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.