Towards the improved monitoring of bacterial infections by the isolation of DNA from human serum using ionic-liquid-based aqueous biphasic systems

Abstract

Early infection diagnosis is crucial to decrease morbidity and mortality rates. However, complex biological samples, like human blood or serum, contain high abundance proteins and metabolites that reduce the sensitivity of methods used to identify and quantify nucleic acids in bacterial infections diagnosis. To address this issue, we investigated aqueous biphasic systems (ABS) composed of polypropylene glycol 400 and cholinium-based ionic liquids (ILs) at different pH values for the pre-treatment of human serum, aiming the separation of DNA from human serum albumin (HSA) to reduce the interference on the DNA quantification by real-time PCR (qPCR). Remarkable extraction efficiencies of DNA to the IL-rich phase were obtained with all investigated systems, ranging between 90 and 100% in a single-step, with no significant losses of DNA observed (yield at the IL-rich phase > 90%). At low pH values HSA precipitates, whereas at neutral pH no HSA precipitation is observed. This trend suggests that IL-based ABS can be tuned to selectively isolate DNA from HSA by adjusting the pH. The most effective ABS identified is composed of cholinium glycolate at pH 5, allowing to completely precipitate HSA at the ABS interface and leading to an IL-rich phase enriched in DNA with high purity (>98%) that can be quantified by qPCR. Finally, it is shown that the IL-rich phase is able to maintain the DNA’s structural integrity at room temperature, for up to six months, implying that the IL-rich phase of the selected ABS could also be a suitable DNA storage medium. In summary, designed IL-based ABS can be applied as a pretreatment strategy of human serum, allowing to isolate bacterial DNA and opening new perspectives in the monitoring of bacterial infections.