Elsevier

Analytica Chimica Acta

Volume 634, Issue 2, 23 February 2009, Pages 255-261
Analytica Chimica Acta

A capacitive immunosensor for detection of cholera toxin

https://doi.org/10.1016/j.aca.2008.12.035Get rights and content

Abstract

Contamination of food with biological toxins as well as their potential use as weapons of mass destruction has created an urge for rapid and cost effective analytical techniques capable of detecting trace amounts of these toxins. This paper describes the development of a sensitive method for detection of cholera toxin (CT) using a flow-injection capacitive immunosensor based on self-assembled monolayers. The sensing surface consists of monoclonal antibodies against the B subunit of CT (anti-CT), immobilized on a gold transducer. Experimental results show that the immunosensor responded linearly to CT concentrations in the range from 1.0 × 10−13 to 1.0 × 10−10 M under optimized conditions. The limit of detection (LOD) was 1.0 × 10−14 M. Two more analytical methods were employed for detection of CT using the same antibody namely, sandwich ELISA and surface plasmon resonance (SPR)-based immunosensor. The former had an LOD of 1.2 × 10−12 M and a working range from 3.7 × 10−11 to 2.9 × 10−10 M whereas, the later had an LOD of 1.0 × 10−11 M and a linearity ranging from 1.0 × 10−9 to 1.0 × 10−6 M. These results demonstrate that the developed capacitive immunosensor system has a higher sensitivity than the other two techniques. The binding affinity of CT to the immobilized anti-CT was determined using the SPR-based immunosensor and an association constant (KA) of 1.4 × 109 M−1 was estimated.

Introduction

Cholera, an acute gastrointestinal infection caused by the bacterium Vibrio cholerae, currently affects over 100,000 persons annually [1]. The disease is common in developing countries and can be fatal in 50% of cases where facilities for treatment are not available [2]. It does not only affect impoverished areas but also poses a serious threat as a potential tool for bioterrorism [3], [4]. Cholera toxin (CT) is the major virulent factor of toxigenic strains of V. cholerae[5]. It has a common hetero-hexameric structure consisting of a single, enzymatically active A subunit (CT-A, 27 kDa), non-covalently linked to a pentameric core of five identical receptor-binding B subunits (CT-B, 58 kDa) [6]. The biological action of CT is initiated by the binding of CT-B to the ganglioside GM1 receptor on the intestinal cell membrane followed by internalization of CT-A into the cell where it activates adenylate cyclase [7]. This leads to increased intracellular levels of cyclic AMP, which in turn results in the secretion of copious amounts water and electrolytes into the bowels, causing severe diarrhea and death if treatment is not given promptly [8], [9], [10], [11], [12], [13], [14], [15], [16].

Although most cholera cases are associated with the ingestion of the organism, the detection of CT is increasingly important because existing serological methods may not be able to detect a new serogroup. Furthermore, conventional laboratory methods that depend on the isolation and identification of V. cholerae by biochemical tests are costly and time-consuming [17]. Various sensitive methods, including radioimmunoassay [18], enzyme-linked immunosorbent assays (ELISA) [19], and latex agglutination assays [20], were developed for detection of CT. However, most of these assays take at least several hours, which makes them unsuitable for rapid detection and field screening.

The lethal dose of CT in humans is relatively low (LD50 ∼250 μg kg−1) [21]. Therefore, methods that selectively and sensitively detect extremely low levels of the toxin are highly desirable. Recently, the development of biosensing methods for detection of CT has drawn attention as a result of an increasing demand for fast, sensitive and reliable detection of the toxin in order to prevent outbreaks of the disease and for counter terrorism campaigns [22], [23]. In response, biosensors that can detect CT on the basis of its interaction with surface immobilized anti-CT molecules became more prevalent and commercially available. Examples for these biosensors include: antibody based microarrays [24], [25], [26], [27], [28], quartz crystal microbalance (QCM) based sensors [29], surface plasmon resonance (SPR) based sensors [30], [31], electrochemical immunosensors [32] and ion-selective field-effect transistor sensor coupled with SPR [33]. However, public health officials and cholera researchers have an ongoing need for improvements of cholera detection methodologies.

Over the last years, capacitive immunosensors have been attracting wide-spread attention in clinical, environmental and biotechnological applications. They are particularly sensitive electrochemical tools, which have established a niche for the analysis of proteins and particularly, toxins. The ability of these sensors to quantify trace analyte concentrations in a short time frame, has offered them wide applications in forensics, diagnostics and manufacture settings [34], [35], [36], [37], [38], [39], [40]. In this work, we report the development of a flow-injection immunosensor system with a capacitive transducer for assay of cholera toxin. The detection limit and working range of the system will be compared to those obtained with an SPR based immuosensor and a sandwich ELISA test developed for the same purpose in order to achieve a proper assessment of the newly developed system.

Section snippets

Materials

Cholera toxin from Vibrio cholerae, 1-fluoro-2,4-dinitrobenzene (FDNB) and polystyrene multiwell plates were purchased from Sigma (St. Louis, MO, USA). Monoclonal anti-cholera toxin antibody raised against CT-B (IgG2b) was a generous gift from the Department of Microbiology and Immunology, Göteborg University, Sweden. Horseradish peroxidase type VIa, bicinchoninic acid solution, α lipoic acid, dry acetonitrile 99.8% and o-phenylenediamine dihydrochloride (Sigma FAST OPD peroxidase substrate

Detection of CT by sandwich ELISA technique

A reconstituted monoclonal anti-CT solution with a total protein concentration of 1.41 mg mL−1, measured by BCA method, was employed successfully for the preparation of peroxidase-conjugated anti-CT (data not shown). A calibration curve obtained by ELISA, at several concentrations of CT, has shown a linearity range between 3.1 and 25 ng mL−1, corresponding to 3.7 × 10−11 and 2.9 × 10−10 M, with the regression equation of y = 0.3248x + 3.605 (R2 = 0.9704), where y is the absorbance at 405 nm and x is the

Conclusions

The current study has successfully demonstrated that the developed flow-injection capacitive immunosensor system provides a highly sensitive method for assay of picomolar concentrations of cholera toxin with an attractive analytical performance. The method proved to be more sensitive than the other two techniques employed in this study namely, sandwich ELISA and SPR-based immunosensor. Our data further revealed the high binding affinity of the toxin to the immobilized monoclonal antibody,

Acknowledgments

Ministry of Higher Education, Egypt is gratefully acknowledged for financial support to Mahmoud Labib during his study at the Department of Biotechnology, Lund University. The authors express their gratitude to Professor Ann-Mari Svennerholm, Department of Microbiology and Immunology, Göteborg University for her keen interest, providing them with the anti-CT used in this work and Dr. Bengt Danielsson, Department of Pure and Applied Biochemistry, Lund University for access to their instruments

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