Efficient capture of target analyte on biosensor platforms is a prerequisite for reliable and specific detection of pathogenic microorganisms in a microfluidic chip. Antibodies have been widely used as ligands; however, because of their occasional unsatisfactory performance, a search for alternative receptors is underway. Heat shock protein 60 (Hsp60), a eukaryotic mitochondrial chaperon protein is a receptor for Listeria adhesion protein (LAP) during Listeria monocytogenes infection. This paper reports application of biotinylated Hsp60 as a capture molecule for living (viable) L. monocytogenes in a microfluidic environment. Hsp60, immobilized on the surface of streptavidin-coated silicon dioxide exhibited specific capture of pathogenic Listeria against a background of other Listeria species, Salmonella, Escherichia, Bacillus, Pseudomonas, Serratia, Hafnia, Enterobacter, Citrobacter, and Lactobacillus. The capture efficiency of L. monocytogenes was 83 times greater than another Listeria receptor, the monoclonal antibody, mAb-C11E9. Additionally, the capture rate was further increased on a Hsp60-coated biochip by 60% when a dielectrophoresis force was applied for 5 min at the beginning of the final 1 h incubation step. Our data show that Hsp60 could be used for specific detection of L. monocytogenes on a biochip sensor platform.

In this paper, we present a new impedance-based method to detect viable spores by electrically detecting their germination in real time within microfluidic biochips. We used Bacillus anthracis Sterne spores as the model organism. During germination, the spores release polar and ionic chemicals, such as dipicolinic acid (DPA), calcium ions, phosphate ions, and amino acids, which correspondingly increase the electrical conductivity of the medium in which the spores are suspended. We first present macro-scale measurements demonstrating that the germination of spores can be electrically detected at a concentration of 10(9) spores ml(-1) in sample volumes of 5 ml, by monitoring changes in the solution conductivity. Germination was induced by introducing an optimized germinant solution consisting of 10 mM L-alanine and 2 mM inosine. We then translated these results to a micro-fluidic biochip, which was a three-layer device: one layer of polydimethylsiloxane (PDMS) with valves, a second layer of PDMS with micro-fluidic channels and chambers, and the third layer with metal electrodes deposited on a pyrex substrate. Dielectrophoresis (DEP) was used to trap and concentrate the spores at the electrodes with greater than 90% efficiency, at a solution flow rate of 0.2 microl min(-1) with concentration factors between 107-109 spores ml(-1), from sample volumes of 1-5 microl. The spores were captured by DEP in deionized water within 1 min (total volume used ranged from 0.02 microl to 0.2 microl), and then germinant solution was introduced to the flow stream. The detection sensitivity was demonstrated to be as low as about a hundred spores in 0.1 nl, which is equivalent to a macroscale detection limit of approximately 10(9) spores ml(-1). We believe that this is the first demonstration of this application in microfluidic and BioMEMS devices.

We present a novel, on-chip system for the electrokinetic capture of bacterial cells and their identification using the polymerase chain reaction (PCR). The system comprises a glass-silicon platform with a set of micro-channels, -chambers, and -electrodes. A platinum thin film resistor, placed in the proximity of the chambers, is used for temperature monitoring. The whole chip assembly is mounted on a Printed Circuit Board (PCB) and wire-bonded to it. The PCB has an embedded heater that is utilized for PCR thermal cycle and is controlled by a Lab-View program. Similar to our previous work, one set of electrodes on the chip inside the bigger chamber (0.6 microl volume) is used for diverting bacterial cells from a flowing stream into to a smaller chamber (0.4 nl volume). A second set of interdigitated electrodes (in smaller chamber) is used to actively trap and concentrate the bacterial cells using dielectrophoresis (DEP). In the presence of the DEP force, with the cells still entrapped in the micro-chamber, PCR mix is injected into the chamber. Subsequently, PCR amplification with SYBR Green detection is used for genetic identification of Listeria monocytogenes V7 cells. The increase in fluorescence is recorded with a photomultiplier tube module mounted over an epifluorescence microscope. This integrated micro-system is capable of genetic amplification and identification of as few as 60 cells of L. monocytogenes V7 in less than 90 min, in 600 nl volume collected from a sample of 10(4) cfu ml(-1). Specificity trials using various concentrations of L. monocytogenes V7, Listeria innocua F4248, and Escherichia coli O157:H7 were carried out successfully using two different primer sets specific for a regulatory gene of L. monocytogenes, prfA and 16S rRNA primer specific for the Listeria spp., and no cross-reactivity was observed.