Automated flow-through amperometric immunosensor for highly sensitive and on-line detection of okadaic acid in mussel sample
Highlights
► In this study, we developed a fully automated flow based immunosensor for OA detection. ► The system was fabricated by injecting modified MBs into the flow cell as solid support to immobilize OA. ► An indirect competitive immunoassay format under automated flow conditions was performed for OA analysis. ► The developed flow immunosensor increased the sensitivity of system as compared to batch systems. ► The developed immunosensor was validated with mussel sample.
Introduction
The detection of okadaic acid (OA) is a challenging and important issue for shellfish industries worldwide. Okadaic acid (OA) is a lipophilic marine biotoxin produced by Dinophysis and Prorocentrum Dinoflagellates [1]. OA intoxication is considered as the most of concern diarrhetic shellfish poisoning (DSP) for human health. Studies carried out on animals have also proved the cancerogenic, mutagenic and immunotoxic effects of OA [2]. The European commission (EC) has implemented regulation on the concentration of OA, and the maximum permitted level is 160 μg/Kg of mussels (EC No.853/2004 15). The European Food Safety Authority (EFSA) has suggested to decrease the maximum permitted level from 160 μg/Kg to 45 μg/Kg of mussels [3]. Mouse bioassay was recommended as a reference method by legislation [4]. However, its ethical problems in addition to poor selectivity and accuracy led the European Commission to look for alternative methods such as conventional chromatographic method, enzymatic biosensor and immunoassay [5], [6], [7], [8]. Nevertheless, there is still need of simple, sensitive and consistent methods to perform rapid monitoring of real samples for field analysis.
An alternative and interesting approach is the use of flow injection analysis system (FIA) for rapid, sensitive and on-line detection of the target molecules. The potential advantages of the FIA are rapidity, precision, and accuracy due to the high degree of control and constancy of analytical parameters. Furthermore automation makes routine tasks easier and less cumbersome [9], [10]. There are numerous examples of immunoassay automation with colorimetric, fluorescent, chemiluminescent and electrochemical signal transduction steps [11], [12]. Among all these, on-line monitoring by electrochemical means has been emerged as a powerful tool both in research and industrial settings [13], [14]. There are reports of flow systems which combine an immunoassay with an electrochemical detection step using screen printed carbon electrode (SPCE) [15], [16]. The preparation of SPCEs is simple, inexpensive, versatile, mass produced with the possibility of miniaturization. The SPCE based biosensor offers the advantages over the conventional electrochemical biosensors for disposability and portability, and have been extensively used in the fabrication of electrochemical biosensors [17], [18], [19]. The flow immunoassay systems have been reported with micro-plate, bead, membrane and capillary immunoassay formats. However, micro-particles are the most commonly used solid surface for the immobilization of antibody, antigen or relevant reagent. The system based on micro-particle is also known as bead based immunoassay [20]. One of the benefits of the bead based immunoassay is the high surface area per volume of the micro-particles and the possibility to increase the immobilization surface as compared to the restricted area of the fixed size micro-well. The ability to immobilize higher numbers of binding molecules helps to improve the sensitivity and detection limit of the assay [21], [22]. Beads made of various materials are commercially available. However, magnetic beads (MBs) have gained much attraction due to their ease in being retained in the flow system during different steps of biosensor fabrication as compared to non MBs [23]. The effectiveness of online magnetic trapping system in some application of flow injection bead based immunoassay using chemiluminescence and electrochemical detection has been reviewed. However, the development of bead-based immunoassay with electrochemical transduction step has been increased [24], [25].
In previous study, we have shown that a SPCE could be tailored with OA modified streptavidine MBs to produce an indirect competitive immunosensor. This device was successfully used in the batch mode for low level determination of OA in mussel samples [26]. However, the incorporation of the MB based immunosensor into an automated flow system could offer attractive advantages over batch system [27]. To our knowledge, this is the first approach using electrochemical automated continuous flow system for the analysis of OA in mussel. The ultimate goal of this work is the development of a biosensor for OA analysis, which can be incorporated into field analysis to provide online monitoring of the OA in mussel samples. The fully automated flow system is based on the incorporation of OA modified MBs into the central flow cell, which were retained there by the application of an external magnet. The device was connected with a flow injection system and amperometric detection based on an indirect competitive immunoassay was performed for the sensitive and on-line detection of OA.
Section snippets
Reagents
OA potassium salt from Sigma was dissolved in ethanol (0.1 g/L) and subsequently diluted in phosphate buffer saline (PBS 1x). Buffer components, tween 20, bovine serum albumin (BSA), diethanolamine (DEA), 1-naphthyl phosphate (1-NP), alkaline phosphatase (ALP)-labeled goat anti-mouse IgG antibody, N- hydroxysuccinimide (NHS), N-(3-dimethylaminopropyle)-N’-ethylecarbodiimide hydrochloride (EDC) were purchased from sigma (France). EZ-link amine-PEO3-biotin was from Pierce (France). Monoclonal
Optimization of parameters for automated flow detection system
Several factors that affect the biochemical reaction must be considered because the reaction conditions in automated flow immunosensors are different than those of conventional batch immunosensors. Optimization and validation of an analytical system are key factors in the development of an automated flow system. Before using the system for OA detection, it was necessary to optimize the system. Since our flow system is able to perform continuous and stop flow, both techniques were applied to the
Conclusion
In this work, we have extended our previous studies into the use of automated flow system for OA detection. Modified super paramagnetic beads were injected into the flow cell as solid support to immobilize OA and subsequently an indirect competitive immunoassay format was performed for electrochemical detection. This paper presented a novel simple fully automated continuous/stop flow immunoassay method for OA based on a disposable immunosensor integrated to a flow injection system. The flexible
Acknowledgment
Rocio B. Dominguez and Gustavo A. Alonso are very thankful to the National Council of Science and Technology (CONACyT) of Mexico for providing scholarship. Akhtar Hayat is very grateful to Higher Education Commission of Pakistan for financial support. This study was carried out as the part of the research project BIOKA.
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