2 research outputs found
Detection of Low-Abundance KRAS Mutations in Colorectal Cancer Using Microfluidic Capillary Electrophoresis-Based Restriction Fragment Length Polymorphism Method with Optimized Assay Conditions
<div><p>Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient’s age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy.</p> </div
SERS–Fluorescence Dual-Mode pH-Sensing Method Based on Janus Microparticles
A surface-enhanced
Raman scattering (SERS)–fluorescence dual-mode pH-sensing method
based on Janus microgels was developed, which combined the advantages
of high specificity offered by SERS and fast imaging afforded by fluorescence.
Dual-mode probes, pH-dependent 4-mercaptobenzoic acid, and carbon
dots were individually encapsulated in the independent hemispheres
of Janus microparticles fabricated via a centrifugal microfluidic
chip. On the basis of the obvious volumetric change of hydrogels in
different pHs, the Janus microparticles were successfully applied
for sensitive and reliable pH measurement from 1.0 to 8.0, and the
two hemispheres showed no obvious interference. The proposed method
addressed the limitation that sole use of the SERS-based pH sensing
usually failed in strong acidic media. The gastric juice pH and extracellular
pH change were measured separately in vitro using the Janus microparticles,
which confirmed the validity of microgels for pH sensing. The microparticles
exhibited good stability, reversibility, biocompatibility, and ideal
semipermeability for avoiding protein contamination, and they have
the potential to be implantable sensors to continuously monitor pH
in vivo