A reusable capacitive immunosensor for detection of Salmonella spp. based on grafted ethylene diamine and self-assembled gold nanoparticle monolayers
Introduction
Immunoassays, which have been proved to be a major analytical tool in the fields of clinical diagnostics and environmental analysis [1], are used to detect antibody or antigen concentrations based on biospecific recognition interaction. Although immunoassays have high sensitivity and veracity, the methods of radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), immunoaffinity column assay (ICA) and immunoaffinity fluorimetric biosensor have some limitations, such as the labeled antibody, the radiation hazards, the complicated wash procedure, and a long analysis time, expensive and cumbersome instruments and/or skillful operators [2], [3].
Nowadays, several direct immunosensors based on electrochemical transducers, such as potentiometric [4], [5], amperometric [6], conductimetric [7] and impedimetric [8] sensors, have been developed. Among the various types of immunosensors, the capacitive immunosensor has been extensively investigated in recent years, which is based on the determining small changes of capacitance at the electrode–electrolyte interface after the interaction between an antibody (Ab) and an antigen (Ag). The change of capacitance is due to the change of the thickness and/or dielectric behavior after the analyte adsorbs on or binds to the biorecognition element on the electrode surface according to the electrical double-layer theory [9]. The capacitive immunosensor has good characteristics of high sensitivity and label-free, and the measurement can be made by using simple instrumentation [10]. The change of capacitance can be detected by numerous techniques, such as perturbation with potentiostatic step [11], field-effect capacitor [12] and electrochemical impedance spectroscopy (denoted as EIS) [8], [13]. EIS in the biorecognition process, such as antigen–antibody [14], oligonucleotide–DNA interactions [15], is regarded as an effective technique for detecting the features of the interfacial state.
Gold nanoparticles, which have good characteristics of easy preparation, good biocompatibility and relatively large surface, are widely used in the field of biological studies [16], [17]. They have been directly linked onto the surface of electrode via various strategies, such as covalent linking, electrodeposition, electroless deposition, sol–gel [18], [19], [20], [21], and have been used to immobilize DNA [22], [23], cells [24], cytochrome [25], [26] and enzyme [27], [28], and so on. Recently, gold nanoparticles have been used to immobilize the antigen or antibody onto the surface of electrode to manufacture the electrochemical immunosensor [29], [30].
Salmonellosis is a serious health concern and a major cause of many food poisoning cases. Detection of Salmonella is of outmost importance in the food industry [31]. Conventional culture methods for detection of salmonella in foods include blending of the food product in a non-selective medium to increase the population of the target organism, followed by plating onto selective or differential agar plates to isolate pure cultures [32], and then examining the cultures by phenotypic analysis or metabolic markers. A major drawback is that these methods are labor-intensive, take 2–3 days for results and up to 7–10 days for confirmation. Various validated immunoassays and biochemical analysis, as well as other “rapid” analytical methods, also require pre-enrichment of samples and a minimum time between 24 and 48 h for the assay. These techniques, which are mostly based on either polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA), involve the use of various enzymes and reagents for colorimetric or fluorescent end-point detection, and require additional equipment. Salmonella is a common, important pathogen of zoonosis. It ranks first among all food poisoning agents in terms of number of cases, causing great damage to national economies. Detection of Salmonella is of great importance in public health, food hygiene, animal science and veterinary service, and import–export inspection and quarantine. At present, some methods of detecting salmonella have been reported, which mainly include piezoelectric immunosensor [33], surface plasmon resonance immunosensor [34], [35], direct-binding optical grating coupler immunosensor [36], immunosensor based on imaging ellipsometry [37], [38], amperometric immunosensor [39], quartz crystal microbalance (QCM) immunosensor [40], immunosensor based on electrochemical impedance spectroscopy [14], [41].
In this paper, a novel immobilization procedure for antibody based on ethylene diamine and gold nanoparticles (denoted as NGs) monolayers was investigated. Due to the formed carbon–nitrogen bonds on the surface of glassy carbon electrode by electrochemical method, the organic monolayer film of ethylene diamine was obtained [42]. With NGs assembled by the other amino groups of film [43], [44], the antibody of McAbs could be immobilized on the electrode surface. When the immobilized antibody was immersed in the solution which contained the specific antigen, the formed immuno-complexes resulted in the increase of the dielectric layer, and the relative change in capacitance was then obtained. Under the optimized conditions, the proposed immunosnensor showed the linearity between the relative change in capacitance and the content of antigen in the range of 1.0 × 102 to 1.0 × 105 CFU mL−1 with the detection limit of 1.0 × 102 CFU mL−1. Moreover, the reproducibility and stability of immunosensor were also investigated in details. Finally, the proposed immunosensor was also successfully used for the detection of Salmonella spp. in lab-processed commercial pork samples.
Section snippets
Chemicals and solutions
Antibody and antigen were made by Jiangsu Key Lab of Zoonosis, Yangzhou University. Bovine serum albumin (BSA) was purchased from Sino-American Biotechnology Company (Shanghai, PR China). Hydrogen tetracholoroaurate tetrahydrate (HAuCl4·4H2O) and anhydrous ethanol (A.R.) were purchased from Shanghai Chemical Reagent Co. (Shanghai, PR China). 0.1 M ethylene diamine solution containing 0.1 M LiClO4 was prepared with ethanol. 0.1 M phosphate buffer solution (PBS, pH 7.0) containing 0.1 M KNO3 was
Characterization of the modifying process
Fig. 1 exhibits the cyclic voltammograms of 0.10 M ethylene diamine in 0.1 M LiClO4 ethanol solution on the surface of GCE in the potential of 0.0–1.5 V at the scan rate of 20 mV s−1. An obvious irreversible anodic peak at about +1.2 V was observed at the first cyclic sweeping. The reaction process was attributed to the electrochemical oxidation of one amino group of ED molecule to amino cation radical, and then carbon–nitrogen bonds on the surface of glassy carbon electrode formed [31]. The other
Conclusions
In this study, the simple method of fabricating the capacitive immunosensor and the capacitive sensing method for the detection of Salmonella spp. was described. Compared with the methods mentioned in literatures which was used to only detect one of Salmonella spp., namely Salmonella typhimurium [42], [43], the novel capacitive immunosensor could recognize the Salmonella spp. by the electrochemical impedance spectroscopy technique. At the same time, the capacitive immunosensor performs the
Acknowledgements
This project was financially supported by the National Natural Science Foundation (No. 20675071) of China and Fund of Key Lab of Analytical Chemistry for Life Science, Ministry of Education (KLACLS07005), the Hi-tech Research and Development Program grant 2007AA02Z419 from the Ministry of Science and Technology of China, and Grant BE2008655 and BE2008011 from Jiangsu Provincial Government.
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Gong-Jun Yang and Jin-Lin Huang contributed equally to the present study.