Investigating the Impact of Blocking Monolayer Architecture on the Performance of Electrochemical DNA Biosensors in Microfluidic Channels
Authors: Freisa Martina , Poujouly Claire , Le Potier Isabelle , Dinh Thi-Hong-Nhung , Couraud Laurent , Bouville David , Sella Catherine , Thouin Laurent , Gamby Jean 
Published: December 2024

An electrochemical nucleic acid biosensor typically involves the immobilization of DNA strands onto a surface, which serves as a recognition element for specific DNA or RNA target molecules.1-3 DNA strands can be single-stranded or double-stranded, and their immobilization onto a surface is crucial for sensing applications. This allows the formation of monolayers which is often used to prevent nonspecific adsorption of interfering molecules onto the surface. This layer acts as a barrier, allowing only specific interactions with the DNA target molecules or analytes of interest. Traditionally, redox mediators such as ferrocyanide/ ferricyanide that undergo reversible oxidation-reduction reactions are often used to detect hybridization of DNA targets with immobilized complementary DNA probes. Under these conditions, the heterogeneous charge transfer rate of redox mediators at the electrode surface is often a key factor taken into account to interpret the sensitivity and efficiency of the biosensor.3-5

In this work, with a set of experiments carried out by hydrodynamic voltammetry and impedance spectroscopy at channel microelectrodes, we propose a new approach supported by numerical modeling to highlight the influence of the mass transport of redox mediators on the response of DNA biosensors. Contrary to what is commonly accepted in the literature, we showed that the presence of DNA monolayers acting as blocking monolayers does not affect the charge transfer rate of redox mediators, regardless of the electrode accessibility (surface coverage). We demonstrated that under flow conditions, the current response of the biosensors was controlled by the mass transport rather than the charge transfer rate of redox mediators. This phenomenon was attributed to the nonlinear effects of diffusion-convection of the redox mediators at the remaining active sites, thereby affecting the overall kinetics of the transduction signal. These experiments, coupled with numerical calculations, allowed us to compare the performance of DNA biosensors obtained with different blocking monolayers and to evaluate the average size of the active sites. In this model, they were assumed to be small compared to the electrode size.
This study constitutes an important advance in the field of sensors or biosensors, in the broad sense, as it points out the influence of parameters controlling mass transport and therefore the performance of electrochemical DNA biosensors, such as the density of DNA monolayers, the composition of the blocking agents, and size of holes (active sites).

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