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Defense & Security

Lab-on-a-chip detector developed for Sarin

Diagnostic techniques for Sarin, a highly toxic nerve agent, have been translated to a real-time, low-cost microfluidics device.
12 February 2007, SPIE Newsroom. DOI: 10.1117/2.1200702.0658

Sarin, a potent chemical warfare agent, is an organophosphate that is highly toxic to humans as it acts as a cholinesterase inhibitor that disrupts neuromuscular transmissions. Because such nerve agents are also colorless and odorless, they can be introduced into drinking water as a means of terrorist sabotage.1–3 Hence, there is a need for on-site, portable detectors such as ‘lab-on-a-chip’ devices. The greatest challenge in this approach is the integration of a microfluidic device into a fully miniaturized system.

Microfluidics presents new possibilities for controlling small amounts of fluids for precise dispensation. This reduces reagent consumption for on-line chemical analysis and real-time process monitoring, and is therefore simpler and cheaper than laboratory-based instruments. Many ways of detecting organophosphates have been tested using enzymes and fluorescent microbeads/microspheres as the recognizing element.4–13 In this project, we use an enzyme recognition cum optical method, i.e., embedding optical fibers in the microchip to detect Sarin in suspect water samples.

The diagnostic technology we currently use was incorporated into a microfluidic platform. We divided our research into four design and fabrication segments leading to a lab-on-a-chip design: enzyme-nerve agent reaction, optical detection, sample cleanup, and in-situ nerve agent regeneration and stabilization. In this paper we report on the first and second phases. The latter two are to be implemented later.

In the present set-up (see Figure 1), we indirectly detect Sarin by measuring the inhibition of cholinesterase introduced into suspect water samples. The microfluidic device was fabricated by polymeric micromachining with PMMA (polymethyl methacrylate) as the substrate. A chromophore, DTNB (5,5′-dithio-bis-2-nitobenzoic acid), was used to measure remnant cholinesterase activity (see Figure 2), which is inversely related to the amount of Sarin present in the sample. Because the software that registers the data for the microchip cannot convert voltage values into absorbance, we conducted an experiment using gluthathione (GSH), an enzyme, to convert the chromophore DTNB to a colored anion. This provided a calibration plot relating voltage to absorbance (see Figure 3).

Figure 1. a) The desktop-based optical setup and b) the microfluidic chip.

Figure 2. Chemical equation of gluthathione (GSH, GSSG) reacting with the chromophore, DTNB (yellow anion).

Figure 3. Calibration chart of voltage against absorbance, the latter calculated using the equation: y=11.837x−0.272.

Several design aspects had to be optimized to fabricate the microchip, including optical detection path-length, flow rate, enzyme concentration (to determine the lowest detectable enzymatic reaction), substrate concentration, and chromophore concentration.

We then conducted experiments with Sarin to determine the feasibility, and hence the detection limit, with the optimized microchip. Sarin inhibits the enzyme, thereby reducing its activity. Changes in the optical absorbance of the chromophore DTNB in the assay were used to determine the activity of the enzyme and hence indirectly correlate the level of Sarin in the solution. A linear correlation between the logarithm of the remaining uninhibited enzyme activity and Sarin concentration could be calculated by the following equation (assuming a pseudo-first order reaction): This latter condition is achieved when the level of Sarin is much greater than the amount of enzyme used in the assay. The equation thus determines log percentage enzyme activity against Sarin concentration (i.e., percentage activity=inhibited enzyme activity/original enzyme activity). As shown in Figure 4, an increase in the Sarin concentration reduces enzyme activity. Using 20% enzyme inhibition as the cut-off level for positive detection, the lowest concentration of Sarin detectable by the microfluidic set-up is 2.5nM (nanomoles).

Figure 4. % Enzyme activity activity correlating to Sarin concentration.

We have translated part of the current agent diagnostic technology into a microfluidic device for rapid, sensitive detection of the chemical warfare agent, Sarin. The method is simple, fast, and as sensitive as gas chromatography. Future work will enhance the optical detection and the specimen mixing efficiency in the microchip, and will produce a portable device with a built-in light source and detector.

Hsih-Yin Tan, Weng-Keong Loke, Yong-Teng Tan
DSO National Laboratories
Nam-Trung Nguyen
Nanyang Technological University