(Digital Presentation) An Electrochemical Sensor in a Disc for Real-Time PCR Assisted with Thermal Convective Flow

Abstract

Nowadays, the polymerase chain reaction (PCR) has extensively adapted to microfluidic devices for several features such as the low consumption of reagents and sample, portability, high-throughput, and low fabrication costs [1]. The reduction of the amplification time has been a relevant parameter since the invention of the PCR, where rapid microfluidic PCR is especially preferred for the detection of infectious diseases [1,2]. The continuous-flow-based PCR is a type of microfluidic PCR that proposes the mobilization of the PCR mixture through a microchannel with different fixed temperature zones to achieve the required thermal-cycling [3]. This approach has smaller thermal inertia because only the PCR mixture needs to be heated and cooled instead of the entire microfluidic device. The continuous-flow approach allows rapid-thermal cycling with lower power consumption and, therefore, reduces the overall amplification time. The Lab-on-a-disc, or centrifugal microfluidic platform, has adapted the continuous-flow PCR by utilizing the flow generated by thermal convection in a ring-structured microchannel [4]. During spinning, embedded heaters in the bottom of the CD heat specific section of the microchannel (see Figure 1) at a fixed temperature and, subsequently, create a natural convection flow in the PCR mixture and move through the microchannel. The centrifugal-assisted thermal convection (CATC) technique not only enables the recirculation pathway for the continuous-flow PCR but overcomes the unidirectional nature of centrifugation force on the CD. Despite the advantage of rapid PCR on Lab-on-a-Disc, the CATC technique lacks the implementation of a detection method with high sensitivity and resolution, aiming for a fully integrated microfluidic platform. Electrochemical detection can provide fast responses with minimum power consumption for miniaturized devices. This work proposes the integration of an electrochemical sensor to the CATC technique in a circular microchannel for the real-time monitoring of DNA amplification. The electrochemical sensor consists of interdigitated gold electrodes located above the ring microchannel to quantify the amplicon concentration after each cycle mediated by the intercalation of Methylene Blue between the newly-generated DNA (see figure 1A). The characterization of the sensor is performed through voltammetric techniques such as cyclic voltammetry (CV) and linear sweep voltammetry (LSV), where the latter is utilized for amplicon quantification. To optimize the CATC technique combined with the electrochemical sensor, a COMSOL simulation is performed to improve the amplification ratio and amplification time in the CD by analyzing the microchannel geometry and flow rate (see figure 1B). Both the thermal control for the thermal-assisted microchannel and the electrochemical sensor are powered and controlled via the “electrified Lab-on-a-Disc” (eLoaD) board [5] (see figure 1C). [1] Y. Zhang, P. Ozdemir, Anal. Chim. Acta 638 (2009) 115–125. [2] X. Dong, L. Liu, Y. Tu, J. Zhang, G. Miao, L. Zhang, S. Ge, N. Xia, D. Yu, X. Qiu, TrAC - Trends Anal. Chem. 143 (2021) 116377. [3] M.B. Kulkarni, S. Goel, Eng. Res. Express 2 (2020). [4] M. Saito, K. Takahashi, Y. Kiriyama, W.V. Espulgar, H. Aso, T. Sekiya, Y. Tanaka, T. Sawazumi, S. Furui, E. Tamiya, Anal. Chem. 89 (2017) 12797–12804. [5] S.M. Torres Delgado, J.G. Korvink, D. Mager, Biosens. Bioelectron. 117 (2018) 464–473. Figure 1