The use of DNA methylation to predict chronological age has shown promising potential for obtaining additional information in forensic investigations. To date, several studies have reported age prediction models based on DNA methylation in body fluids with high DNA content. However, it is often difficult to apply these existing methods in practice due to the low amount of DNA present in stains of body fluids that are part of a trace material. In this study, we present a sensitive and rapid test for age prediction with bloodstains based on pyrosequencing and random forest regression. This assay requires only 0.1 ng of genomic DNA and the entire procedure can be completed within 10 hours, making it practical for forensic investigations that require a short turnaround time. We examined the methylation levels of 46 CpG sites from six genes using bloodstain samples from 128 males and 113 females aged 10 to 79 years. A random forest regression model was then used to construct an age prediction model for males and females separately. The final age prediction models were developed with seven CpG sites (three for males and four for females) based on the performance of the random forest regression. The mean absolute deviation was less than three years for each model. Our results demonstrate that DNA methylation-based age prediction using pyrosequencing and random forest regression has potential applications in forensics to accurately predict the biological age of a bloodstain donor. This article is protected by copyright. All rights reserved.

Pertaining to the mixing of the non-Newtonian Carreau fluid under electrokinetic actuation inside a plane microchannel, we propose a new design of micromixer that involves inserting a two-part cylinder bearing zeta potential of the same sign but different magnitude in the upstream and downstream directions. We numerically solve the transport equations to predict the underlying mixing characteristics. We demonstrate that a substantial momentum difference between the microchannel's plane wall and cylinder leads to the development of a vortex in the flow pathway, which in turn, enhances mixing substantially. As shown, for a fluid having a highly shear-thinning nature, the vortex-assisted convection mixing strength increases with diffusivity of the candidate fluids. Moreover, it is shown that for the higher shear-thinning nature of the candidate fluid, an increase in cylinder radius enhances mixing efficiency and flow rate simultaneously, resulting in a "quick and efficient" mixing condition. Additionally, the fluid rheology significantly alters the kinetics of shear-induced binary aggregation. Our findings show that the shear-induced aggregation characteristic time sharply increases with increasing shear-thinning behavior of the fluid.

In this study, the capillary electrophoresis-photodiode array detector (CE-PAD) was employed for the analysis of four iridoid compounds in Gentiana macrophylla Radix (RGM), and the method was optimized based on the concept of Analytical Quality by Design (AQbD). The peak areas RSD (n = 3) and resolution of the four analytes were selected as critical method attributes. Fractional factorial design test combined with Pareto analysis were employed to screen critical method parameters (buffer concentration, pH, SDS micelle concentration, temperature, and voltage). Subsequently, three main factors (buffer concentration, buffer pH, SDS concentration) were selected by central composite design test for constructing the design space. The optimal separation conditions as follows: capillary column (50.2 cm × 50 μm, detection length 40 cm). Working background electrolyte consisted of 51 mmol/L borax solution (pH = 9.47) and 40 mmol/L SDS. The samples were injected by pressure (5 s at 0.5 psi) and the detection was performed at 254 nm. Applied voltage was 20 kV and column temperature were 23°C. The developed method is rapid and reliable for the quantitative analysis of four iridoid compounds in RGM, providing a reference for the application of AQbD concept in the analysis of natural products. This article is protected by copyright. All rights reserved.

A capillary electrophoresis method is proposed to analyze the four most well-known Growth Hormone Releasing Hormone (GHRH) analogs that are misused by athletes. Dimethyl-β-cyclodextrin used as a chiral selector allowed, for the first time, the separation of those basic peptide analogs, including enantiopeptides (sermorelin and CJC-1293) that differ by the chirality of only one amino acid. To increase the method sensitivity, electrokinetic preconcentration methods have been investigated. The Large Volume Sample Stacking with polarity switching (PS-LVSS) method with an injected sample volume corresponding to 80% of the capillary one was found superior to the sweeping in terms of signal enhancement factor (SEF). Acid and organic solvent addition to the sample (0.1 mM phosphoric acid with 30% methanol) led to a two-fold signal improvement, when compared to water as a matrix. We increased capillary dimensions to provide a signal enhancement through the injection of a larger sample volume. Finally, using a combination of the optimized PS-LVSS preconcentration with the chiral CZE, the GHRH analogs were separated and LODs between 75 to 200 ng/mL were reached. This method was successfully applied to urine after a desalting step. An optimized C18 SPE was used for that purpose in order to provide low sample conductivity (<130 μS/cm) and preserve the efficiency of LVSS preconcentration. SEF of 640 was obtained with desalted urine spiked with sermorelin by comparison to the CZE (without preconcentration) method. This article is protected by copyright. All rights reserved.

Polydimethylsiloxane (PDMS) based microfluidic devices have found increasing utility for electrophoretic and electrokinetic assays because of their ease of fabrication using replica molding. However, the fabrication of high-resolution molds for replica molding still requires the resource-intensive and time-consuming photolithography process, which precludes quick design iterations and device optimization. We here demonstrate a low-cost, rapid microfabrication process, based on electrohydrodynamic jet printing (EJP), for fabricating non-sacrificial master molds for replica molding of PDMS microfluidic devices. The method is based on the precise deposition of an electrically stretched polymeric solution of polycaprolactone in acetic acid on a silicon wafer placed on a computer-controlled motion stage. This process offers the high-resolution (order 10 μm) capability of photolithography and rapid prototyping capability of inkjet printing to print high-resolution templates for elastomeric microfluidic devices within a few minutes. Through proper selection of the operating parameters such as solution flow rate, applied electric field, and stage speed, we demonstrate microfabrication of intricate master molds and corresponding PDMS microfluidic devices for electrokinetic applications. We demonstrate the utility of the fabricated PDMS microchips for nonlinear electrokinetic processes such as electrokinetic instability and controlled sample splitting in ITP. The ability to rapid prototype customized reusable master molds with order 10 μm resolution within a few minutes can help in designing and optimizing microfluidic devices for various electrokinetic applications. This article is protected by copyright. All rights reserved.