Colorimetric detection of both total genomic and loci-specific DNA methylation from limited DNA inputs

Background: Aberrant DNA methylation marks are potential disease biomarkers, and detecting both total genomic and gene-specific DNA methylation can aid in clinical decisions. While a plethora of methods exist in research, simpler, more convenient alternatives are needed to enhance both routine diagnostics and research. Results: Herein, we describe colorimetric assays using methyl-binding domain (MBD) proteins for rapid and convenient evaluation of total genomic and gene-specific methylation from 50\ua0ng or less DNA input in under 2\ua0h. As little as 5\ua0% methylation differences can be detected and are enhanced by a novel MBD protocol for improved specificity. Our assays could differentiate naïve from de-methylating drug-treated cells and detect the presence of a methylated prostate cancer biomarker in the urine. Finally, the assay was evolved onto disposable screen-printed electrodes for convenient detection of gene-specific methylation in urine. Conclusions: Rapid MBD-based colorimetric and electrochemical approaches to detect DNA methylation from limited samples were successfully demonstrated and applied to clinical samples. We envision that the ease, low sample requirements and speed of these assays could have both clinical and research-wide applications

A simple bridging flocculation assay for rapid, sensitive and stringent detection of gene specific DNA methylation

The challenge of bringing DNA methylation biomarkers into clinic is the lack of simple methodologies as most current assays have been developed for research purposes. To address the limitations of current methods, we describe herein a novel methyl-protein domain (MBD) enrichment protocol for simple yet rapid and highly stringent selection of highly methylated DNA from limiting input samples. We then coupled this with a DNA-mediated flocculation assay for rapid and low cost naked-eye binary evaluation of highly methylated genes in cell line and blood DNA. The low resource requirements of our method may enable widespread adoption of DNA methylation-based diagnostics in clinic and may be useful for small-scale research

Non-coalescence of oppositely charged drops

Oppositely charged drops have long been assumed to experience an attractive force that favors their coalescence. Here we demonstrate the existence of a critical field strength above which oppositely charged drops do not coalesce. We observe that appropriately positioned and oppositely charged drops migrate towards one another in an applied electric field; but whereas the drops coalesce as expected at low field strengths, they are repelled from one another after contact at higher field strengths. Qualitatively, the drops appear to ‘bounce’ off one another. We directly image the transient formation of a meniscus bridge between the bouncing drops, and propose that this temporary bridge is unstable with respect to capillary pressure when it forms in an electric field exceeding a critical strength. The observation of oppositely charged drops bouncing rather than coalescing in strong electric fields should affect our understanding of any process involving charged liquid drops, including de-emulsification, electrospray ionization and atmospheric conduction

Experiments on opto-electrically generated microfluidic vortices

Strong microfluidic vortices are generated when a near-infrared (1,064 nm) laser beam is focused within a microchannel and an alternating current (AC) electric field is simultaneously applied. The electric field is generated from a parallel-plate, indium tin oxide (ITO) electrodes separated by 50 mu m. We present the first mu-PIV analysis of the flow structure of such vortices. The vortices exhibit a sink-type behavior in the plane normal to the electric field and the flow speeds are characterized as a function of the electric field strength and biasing AC signal frequency. At a constant AC frequency of 100 kHz, the fluid velocity increases as the square of the electric field strength. At constant electric field strength fluid velocity does not change appreciably in the 30-50 kHz range and it decreases at larger frequencies (\u3e 1 MHz) until at approximately 5 MHz when Brownian motion dominates the movement of the 300 nm mu-PIV tracer particles. Presence of strongly focused laser beams in an interdigitated-electrode configuration can also lead to strong microfluidic vortices. When the center of the illumination is focused in the middle of an electrode strip, particles experiencing negative dielectrophoresis are carried towards the illumination and aggregate in this area