RESUMEN
Reports have indicated that warfarin is the most widely prescribed anticoagulant. However, traditionally prescribed doses for each patient may be too low or too high. The therapeutic effect is often hindered by a lack of evidence-based medical information. Herein, our aim is to provide this information. To accomplish this challenge, we report the development of a novel assay based on biotinylated tetrahedral DNA as a capture probe and fullerene (C60)-based nanomaterial as a redox probe using an ultrasensitivity assay with the Vitamin K epoxide reductase complex, subunit 1 (VKORC1). Platinum porous nanoparticles (PtPNPs) were modified on amino-terminated polyamidoamine (PAMAM)-functionalized C60 nanoparticles (C60NPs). The resultant C60NPs-PAMAM-PtPNPs were used as a redox probe. In this design, C60 exhibited excellent redox activity that was triggered by tetraoctylammonium bromide (TOAB). To improve the immobilization of the tetrahedral DNA capture probe, avidin was introduced during the fabrication of the biosensor because it can provide more active sites for the immobilization capture probe. The free-standing probe on top of the tetrahedral DNA served as a receptor to hybridize with target DNA directly. Different pulse voltammetry (DPV) was applied to record the electrochemical signals, which increased linearly with the target DNA. Under optimal conditions, the prepared biosensor showed a wide linear relationship, from 1 pM to 10â¯nM, with detection limits of 0.33 pM. This strategy demonstrates a new avenue for the determination of tumour-related mutated nucleotides in biosamples.
Asunto(s)
Anticoagulantes/química , Técnicas Biosensibles , Sondas de ADN/química , ADN/análisis , Vitamina K Epóxido Reductasas/análisis , Warfarina/química , ADN/genética , Técnicas Electroquímicas , Fulerenos/química , Humanos , Nanopartículas del Metal/química , Oxidación-Reducción , Tamaño de la Partícula , Platino (Metal)/química , Poliaminas/química , Porosidad , Propiedades de Superficie , Vitamina K Epóxido Reductasas/genéticaRESUMEN
AIMS: Developing genetic and pharmacogenetic panels enhances genetic testing in clinical molecular diagnostics and precision medicine. This study was designed to cross-validate the performance of Canon's multiplex high-resolution DNA melting analysis platform with the Applied Biosystems TaqMan®-based Quant Studio Real-Time PCR System and Pyrosequencing® genotyping platforms for common genetic polymorphisms of the vitamin K epoxide reductase complex 1 (VKORC1) and CYP2C9. MATERIALS AND METHODS: Genomic DNA isolated from 240 blood and saliva samples was used to genotype the VKORC1-1639 G/A (rs9923231), CYP2C9*2 (430C>T, rs28371674), and CYP2C9*3 (1075A>C, rs1057910) single-nucleotide polymorphisms (SNPs) on the three above-mentioned genotyping platforms. RESULTS: There was 99.2%, 100%, and 100% concordance among the Canon DNA analyzer, the TaqMan-based QuantStudio, and the Pyrosequencing genotyping results for the VKORC1 (rs9923231), CYP2C9*2, and CYP2C9*3 SNPs, respectively, in DNA samples isolated from blood. The DNA samples isolated from saliva showed 100% concordance among the three test platforms for the three tested SNPs. CONCLUSION: These results show that, the DNA analyzer performed very well when compared with two commonly used genotyping platforms. The reliability, multiple genetic variant testing capability, and short turnaround time for up to eight samples make the DNA analyzer an ideal genotyping platform for genetic testing in the clinical practice setting, where efficient genotyping is important to prevent delays in optimizing drug therapy.