使用荧光氮-空位中心纳米金刚石从人黑色素瘤细胞系和FFPE组织中灵敏检测microRNA

Diamond magnetometry (DM), using fluorescent nitrogen-vacancy center nanodiamonds (FND), is an enzyme-free, quantum-based platform that can detect and monitor cancer biomarkers, such as microRNAs. Fluorescence is due to the nitrogen vacancy center (NV-center), a crystallographic defect where a nitrogen atom is adjacent to a vacant (empty) site within the diamond lattice. Importantly, NV-center fluorescence can be optically manipulated and is sensitive to nanoscale magnetic fields. In the present study, we used diamond magnetometry to rapidly detect picomolar amounts of microRNAs in human melanoma cell lines without requiring the Polymerase Chain Reaction (PCR) or amplification or preamplification steps. Method. FNDs and magnetic nanoparticles were functionalized with biomolecules to create NV-Biosensors for miR-486-5p, miR-363-3p, miR-196-5p, miR-135b-5p, and miR-21-5p. Synthetic RNA and DNA oligonucleotides were synthesized by IDT and quantified on a Nanodrop spectrophotometer. Total RNA was prepared from the melanoma cell lines A375, MEL-39, CHL-1 HT-144, 18105 Mel, and MEL1174. Following isolation, nucleic acid sensors were mixed with 2 μL of total RNA in TBS reaction buffer containing 0.05% SDS, 5 mM MgCl 2, and 1 nM of a random 50-mer DNA sequence. Reactions were run for 15 minutes, then spotted onto a glass coverslip, gently rinsed, then mounted on a widefield fluorescence microscope set-up, and analyzed by a technique called optically detected magnetic resonance (ODMR). In the absence of a microRNA target, the FND and magnetic particle are associated with DNA hybridization and show low ODMR contrast. If a microRNA target is present, it displaces the FND-MNP hybrid, and the FND shows increased ODMR contrast. Results were collected in a few minutes at room temperature, with between 200 and 600 independent ODMR data points recorded for each sample. The large number of data points allowed robust data analysis. Results. We demonstrated the specific detection of synthetic RNA and DNA oligonucleotides for miR-21-5p and miR-486-5p down to a concentration of 1 pmole. Our NV-Biosensors also detected miR target molecules in total RNA preparations from cell lines. We could not detect miR-486-5p in total RNA from HT-144 and this was consistent with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) results. We used total RNA from HT-144 in spiking studies to demonstrate specific detection of miR-486-5p. Finally, we benchmarked our technology against the gold standard, RT-PCR, and showed that DM was as sensitive as RT-PCR for microRNA detection. DM has the advantage of using single microliter sample volumes and is a simple, accurate assay that can be performed in a fraction of the time needed for RT-PCR