PCR-based detection in a micro-fabricated platform

Abstract

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.

Review: Microbial analysis in dielectrophoretic microfluidic systems

“…In a similar process described by Perch-Nielsen et al 165 , pDEP was used to hold Saccharomyces cerevisiae cells, while haemoglobin and heparin were removed. In another integrated system reported by Bhattacharya et al 166 , one set of electrodes inside the bigger chamber (0.6 μL volume) was used to divert bacterial cells from a flow stream into a 0.4 nL chamber, wherein a set of interdigitated electrodes were used to specifically capture and enrich the bacterial cells by DEP. In this manner, Listeria monocytogenes V7 cells were identified using as few as 60 cells collected from a sample of 10 4 CFU/mL in less than 90 min.…”

Focusing of sub-micrometer particles and bacteria enabled by two-dimensional acoustophoresis

“…Thus, considerations such as initial sample volume or batch volume are no longer relevant. Previously used methods for handling of sub-micrometer particles included filters, 4,5 dielectrophoresis, [6][7][8] inertia in combination with hydrodynamic forces, 9 magnetophoresis, 10,11 deterministic lateral displacement, 12 and surface acoustic waves (SAW). 13 These methods have been mainly used for handling of bacteria and particles of around 1 μm in diameter.…”

On a Chip

“…Taking advantage of the small volume of the biochip, 10 4 to 10 5 fold concentrations of bacterial cells from a dilute sample can be achieved. Similar platforms can be used to perform Polymerase chain reaction amplification and detection of as few as 10-50 cells of Listeria monocytogenes has been performed using optical fluorescence detection [27]. …”

“…Blood-borne metastasis is initiated by cancer cells that are transported through the circulation from the primary tumor to vital distant organs, and it is directly responsible for most cancer-related deaths [27]. However, CTCs are extraordinarily rare (estimated at one CTC per billion normal blood cells in the circulation of patients with advanced cancer) and they have proven very difficult to isolate in sufficient numbers to be clinically useful.…”