Electrokinetic mixing in electrode-embedded multiwell plates to improve the diffusion limited kinetics of biosensing platforms

Rapid and accurate biosensing with low concentrations of the analytes is usually challenged by the diffusion limited reaction kinetics. Thus, as a remedy, long incubation times or excess amounts of the reagents are employed to ensure the reactions to go to completion. Therefore, mixing becomes both a serious problem and necessity to overcome that diffusion limitation and homogenize the samples, especially for the biochemical reactions that take place in multiwell plates. Because the current mixing platforms such as shakers/vortexers, sonicators, magnetic stirrers and acoustic mixers have disadvantages including, but not limited to, being invasive/harfmul to the samples, causing the samples to splash out or stick to the walls of the wells and allowing foreign compartments to enter the solutions in the wells. Here we propose a noninvasive and safer (considering the risk of sample loss) technology that provides electrokinetic-mixing (EKM) of the reagents placed in electrode-embedded multiwell plates where the incubation times, or in other words, the time required for the desired molecules to meet in stationary solutions, can be reduced substantially. In order to demonstrate the power of this innovation, in this specific case, a simple Förster resonance energy transfer (FRET) based quenching bioplatform was adopted, where a molecular beacon DNA (MB) modified with sulfhydryl (-SH) and fluorescein (FITC) dye at opposite terminals was incubated with 10 nm sized gold nanoparticles (AuNPs) in the wells of an electrode-embedded multiwell plate, in which a printed circuit board (PCB) was attached at the bottom to control the liquid flows by EKM. When the MB binds to AuNPs through thiolate chemistry in the solution, FITC dye comes in close proximity to the AuNP surface and the emission is quenched via FRET principle. Thus, this quenching percentage over time was our comparison parameter for the mixing and no mixing cases to demonstrate the impact of mixing on the quenching kinetics. This reaction was conducted with different concentrations of AuNPs to observe the impact of mixing on MB quenching kinetics when the concentrations of the AuNPs were increased. Total quenching efficiency could go up to 90% in the presence of the AuNPs and it took about 60 min to reach stability. When the EKM was involved, fluorescence quenching time for the MBs could be reduced by up to 4.1 times. Thus, it was demonstrated that this technology may improve the kinetics of the diffusion limited biological reactions take place in multiwell plates substantially so that it may be adopted in various different sensing platforms for rapid measurements.