Isoelectric trapping and discrimination of histones from plasma in a microfluidic device using dehydrated isoelectric gate.

Abstract

Histones are basic proteins with an isoelectric point around 11. It has been shown that the level of plasma circulating histones increases significantly during sepsis, and circulating free histones are associated with sepsis severity and mortality. It was found that the median plasma total free histone concentration of sepsis ICU non-survivors is higher compared to survivors. Therefore, histone concentration can serve as a prognostic indicator and there is a need for a simple, low-cost, and rapid method for measuring histone levels. In this work, we have developed a microfluidic device containing an isoelectric membrane made of dehydrated agarose gel of a specific pH embedded in a porous membrane for isoelectric trapping of histones rapidly. Although isoelectric gates have been used for trapping proteins before, they have to be introduced at the time of the experiment. Here, we show that isoelectric gates formed by gels loaded in a scaffold can be integrated directly into the fabrication process flow, dehydrated for storage, and rehydrated during the experiment and still function effectively to achieve isoelectric trapping. A low-cost and rapid microfabrication technique, xurography, was used for agarose integration and device fabrication. The integrated device was tested with samples containing buffered histone, histone in the presence of high-concentration bovine serum albumin (BSA), and histone spiked in blood plasma. The results show that the device can be used to distinguish between survivors and non-survivors of sepsis in less than 10min, making it suitable as a point-of-care device for sepsis prognosis.