recovery test The selectivity of the present glycosensor based on the β-galactose modified gold electrode has been examined against possible interfering proteins and lectins such as bovine serum albumin (BSA), wheat germ agglutinin (WGA) and concanavalin A (Con A). As shown in Fig. 7, the relative signals of the present glycosensor to BSA, WGA, and Con A were 20.1%, 2.95%, and 9.07%, respectively, in comparison to that measured for the same concentration of RCA120 (4.17 × 10−8 M). There are two types of RCA including RCA60 and RCA120. Since RCA60, also called ricin, is highly toxic, it has not been included in the present selectivity test. In addition, the recovery test of the spiked RCA120 of 8.33 × 10−10 M in human plasma sample has been performed. The test results were satisfactory with the recovery of 76.8 ± 0.38% (n = 3) for spiked RCA120 solutions. This result strongly suggests that the present glycosensor based on the β-galactose modified gold electrode
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Fig. 7. Selectivity studies of the present glycosensor in the same concentration (4.17 × 10−6 M) of various protein samples. Each sample: BSA, WGA and Con A. Error bar represent the standard deviation of each sample, n = 3.
has good selectivity towards RCA120, and shows a potential to be applied for the determination of RCA120 in real samples. The long-term storage stability of the present glycosensor was examined during 2 weeks by measuring EIS response for 4.17 × 10−10 M RCA120. The present glycosensor has shown good stability, in which 93% of its initial activity was retained after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0. 4. Conclusions
Fig. 5. (A) Nyquist plots for the faradaic impedance measurements in the presence of 5.0 mM K3Fe(CN)6/K4Fe(CN)6 at the glycosensor before (a) and after the binding of 8.33 × 10−11 M (b), 4.17 × 10−10 M (c), 8.33 × 10−10 M (d), 4.17 × 10−9 M (e), 4.17 × 10−8 M (f), 4.17 × 10−7 M (g), 4.17 × 10−6 M (h) RCA120. (B) Calibration curve for RCA120 obtained at the present glycosensor.
This work has demonstrated that the impedimetric glycosensor based on the mixed SAMs of β-galactose and ethylene glycol derivatives can be effectively used for the label-free detection of RCA120 - a surrogate of potential security threat toxin ricin. The surface coverage of SAMs of β-galactose derivative on gold electrode has strongly affected the association constant between the target RCA120 and the βgalactose derivative. The association constant between the RCA120 and the β-galactose derivative with 10% coverage on gold electrode surface has been determined to be ca. 1.54 × 109 M−1, implying that the βgalactose derivative can be used as an effective recognition element for RCA120. The present glycosensor can selectively detect the RCA120 Table 1 The comparison of the present glycosensor with other reported sensors based on different recognition element and detection methods for ricin or RCA120.
Fig. 6. Plot of ΔRet versus binding time of the glycosensor in 4.17 × 10−8 M RCA120.
Recognition element
Detection method
Target
Limit of detection (M)
Reference
Ab Ab Ab Ab Ab Ab Ab/Aptamer GM1 Lactose β-Galactose β-Lactoside β-Galactose β-Galactose β-Galactose
Fluorescence Immunochromatographic Magnetoelastic Radioimmunoassay FRET ECL Impedance QCM Fluorescence FRET SPR Colorimetric Fluorescence Impedance
Ricin Ricin Ricin Ricin Ricin Ricin Ricin Ricin Ricin RCA120 Ricin RCA120 RCA120 RCA120
3.3 × 10−9 8.3 × 10−10 8.3 × 10−11 7.5 × 10−8 1.7 × 10−11 1.7 × 10−10 N/A 8.3 × 10−8 5.0 × 10−10 5.7 × 10−9 5.0 × 10−10 9.0 × 10−9 2.0 × 10−8 1.23 × 10−11
3 4 5 2 6 7 8 9 10 11 12 13 14 This study
Ab: antibody, FL: fluorescence, FRET: fluorescence resonance energy transfer, SPR: surface plasmon resonance, QCM: quartz crystal microbalance, ECL: electrochemiluminescence.
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from 8.33 × 10−11 M to 4.17 × 10−6 M with a detection limit (S/N = 3) of 1.23 × 10−11 M, which is much lower than those of previous reports. Since the proposed glycosensor does not need time-consuming labeling or signal amplification steps commonly carried out in antibody-based immunosensor or RCA120 immunoassays, it provides the potential for a simple and rapid (less than 1 h) on-site detection of a security threat toxin ricin. Author contributions Daeho Jeong: Conceived and designed the analysis (first author designed the RCA detection using glycosensor), collected the data (he collected all data), contributed data or analysis tools (he dealt with EIS data analysis), performed the analysis (he performed electrochemical analysis), wrote the paper (he wrote the first draft), other contribution (he checked and approved the final version). Won-Yong Lee: Conceived and designed the analysis (he developed the idea of RCA detection using glycosensor), performed the analysis (he interpreted all data), wrote the paper (he wrote the final manuscript), other contribution (he worked as a principle investigator in the present work and shared all information with the first author regarding the paper work). Acknowledgements Financial support for this work has been provided by Basic Science Research Program through the National Research Foundation of Korea (NRF-2018R1D1A1A09082076).
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