Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms.

The extremely low limit of detection (LOD) posed by global food and water safety standards necessitates the need to perform a rapid process of integrated detection with high specificity, sensitivity and repeatability. The work reported in this article shows a microchip platform which carries out an ensemble of protocols which are otherwise carried in a molecular biology laboratory to achieve the global safety standards. The various steps in the microchip include pre-concentration of specific microorganisms from samples and a highly specific real time molecular identification utilizing a q-PCR process. The microchip process utilizes a high sensitivity antibody based recognition and an electric field mediated capture enabling an overall low LOD. The whole process of counting, sorting and molecular identification is performed in less than 4 hours for highly dilute samples.

Dielectrophoresis assisted concentration of micro-particles and their rapid quantitation based on optical means.

The detection and counting of micro particles having sizes comparable to biological entities can provide a tremendous impetus to rapid diagnostics and clinical applications. MEMS technology has already been used in capture and detection of such micron size entities in miniscule concentrations. For this purpose a concentration step is normally added prior to the detection process. A variety of methodologies are used for quantization of such micron size particles/entities including change in permittivity, medium impedance, magnetic permeability and other means. Although optical studies have been extensively performed prior to this, it has not been used for quantization of the micro particles. We have designed, developed and characterized a MEMS counter which captures micron size fluorescent beads using delectrophoresis (DEP) and monitors their accumulation in a 12 µm x 230 µm size channel and monitors this accumulation as growth of overall fluorescence. The field is generated by a set of finely placed interdigitated microelectrodes. As we apply an alternating voltage at 10 V(pp) for a range of different frequencies we are able to capture the flowing beads and concentrate them by several orders of magnitude. This is also followed by their quantification in terms of growing fluorescence signal. For quantitating the fluorescence values a CCD (charge couple device) module fitted over an inverted fluorescence microscope is used that visualizes the whole capture process and a Labview based image acquisition software simultaneously calculates the signal intensity over these frames and arranges it temporally. Our work will have tremendous utility in developing a rapid bacterial counting procedure and will be a valuable tool in microbiological laboratories.