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Development of thermally stable coarse water-in-oil emulsions as potential DNA bioreactors

Abstract:

Conventional polymerase chain reaction (PCR) performed in a continuous aqueous phase often shows an inability to detect ultra-low DNA concentration. Droplet digital PCR, where DNA polymerization happens in isolated water droplets of water-in-oil (W/O) emulsions, could significantly improve the detection limit. However, water droplet breakups under multiple thermal cycles necessary for DNA polymerization is preventing the wide-spread application of such technology. The present research aims to develop thermally stable coarse W/O emulsions that could act as DNA microreactors. Span 80, polyglycerol polyricinoleate (PGPR), or sodium bis(2-ethylhexyl) sulfosuccinate (AOT) were dissolved in mixtures of light and heavy mineral oils at different concentrations. The aqueous phase was selected based on PCR protocol, without the presence of enzymes and DNA. Emulsions prepared without bovine serum albumin (BSA) in the aqueous phase was used as a control to understand its effect on stability. Emulsions were formed using a vortex mixer for 2 minutes at 3200 rpm. Only the emulsions without visible aqueous phase separation were exposed to PCR thermal cycling. The volume mean diameters of water droplets were calculated by image analysis. Span 80-stabilized emulsions destabilized upon thermal cycling, while PGPR-stabilized emulsions remained stable with an increase in water droplet size. AOT was able to form thermally stable emulsions, with an average droplet size around 119 µm without any significant change. The influence of emulsifiers concentration, surface saturation, molecular interaction with BSA at the interface, and continuous oil phase viscosity was considered to explain the mechanism of emulsion formation and thermal stability.