Biosensors play a crucial role in various fields, including medical diagnostics, environmental monitoring, and food safety. Enhancing the sensitivity of biosensors is essential for improving their performance and reliability. One effective approach to achieve this goal is by reducing the distance of electrodes, a parameter that significantly influences the sensor's response. Employing vertical structures to reduce electrode gaps offers several advantages. the heightened controllability of vertical structures facilitates easy adjustments to element size and shape, catering to specific application requirements and maximizing sensor performance. The choice of vertical structures presents a more accessible, flexible, and cost-effective solution from a manufacturing standpoint, particularly for large-scale sensor production.On the other hands, DNA testing plays a pivotal role in the context of COVID-19, demonstrating its profound significance in the identification, monitoring, and management of the virus. Through the analysis of genetic material, particularly RNA in the case of COVID-19, DNA testing enables accurate and timely diagnosis of infections. This precision is instrumental in differentiating the virus from other respiratory illnesses, facilitating early intervention and containment measures. Moreover, the genetic insights garnered from DNA testing contribute to a better understanding of the virus's behavior, mutations, and potential therapeutic targets, thereby informing public health strategies and vaccine development. The integration of DNA testing in the comprehensive approach to combating COVID-19 underscores its indispensable role in mitigating the impact of the pandemic on global health.In this study, we present the development of a novel biosensor based microwell array structure. The fabrication process employed a layer-by-layer method. To be brief, a 4-inch silicon wafer with a 300 nm thick thermal SiO2 was cleaned in acetone, isopropanol, and water. Subsequently, the bottom electrode was deposited by E-gun evaporator followed by a lift-off process. An extremely thin 45nm aluminum oxide layer was deposited by Atomic Layer Deposition (ALD). Then, a 100nm gold layer was deposited as the top electrode by E-gun. Finally, conclude the fabrication by defining the sensing area of the microwell array through Lithography and several etching steps.For the DNA immobilization process, the Covid-probe ssDNA was incubated with the biosensor at 4℃ within the PDMS wells overnight, followed by rinsed 3 times. And 1 uM Covid-target ssDNA was incubated after immobilization of probe DNA. The condition for the reaction of target and probe was 1 hour at room temperature, followed by rinsed 3 times in DI water.The detection was achieved using a combination of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), collectively offering a comprehensive approach for accurate and reliable measurements. The electrolyte consists of 1 mM KCl solution as supporting electrolytes and 5 mM ferricyanide/ferrocyanide as redox pair. The Rct charge transfer resistance of bare device is around 1320 Ohm due to the small sensing area of around 500 * 500 um2. After incubation with the probe DNA, the Rct is around 2150 Ohm indicating that the probe DNA binds to the Au surface successfully. Target DNA was quantitatively measured over a wide concentration range, spanning from 10 pM to 10 nM. The Rct variance from 2260 to 4970 Ohm.Our results demonstrate the biosensor's exceptional performance in terms of sensitivity and dynamic range, showcasing its potential application in the early and accurate diagnosis of Covid-19. The micro-well array biosensor, with its unique design and integrated electrochemical measurement strategy, holds promise for future advancements in point-of-care diagnostics and environmental monitoring. Figure 1
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