Multi-analyte explosive detection using a fiber optic biosensor
Introduction
Accurate and rapid monitoring of the environment for pollutants has become increasingly important in the last few years. Emphasis on reducing analysis time, sample size and overall cost has resulted in the development of multi-analyte immunosensors for simultaneous detection of multiple antigens. There are numerous approaches that are being actively examined for multi-analyte detection. Most commonly, antibodies or antigens are localized in the same assay zone labeled with distinct labels such as radioactive markers [1], different fluorescent molecules [2], different enzymes [3], colored latex particles [4], or particles of different sizes [5]. However, a loss of sensitivity is usually observed in the multi-analyte format as compared to the single-analyte detection format. The cause is attributable primarily to difficulties in discriminating between the signals generated by the different labels. An alternative approach utilizes spatially separated assay zones applying the same or different labels [6], [7].
The two most commonly found explosives in environmentally contaminated sites are TNT and RDX. Current U.S. EPA methodology for their detection utilizes reverse phase HPLC. Although very accurate, this technique usually requires pre-concentration of the samples, followed by the extraction of the explosive material, resulting in long analysis time and high cost [8], [9], [10]. Moreover, HPLC has been limited to the laboratory only. Recently, commercially available on-site detection kits for explosives have been introduced [11], [12], [13]. Some of these are antibody assays, based on modified versions of an enzyme-linked immunosorbent assay (ELISA). However, several washing steps, significant waste generation, long analysis time, and the higher cost associated with this methodology, restrict the application of such kits.
The need for accurate, on-site, fast and economical monitoring of the environment for contaminants has triggered the development of a variety of portable, easy to operate, low-cost biosensors. The fiber optic biosensor possesses these characteristics. It employs evanescent wave sensing and molecular recognition to specifically detect explosive analytes [14], [15], [16]. Optical fibers, which have the cladding removed from the distal end to form the sensing region, are attached to a small, portable, fiber optic biosensor, the Analyte 2000 [17]. This sensor is capable of monitoring four optical fiber probes simultaneously. This ability permits duplicate analysis for improved reliability and confidence. Field demonstration of the fiber optic biosensor for TNT has been documented [16].
In this study, the fiber optic biosensor [17] and the competitive immunoassay were developed further to detect RDX and then to perform multi-analyte explosive detection. Two fiber probes coated with antibodies against TNT and two fiber probes coated with antibodies against RDX were connected in series, thus creating spatially separated assay zones. A mixture of the two fluoroscent explosive analogs, Cy5-ethylenediamine-RDX hapten (Cy5-EDA-RDH) and Cy5-ethylenediamine-trinitrobenzene (Cy5-EDA-TNB) was run over the fibers in a competitive immunoassay. Initial work demonstrated the need for changing the detergent employed in the buffers [18]. For a multi-analyte sensor to be useful, it must retain the analytical capacity of the individual sensor. Therefore, we compared the performance of the multi-analyte fiber optic immunosensor for simultaneous detection of TNT and RDX to the performance of the fiber optic immunosensor for detection of a single explosive only. Specifically, limit of detection, standard curve characteristics, and cross-reactivity effects were investigated. Our results clearly demonstrated that the individual fiber optic probes can be connected in series to form a multi-analyte immunoassay with minimal cross-reactivity and achieve analytical performance as accurate as that of the single analyte assays.
Section snippets
Materials
N-succinimidyl-4-maleimidobutyrate (GMBS) and 3-mercaptopropyltrimethoxysilane (MTS) were obtained from Fluka. Anti-TNB monoclonal antibodies (α-TNT-Ab, monoclonal antibody no. IgG50359), RDX hapten, and anti-RDX-hapten (RDH) monoclonal antibodies (a-RDH-Ab, monoclonal antibody no. IgG50591) were purchased from Strategic Diagnostics (Newark, DE). Standard solutions of TNT and RDX were purchased from Radian International (Austin, TX). Cy5-EDA-TNB was synthesized as described before [14]. The
TNT detection: single analyte assay
Fig. 1 represents the percent inhibition of the reference signal as a function of TNT concentration (1–1000 ng/ml), after performing competitive immunoassays on anti-TNT fibers employing the fluorescent analog Cy5-EDA-TNB in either DOC or Tween 20. As mentioned earlier, all previously reported work for explosive detection used Tween 20 in the assay buffers but to prevent possible fluorophore aggregation, DOC is the detergent of choice. The limit of detection is defined as the signal value
Discussion
In this paper, individual assays for TNT and RDX employing an ionic detergent were characterized for range and limits of detection. The performance of a multi-analyte assay using the fiber optic immunosensor for the detection of TNT and RDX was compared to the performance of the individual assays. Specifically, limits of detection, standard curve characteristics, and cross-reactivity effects were investigated.
In order to prevent fluorophore aggregation on the fiber surface, the ionic detergent
Acknowledgements
This work was funded by the Environmental Security Technology Certification Program (ESTCP) and by the Security Environmental Research and Development Program (SERDP). The opinions and assertions contained herein are not to be construed as official policy or reflecting the views of the U.S. Navy or Department of Defense.
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