Stephen S. W. Yeung, Chemical Engineering, Hong Kong University of Science and Technology, HKUST, Clear Water Bay, Kowloon, Hong Kong, Thomas Ming-Hung Lee, Department of Chemical Engineering, Hong Kong University of Science and Technology, HKUST, Clear Water Bay, Kowloon, Hong Kong, and I.-Ming Hsing, Department of Chemical Engineering and Bioengineering Postgraduate Program, Hong Kong University of Science and Technology, HKUST, Clear Water Bay, Kowloon, Hong Kong.
We report, to our knowledge, the first electrochemistry-based real-time polymerase chain reaction (PCR) technique for simultaneous DNA amplification and sequence-specific detection. This new method has the advantages (e.g., a short assay time) of the well-established fluorescence-based counterpart. On-chip implementation of this technique in a microreactor will be demonstrated. The underlying principle of our EC real-time PCR method is based on the progressive amplification and incorporation of ferrocene-dUTP (2'-deoxyuridine 5'-triphosphate) redox markers to the oligonucleotide capture probes during the solid-phase extension process. Prior to the PCR, an oligonucleotide probe specific to the target amplicon is immobilized onto the electrode surface within the PCR amplification chamber. During the PCR denaturation step (~ 95 „aC), the double-stranded amplicon is first denatured into a single-stranded one and followed by the hybridization with the immobilized capture probe at annealing temperature (~ 55 „aC). Simultaneously, PCR reactions occurring at the solution phase increase the number of target amplicon copies available for hybridization with the capture probe at the solid electrode. The capture probe is extended with the incorporation of ferrocene-dUTP by the polymerase in each PCR cycle and the redox signals of ferrocene reporters build up in proportion to the amount of amplicons produced in PCR thermal cycles. The most prominent feature of this strategy is the possibility to detect the amplicon signal electrochemically cycle-by-cycle, enabling accurate target quantification over a wide dynamic range. The method presented in this study offers a significant advantage over the end-point sequence-specific detection in an integrated PCR-EC microdevice, previously reported in our group [1]. The microdevice with the ability to carry out electrochemistry-based real time PCR could easily rival with the fluorescence-based equipments for the point of care diagnostic applications. (REFERENCE: [1] Lee, T. M. H.; Carles, M. C.; Hsing, I. M., "Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection", Lab Chip, 2003, vol. 3, no. 2, 100 - 105.)