Muhammad J. A. Shiddiky Md. Aminur Rahman Jang-Su Park Yoon-Bo Shim Department of Chemistry and Center for Innovative BioPhysio Sensor Technology, Pusan National University, Keumjeong-ku, Busan, South Korea Received September 12, 2005 Revised December 30, 2005 Accepted December 30, 2005 Research Article Analysis of polymerase chain reaction amplifications through phosphate detection using an enzyme–based microbiosensor in a microfluidic device An electrochemical method was developed for analyzing PCR amplification through the detection of inorganic phosphates (Pi). This method coupled a microchip to a nanoparticle comprising poly-5,2’-5’,2’’-terthiophene-3’-carboxylic acid (poly-TTCA)/ pyruvate oxidase (PyO) modified microbiosensor. It detects Pi produced from the pyrophosphate (PPi), which is released as a byproduct of PCR. After completion of PCR, PPi is hydrolyzed to Pi by inorganic pyrophosphatase. On the microbiosensor surface, pyruvate was converted to H 2 O 2 by PyO in the presence of Pi and oxygen, and subsequently, the anodic current of enzymatically generated H 2 O 2 was detected at 10.5 V versus Ag/AgCl. The CE-EC analysis was completed within 2 min in a coated channel with 75.0 mm separation length at the field strength of 2200 V/cm. Excellent operation stability of poly-TTCA/PyO was observed for a long period of analysis. The reproducibility of the analysis yielded an RSD of 3.4% (n = 22) for the peak areas and 1.8% (n = 22) for the migration times. The sensitivity of the analysis was 0.59 6 0.01 nA/cycle with a regression coefficient of 0.971. Keywords: Electrochemical detection / Inorganic phosphate / Microbiosensor / PCR / Pyrophosphate DOI 10.1002/elps.200500679 1 Introduction The PCR technique is a powerful tool for amplifying trace amounts of nucleic acids [1]. At the primer extension step of PCR, onemole of pyrophosphate (PPi) is generated for each mole of deoxyribonucleotide monophosphate incor- porated by the DNA polymerase [2–4]. The produced-PPi can be hydrolyzed into two equivalent inorganic phosphate (Pi) ions by inorganic pyrophosphatase (PPase) [4]. Thus, detection of the total amount of Pi is described as a measure of PCR amplification. Amplification of nucleic acids in biomedical and biochemical researches has been used for diagnosing disease, sequencing, genotyping, and evolutionary studies [5, 6]. Such applications of PCR require highly sensitive, fast, selective, and accurate detection methods. Therefore, there has been recent interest in developing an accurate, sensitive, selective, and fast detection method for PCR amplification. CE with electrochemical detection (CE-EC) is a suitable technique for the analysis of electroactive analytes be- cause of its high sensitivity, high separation efficiency, speed, portability, disposability, cost, and minimal con- sumption of analytes/reagents [7]. The CE-EC method has other advantages, including inherent miniaturization of the electrode and supporting instruments, compat- ibility with advanced micromachining technology, and independence of the optical path length or sample tur- bidity. Therefore, PCR products are analyzed by several CE-EC methods coupled with plasma-sputtered thin-film Pt electrode [8], gold- and silver-plated electrodes [9], and conducting polymer (CP)-modified screen-printed electrode [10]. All of these methods rely on detecting amplified-dsDNA as a product of the PCR. However, there has been no report of PCR amplification based on EC of amplified-Pi or PPi as a byproduct of the PCR fol- Correspondence: Professor Yoon-Bo Shim, Department of Chemis- try and Center for Innovative BioPhysio Sensor Technology, Pusan National University, Keumjeong-ku, Busan 609–735, South Korea E-mail: ybshim@pusan.ac.kr Fax: 182-51-514-2430 Abbreviations: AFM, atomic force microscope; BR, buffer reservoir; CP , conducting polymer; DR, detection reservoir; EDC, 1-ethyl-3(3- (dimethylammonium)-propyl)carbodiimide; FAD, flavin adenine dinu- cleotide; Pi, inorganic phosphates; poly-TTCA, poly-5, 2’-5’,2’’- terthiophene-3’-carboxylic acid; PPase, pyrophosphatase; PPi, pyrophosphate; PyO, pyruvate oxidase; SR, sample reservoir; TTP , thiamine pyrophosphate chloride Electrophoresis 2006, 27, 2951–2959 2951  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com