Immuno-cytometric detection of circulating cell free methylated DNA, post-translationally modified histones and micro RNAs using semi-conducting nanocrystals.

The diagnostic potential of cell free epigenomic signatures is largely driven by the fact that manifold quantities of methylated DNA, post-translationally modified histones and micro RNAs are released into systemic circulation in various non-communicable diseases. However, the time-consuming and specificity-related complications of conventional analytical procedures necessitate the development of a method which is rapid, selective and sensitive in nature. The present work illustrates a novel; prompt; "mix and measure" cytometric-based nano-biosensing system that offers direct quantification of cell-free circulating (ccf) epigenomic signatures (methylated ccf-DNA, tri-methylated histone H3 at lysine {4, 9, 27 & 36} and argonaute 2 protein-bound ccf-micro RNAs) using triple nano-assemblies in a single tube format. Each assembly with unique structural and spectral properties comprised of n-type semiconducting nanocrystals conjugated to a specific monoclonal antibody. Our results suggested that the developed combinatorial approach may offer simultaneous detection of three distinct yet biologically interrelated signatures with high selectivity and sensitivity using flow cytometry and fluorometry in the enriched and test samples. The proposed novel nano-assembly based detection system has a considerable potential of emerging as a minimal invasive easy-to-use method that could possibly permit real-time, rapid and reproducible monitoring of epigenomic markers in clinical and field settings.

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