Enzyme-free and isothermal discrimination of microRNA point mutations using a DNA split proximity circuit with turn-on fluorescence readout.

MicroRNAs (miRNAs) hold immense potential as disease biomarkers, yet their short lengths and high sequence homology pose unique challenges in detection. Conventional methods such as the gold standard qRT-PCR and other isothermal amplification methods require sophisticated primer designs and use of enzymes which add uncertainties to the assay robustness. In this work, we demonstrate the use of a plug-and-play molecular detection platform, termed split proximity circuit (SPC), to achieve a selectivity comparable to qRT-PCR in differentiating point mutations using several miRNAs as proof-of-concept models. The analytical sensitivity of SPC has been improved by a hundred-fold over our previous work and matches/outperforms the enzyme-free assays reported in the literature by evolving the core signal-generating domains. Key design changes include improved hybridization chain reaction (HCR) hairpin sequences and the incorporation of a turn-on fluorescence signal based on fluorophore-quencher format. The core domains were then kept constant while redesigning the target recognition region to be complementary to various target sequences, all of which yield similar analytical performance. Notably, SPC maintained robust signal recovery with low variance even in complex biological matrices. With its enzyme-free and single room temperature operation, SPC presents a promising platform for quick and easy miRNA quantification.

Dielectrophoretic capture voltage spectrum for measurement of dielectric properties and separation of cancer cells.

In this paper, a new dielectrophoresis (DEP) method based on capture voltage spectrum is proposed for measuring dielectric properties of biological cells. The capture voltage spectrum can be obtained from the balance of dielectrophoretic force and Stokes drag force acting on the cell in a microfluidic device with fluid flow and strip electrodes. The method was demonstrated with the measurement of dielectric properties of human colon cancer cells (HT-29 cells). From the capture voltage spectrum, the real part of Clausius-Mossotti factor of HT-29 cells for different frequencies of applied electric field was obtained. The dielectric properties of cell interior and plasma membrane were then estimated by using single-shell dielectric model. The cell interior permittivity and conductivity were found to be insensitive to changes in the conductivity of the medium in which the cells are suspended, but the measured permittivity and conductivity of cell membrane were found to increase with the increase of medium conductivity. In addition, the measurement of capture voltage spectrum was found to be useful in providing the optimum operating conditions for separating HT-29 cells from other cells (such as red blood cells) using dielectrophoresis.

The induction of epigenetic regulation of PROS1 gene in lung fibroblasts by gold nanoparticles and implications for potential lung injury.

Advances in nanotechnology have given rise to the rapid development of novel applications in biomedicine. However, our understanding in the risks and health safety of nanomaterials is still not complete and various investigations are ongoing. Here, we show that gold nanoparticles (AuNPs) significantly altered the expression of 19 genes in human fetal lung fibroblasts (using the Affymetrix Human Gene 1.0 ST Array). Among the differentially expressed genes, up-regulation of microRNA-155 (miR-155) was observed concomitant with down-regulation of the PROS1 gene. Silencing of miR-155 established PROS1 as its possible target gene. DNA methylation profiling analysis of the PROS1 gene revealed no changes in the methylation status of this gene in AuNP-treated fibroblasts. At the ultrastructural level, chromatin condensation and reorganization was observed in the nucleus of fibroblasts exposed to AuNPs. The findings provide further insights into the molecular mechanisms underlying toxicity of AuNPs and their impact on epigenetic processes.