Microfluidic PCR devices with electrochemical detection of DNA
This research undertaken involved designing, fabricating and testing of a microfiuidic peR micro device with real-time electrochemical detection. The aim was to provide an analytical device which could lower the cost and the time taken for running DNA amplification. The later addition of automated sample handling and detection would thereby reduce the time taken and consequently the overall cost. The real-time electrochemical detection utilised an electrochemical assay for the detection of DNA invented by Molecular Sensing Ltd. It used a single strand ferrocenylated probe DNA molecule which could be detected with an electrochemical cell. The integration of an electrochemical cell was a key feature of this work, along with the immobilisation of the Taq polymerase at its working temperature. Taq polymerase enzyme and T7 polymerase enzyme were immobilised on to microspheres. Taq polymerase was immobilised in four ways and T7 polymerase was immobilised in only one method. After immobilisation the enzymes were unable to amplify DNA within peR experiments. Microfiuidic peR devices, which incorporate the above two features, were designed and fabricated. 3 basic ideas of devices were investigated, fiowthrough, straight line and cyclic triangle device. All the devices had fundamental problems which inhibited there ability to successfully amplify DNA. An electrochemical assay was used within a microfiuidic device with internal electrochemical detection, which utilised a filter to bring about sequence specific DNA detection. Using biotinylated complementary probe DNA attached to streptavidin coated beads to hybridise to the sample DNA. This device incorporated a solid phase extraction and clean up step as well as producing sequence specific DNA detection.