RESUMO
Micromolded carbon paste electrodes are easily fabricated, disposable, and can be integrated into microfluidic devices to fabricate inexpensive sensors and biosensors. In this work, carbon paste microelectrodes were fabricated in poly(dimethylsiloxane) using micromolding techniques and were coupled to a microfluidic channel to fabricate electrogenerated chemiluminescent (ECL) sensors. ECL was generated using both the tris(2,2'-bipyridyl)ruthenium(II)-tripropylamine system and the hydrogen peroxide and luminol system. For each of these ECL systems, the sensor fabrication method was optimized, along with key experimental parameters (applied voltage, solution flow rate, buffer species and luminol concentration). The limit of detection (S/N = 3) for TPrA was ~2.4 µM with a linear range of 10-100µM. For hydrogen peroxide the LOD was ~11 µM and the electrodes gave a linear response between 30 µM and 200 µM hydrogen peroxide. Electrodes containing glucose oxidase were fabricated using this new method, demonstrating that glucose could be indirectly detected via generation of hydrogen peroxide by the enzymatic reaction at the micromolded biosensor.
RESUMO
BACKGROUND: Most PCR-based diagnostics are still considered time- and labor-intensive due to disparate purification, amplification, and detection steps. Advancements in PCR enzymes and buffer chemistry have increased inhibitor tolerance, facilitating PCR directly from crude samples. Obviating the need for DNA purification, while lacking a concentration step, these direct sample methods are particularly apt for human genetic testing. However, direct PCR protocols have traditionally employed thermal cyclers with slow ramp rates and conservative hold times that significantly increase an assay's time-to-result. For this proof-of-principle study, our objective was to significantly reduce sample preparation and assay time for a PCR-based genetic test, for myotonic dystrophy type 1 (DM1), by pairing an inhibitor-resistant enzyme mix with a rapid thermal cycler to analyze samples directly in whole blood. METHODS: DM1 genetic screening was done with an adapted conventional PCR approach that employed the Streck Philisa® Thermal Cycler, the inhibitor-resistant NEBNext® High-Fidelity 2X PCR Master Mix, and agarose gel electrophoresis or an Agilent 2100 Bioanalyzer for detection. The Gene Link™ Myotonic Dystrophy Genemer™ Kit was used as a reference assay kit to evaluate the rapid assay. RESULTS: In this work, a rapid and direct PCR assay testing 10% whole blood as template has been developed as an exclusionary screening assay for DM1, a triple-repeat genetic disorder. PCR amplification was completed in 15 minutes using 30 cycles, including in situ hot-start/cell lysis. Out of the 40 donors screened, this assay identified 23 (57.5%) as DM1 negative suggesting no need for further testing. These data are 100% concordant with data collected using the commercially available Gene Link Genemer™ Kit per the kit-specific PCR protocol. CONCLUSIONS: The PCR assay described in this study amplified DM1 short tandem repeats in 15 minutes. By eliminating sample purification and slower conventional PCR protocols, we demonstrated how adaptation of current PCR technology and chemistries can produce a simple-to-use exclusionary screening assay that is independent of up-front sample prep, improving a clinical lab technician's time-to-result. We envision this direct and rapid methodology could be applied to other conventional PCR-based genetic tests and sample matrices where genomic DNA is targeted for analysis within a given molecular diagnostic platform.
