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Miniaturized PCR-based biosensors

Microarrays
Edited by: Zhili He
Focused on current microarray technologies and their applications in environmental microbiology.
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Within the last ten years there have been significant efforts to take PCR out of the laboratory and into the field. This is partially due to an increased demand for rapid and accurate methods of detecting pathogenic bacteria, viruses and other disease-causing agents. Due to its relative accuracy and sensitivity, the polymerase chain reaction (PCR) is well suited to these needs. Miniaturized PCR-based biosensors have been developed utilizing a variety of manufacturing technologies. At least four commercial entities have developed miniaturized PCR-based detection systems with corresponding detection assays targeting DNA from tissue, blood, environmental, or food samples (Herold and Rasooly, 2009. Lab-on-a-Chip Technology. ISBN: 978-1-904455-47-9). These PCR-based systems are both sensitive and robust, but a variety of contaminants can inhibit successful amplification and diminish their sensitivity.

In order to circumvent this problem, DNA is typically extracted and purified from a sample through a variety of lysis protocols and purification techniques. One of the most common methods is chemical lysis followed by DNA purification using silica-based resins. DNA in chaotropic salt containing buffers, such as those containing guanidinium or sodium iodide salts, preferentially binds to silica surfaces, while other macromolecules, such as protein and lipids, remain free in solution. These unwanted components can be removed by various methods, including centrifugation and subsequent alcohol based washing steps. The relatively pure DNA is then eluted in low-ionic strength buffer or water. Simple kits are commercially available based upon particulate matrices with problematic flow rates and no direct integration into chip-based devices. At least one group has reported the incorporation of silica-based resins into a micro-flow device while others have used microfabricated silica pillar structures for the same purpose.

Integrating DNA purification and subsequent reactions on the same chip reduces sample size, conserves sample volume and eliminates complicated steps for the technician. Once DNA has been extracted and purified from a given sample, PCR can be employed to amplify target DNA sequences. For pathogen detection, specific PCR primers can be designed to amplify known pathogenesis genes or other identifying sequences within the chromosome or associated DNA. Various strategies have been used to cyclically heat and cool the chip to the required temperatures using infrared light, thermoelectric coolers, and resistive electrodes (Herold and Rasooly, 2009. Lab-on-a-Chip Technology. ISBN: 978-1-904455-47-9). In addition to changing the temperature of the entire reaction chamber, other methods have used so-called 'flow-through' PCR in which the sample is passed through different thermal regions on the chip. Another approach employs a thermoelectric cooler (TEC) that can alternately heat and cool, depending on the direction of electrical current. These miniature heat pumps are easily controlled with electronic feedback controllers and have relatively low power requirements (5 - 10 watts).

A method for performing microchip-based PCR with an external heat source such as a TEC or resistive heater has been described. Although flow-through PCR and other novel methods of microchip-based thermocycling have been described, external heating of the microchip is still one of the simplest and most reliable methods of performing PCR in lab-on-a-chip systems. Additionally, external heating of the microchip allows for seamless integration of the PCR amplification component with existing microchip designs. Fabrication steps and amplification methods have been successfully used for chip-based PCR amplification (Herold and Rasooly, 2009. Lab-on-a-Chip Technology. ISBN: 978-1-904455-47-9).