New novel method for fluorescence-based indirect detection of analytes and demonstrate its use for label-free detection of chemical toxins in a hand-held device.
Environmental monitoring efforts, and water quality assessment in particular, would benefit from widely available and inexpensive chemical assays and sensor technologies.1 Gas and liquid chromatography methods, and their coupling to mass spectrometry, currently are standard methods suggested by the United States Environmental Protection Agency (EPA) for the detection of chemical toxins in drinking water While these methods are considered sufficiently sensitive and accurate, their use is mostly confined to laboratory settings, because of their size, weight, power requirement, peripheral equipment, cost, and sample preparation steps.
There is a need for detection techniques that are cost-effective, sensitive, and portable.
Stanford University has developed novel method to Label-Free Detection of Chemical Toxins in Tap Water by leveraging Fluorescent Carrier Ampholytes Assay.
New fluorescently label a mixture of low-concentration carrier ampholytes and introduce it into an isotachophoresis (ITP) separation. The carrier ampholytes provide a large number of fluorescent species with a wide range of closely spaced effective electrophoretic mobilities. Analytes focus under ITP and displace subsets of these carrier ampholytes.
The analytes are detected indirectly and quantified by analyzing the gaps in the fluorescent ampholyte signal. The large number (on the order of 1000) of carrier ampholytes enables detection of a wide range of analytes, requiring little a priori knowledge of their electrophoretic properties.
One approach toward widespread toxin detection is the miniaturization of traditional chromatography systems. Although there have been efforts to reduce size and weight significantly,4 scaling down and integrating the essential system components remains a challenge.
Much of the work is focused on implementation of an efficient stationary phase in microstructures, and in miniaturization of pressure sources, pumps, and valves. An alternative approach to realizing low-cost and portable toxins detection is developing novel assays that have increased functionality, avoid complex sample preparation (e.g., labeling), and are compatible with inexpensive system architectures. Fluorescence-based detection is the most sensitive method for onchip applications, but this methodology typically requires auto- fluorescent analytes (a property that is not possessed by most toxins of interest) or fluorescent labeling (e.g., using immunoassays ).
Kuhr and Yeung demonstrated indirect detection through displacement of fluorescence-detectable background ions in capillary electrophoresis (CE). However, high analyte concentrations were required for the displacement physics, limiting sensitivity to analyte concentrations of ∼100 µM. Recently, several fluorescence-based indirect detection methods based on isotachophoresis (ITP) have been proposed.9,10 In ITP, sample ions simultaneously focus and separate according to their electrophoretic mobilities between a leading electrolyte (LE) and trailing electrolytes (TE). This creates purified, highconcentration, adjacent zones electromigrating at a uniform velocity.
Chambers and Santiago9 developed an indirect detection method called nonfocusing, wherein sample zones are detected by analyzing the local intensity of a nonfocusing fluorescent tracer molecule as it electromigrates through ITP zones of unlabeled analytes. The NFT method requires only a single fluorophore for multiple analytes, but the fluorescence signal strength is on the order of the initial concentration (which can be limited by selfquenching, wall adsorbance, or interaction with analytes).
Credit: Stanford University