Fully Automated Sample Preparation Microsystem for Genetic Testing of Hereditary Hearing Loss Using Two-Color Multiplex Allele-Specific PCR

Abstract

A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-μL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-μL human whole blood followed by amplifications of 59-bp β-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis

“…Compared to other alternative methods, 4,7,8 the proposed microdevice has the following four advantages: (1) because this microfluidic chip does not contain any extra components such as microelectrodes or piezo transducers, the fabrication process is simple and the cost is low; (2) the device eliminates the need for complicated external instruments, aside from two pumps for fluid control, making the device easy to automate and miniaturize; (3) the chip material (PDMS) can be replaced with polymethyl methacrylate or other polymer materials that are easy for mass production, 28 and thus the microfluidic chip has a great potential to be transformed into a disposable product for forensic analysis of sexual assault cases; and (4) the microdevice is easily integrated, and the integration of this microfluidic chip with subsequent sample processing chips (e.g., DNA extraction, PCR, or STR chips 29,30 ) may offer a chance to replace the conventional labor-consuming and time-consuming processes for forensic evidence analysis.…”

Simple and rapid sample preparation system for the molecular detection of antibiotic resistant pathogens in human urine

“…More complex cell concentration methods involve manipulating the flow of cells using size or charge based differentiation methods (Pratt et al 2011 ). Simpler cell capture approaches using a physical substrate, such as a filter, (Baier et al 2009 ; Gulliksen et al 2012 ; Gan et al 2014 ; Zhuang et al 2015a , b ) or beads (Cho et al 2007 ; Ritzi-Lehnert et al 2011 ) have also been demonstrated on microfluidic platforms. However, these methods require electrical power to control the sample flow or the magnetic beads.…”

The Optimization of Electrophoresis on a Glass Microfluidic Chip and its Application in Forensic Science

“…A μCE system capable of multi‐channel confocal laser‐induced fluorescence detection was built in‐house and has been previously described in detail by Zhuangbin et al . Briefly, the microchip was coated with surface coating agent and loaded with polymer solution by pipetting DNA samples into the sample well.…”

“…A lCE system capable of multi-channel confocal laserinduced fluorescence detection was built in-house and has been previously described in detail by Zhuangbin et al (19) the microchip was coated with surface coating agent and loaded with polymer solution by pipetting DNA samples into the sample well. Then, electrophoresis was initiated with split-flow electrokinetic injection (20) of ssDNA molecules from the sample inlet toward the waste well with an electric field of 200 V/cm for 90 s, while grounding the sample well and floating both the buffer well and buffer waste well.…”