Circulating
microRNAs have been identified as potential biomarkers
for early detection, prognosis, and prediction of several diseases.
Their use in clinical diagnostics has been limited by the lack of
suitable detection techniques. Most of the current technologies suffer
from requiring complex protocols, not yet able to deliver robust and
cost-effective assays in the field of clinical diagnostics. In this
work, we report the development of a breakthrough platform for profiling
circulating microRNAs. The platform comprises a novel silicon photomultiplier-based
reader in conjunction with a chemical-based method for nucleic acid
detection. Accurate microRNAs profiling without extraction, pre-amplification,
or pre-labeling of the target is now achievable. We designed and synthesized
a set of reagents that combined the chemical-based method with a chemiluminescent
reaction. The signals generated were read out using a novel,
compact silicon photomultiplier-based reader. The platform sensitivity
was determined by measuring known concentrations of hsa-miR-21-5p
spike-ins. The limit of detection was calculated as 4.7 pmol/L. The
platform was also successfully used to directly detect hsa-miR-21-5p
in eight non-small cell lung cancer plasma samples. Levels of plasma
hsa-miR-21-5p expression were also measured via TaqMan RT-qPCR. The
successful integration of the unique chemical-based method for
nucleic acid detection with the novel silicon photomultiplier-based
reader created an innovative product (ODG platform) with diagnostic
utility, for the direct qualitative and quantitative analysis of microRNA
biomarkers in biological fluids.
This work deals with the design, fabrication, and thermal characterization of a disposable miniaturized Polymerase Chain Reaction (PCR) module that will be integrated in a portable and fast DNA analysis system. It is composed of two independent parts: a silicon substrate with embedded heater and thermometers and a PDMS (PolyDiMethylSiloxane) chamber reactor as disposable element; the contact between the two parts is assured by a mechanical clamping obtained using a Plastic Leaded Chip Carrier (PLCC). This PLCC is also useful, avoid the PCR mix evaporation during the thermal cycles. Finite Element Analysis was used to evaluate the thermal requirements of the device. The thermal behaviour of the device was characterized revealing that the temperature can be controlled with a precision of ±0.5°C. Different concentrations of carbon nanopowder were mixed to the PDMS curing agent in order to increase the PDMS thermal conductivity and so the temperature control accuracy.
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