A Miniaturized Device for Performing PCR, Integrated with an Electrochemical DNA Biosensor for Detection of Corynebacterium Diphtheriae

Abstract

Introduction Corynebacterium diphtheriae by secreting harmful toxin causes lethal disease called diphtheria. It occurs mainly in underdeveloped areas of the world with low vaccination rate [1]. Rapid detection of this pathogen is crucial to prevent serious health complications of the patient and spreading of the epidemic. As an alternative to time-consuming phenotypic analysis, a device has been designed, which allows the fast detection of the pathogen based on biological interactions at the molecular level, which reduces the risk of error. This device consists of a microfluidic system for performing a fast polymerase chain reaction (PCR), which is integrated with a DNA biosensor. It enables fast and unambiguous detection of bacteria in the sample, therefore it can be potentially used as a point-of-care device. Microfluidic PCR device The concept of rapid bacterial analysis is based on the amplification of a DNA fragment specific for a pathogen followed by its fast detection with e.g. biosensor. A miniaturized system for carrying out PCR was developed. The geometry of the microchannels was designed using CAD software and then micromilled in poly(methyl methacrylate). The microsystem was connected with miniaturized peristaltic pump and with a valve to enable automatic introduction, circulation and removal of the solution from the system. Moreover, a set of six heaters was used to divide the microsystem into three temperature zones. One cycle of the solution in a microchip corresponded with one reaction cycle. Temperatures of three zones were measured and controlled by resistance temperature sensor and set to the suitable values for following reaction steps: denaturation, annealing and elongation. Using the microsystem, any number of reaction cycles could be carried out and peristaltic pump enabled the medium flow in wide range (4 - 150µL/min). A number of both standard and asymmetric PCR reactions were carried out in the microchip using various polymerases, of which the Phire polymerase gave the most promising results. The products were analyzed by electrophoretic techniques and compared with the results of reactions carried out in thermocyclers. The ultimate goal is direct detection of the reaction product using a DNA biosensor. DNA biosensor An electrochemical DNA biosensor has been developed as detection part of a device. Its receptor layer was based on a hairpin like DNA probe labeled with methylene blue (MB). After the asymmetric PCR reaction the hybridization process took place which results in formation of double stranded DNA. The changes in hairpin like structure influenced the registered current and the same the biosensor response, proportional to PCR product concentration [2]. Results and Conclusions The miniaturized device consisting of a microchip with designed channel geometry, connected to the pump and valve was constructed. It enabled to automatically control the flow of a solution in a microchip and to carry out any number of reaction cycles. The reaction mixture changed its temperature rapidly when it flowed between different zones of the microchip, which contributed to a significant reduction in the reaction time. Asymmetric PCR was successfully carried out using the designed device in just 15 minutes. The electrochemical genosensor enabled detection of ssDNA, and its electrochemical signal was directly proportional to the concentration of the analyte in 0.0125-0.25µmol/L range. Studies on the effects of different factors, such as probe sequence, temperature, magnesium ion concentration and addition of dimethyl sulfoxide on electrochemical signal of a biosensor have also been performed.The constructed microchip allowed to significantly shorten the PCR reaction time, when compared with the thermocycler. Integration of microPCR with the genosensor might results in construction of fully automatized portable device enabling rapid and reliable pathogen detection. This type of instrument could be used in medical diagnostics as the point-of-care device contributing to the rapid detection of the disease, hence preventing the spread of the epidemic [3].