The COVID-19 pandemic has highlighted the need for new biodetection methods that would enable mass population screening, and help controlling the spread of viral diseases. Such technologies could benefit the prevention of many other infectious diseases. For example, monitoring the circulation of arboviruses is of interest in Europe where tiger mosquitoes are now established. DNA biosensors, which enable high-throughput detection, are interesting candidates for this purpose. These sensors rely on the hybridization of a targeted gene with an immobilized DNA probe on a surface. Different transduction techniques can be used to convert the hybridization event into a readable signal. Among them, fluorescence is one of the most commonly used techniques. Electrochemical techniques, including bioimpedance detection, are also prevalent. The functionalization of the sensing surfaces with the DNA probes must be carefully considered. Indeed, it can have a drastic impact on the final biosensor performance. In particular, the surface properties must be precisely tailored to achieve a high sensitivity, eliminating the need for an amplification step and enabling rapid detection.

This work describes the optimization of DNA-conjugated glass and gold surfaces for fluorescence and bioimpedance detection of viral RNA. These surfaces are designed to be inserted in a microfluidic platform, which is part of an automated device. The final system aims at detecting SARS-CoV-2 RNA in human saliva and Zika virus RNA in mosquito saliva in less than 10 minutes.

This manuscript first details the preparation of DNA-conjugated glass surfaces with optimized properties. The hybridization density, as well as the vertical and lateral space around the DNA probes were successfully tailored using organic spacers and peptides as anchoring units. In particular, the DNA hybridization densities reached were among the highest reported values. Additionally, a thermoresponsive polymer layer was later incorporated to obtain sensing surfaces with antifouling properties. One system was then selected for the fluorescence detection of SARS-CoV-2 RNA, and inserted into the platform, achieving a limit of detection in the attomolar range with high specificity. However, subsequent tests revealed specificity issues, and the detection of Zika virus RNA could not be performed with the developed method.

In parallel, gold electrodes were conjugated with DNA probes via the thiol-gold interactions. These electrodes were incorporated in the microfluidic platform for Zika virus gene bioimpedance detection. Unfortunately, no significant changes in bioimpedance changes were observed upon hybridization of the targeted genes with the immobilized probes.

To address the detection challenges faced, the final part of the work involved the development of DNA-conjugated nanoparticles for sandwich assays. These assays were developed for both fluorescence and bioimpedance detection, and are expected to improve the specificity of the detection techniques and enhance the measured signals. Additionally, these nanoparticles were also designed to allow the differentiation of Zika virus Asian and African lineages. The components of these sandwich assays were successfully developed, and lineages identification was performed in solution. Future work will focus on the implementation and optimization of the sandwich assays, by combining the 2D surfaces and the nanoparticles.