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.

Electrokinetic Mixing for Improving the Kinetics of an HbA1c Immunoassay.

The efficiency of the diagnostic platforms utilizing ELISA technique or immunoassays depends highly on incubation times of the recognition elements or signaling molecules and volume of the patient samples. In conventional immunoassays, long incubation times and excess amounts of the recognition and signaling molecules are used. The technology proposed here uses electrokinetic mixing of the reagents involved in a sandwich immunoassay based diagnostic assay in electrode-enabled microwell plates in such a way that the incubation times and volumes can be reduced substantially. The integration of the electrodes at the bottom of the conventional microwell plates ensures that the motions of the liquid flows in the wells can be controlled through the application of high frequency AC current along these electrodes. The strategy to generate chaotic mixing by modification of standard multiwell plates, enables its use in high throughput screening, in contrast to microfluidic channel-based technologies that are difficult to incorporate into conventional plates. An immunoassay for detection of glycated hemoglobin (HbA1c) that can reveal a patient's average level of blood sugar from the past 2-3 months instead of just measuring the current levels and thereby constitutes a reliable diabetes monitoring platform was chosen as a pilot assay for technology demonstration. The overall incubation time for the assay was reduced by approximately a factor of five when electrokinetic mixing was employed. Furthermore, when the quantity of the reagents was reduced by half, almost no distinguishable signals could be obtained with conventional immunoassay, while electrokinetic mixing still facilitated acquisition of signals while varying concentration of the glycated hemoglobin. There was also a substantial difference in the signal intensities especially for the low concentrations of the HbA1c obtained from electrokinetic mixing assisted and conventional immunoassay when the quantity of the reagents and incubation times were kept constant, which is also an indication of the increase in bioassay efficiency. The electrokinetic mixing technique has the potential to improve the efficiency of immunoassay based diagnostic platforms with reduced assay time and reagent amounts, leading to higher throughput analysis of clinical samples. It may also open new avenues in point of care diagnostic devices, where kinetics and sampling size/volume play a critical role.