Label-Free and Multiplexed Quantification of microRNAs by Mass Spectrometry Based on Duplex-Specific-Nuclease-Assisted Recycling Amplification

Abstract

MicroRNAs (miRNAs) are important biomarker candidates for cancer screening and early detection research. Generally, miRNAs undergo synergistic adjustments in tumor cells. Herein, a mass-spectrometric method based on a duplexspecific-nuclease (DSN)-enzyme-assisted signal-amplification technique was proposed for label-free and multiplexed detection of multiple miRNAs, and applied to the quantification of three miRNAs (i.e., miRNA-141, miRNA-21, and let-7a) in samples of HeLa and MDA-MB231 cell extracts. Experimental results showed that the digestion modes of DSN against three different DNAs complementary to miRNA-141, miRNA-21, and let-7a in their DNA−miRNA heteroduplexes were quite different, verifying the multiplexed-detection capability of the proposed method. Moreover, an advanced calibration model was derived for the quantitative analysis of the complex mass-spectral data measured during the label-free and multiplexed detection of miRNA-141, miRNA-21, and let-7a by the proposed massspectrometric method. With the aid of the advanced calibration model, the proposed mass-spectrometric method achieved quite reliable quantitative results for miRNA-141, miRNA-21, and let-7a in samples of HeLa and MDA-MB231 cell extracts, with recovery rates within the range of 89.2 to 111.6%. The limits of detection (LODs) of the proposed mass-spectrometric method for miRNA-141, miRNA-21, and let-7a in standard samples were estimated to be 42, 41, and 95 pM, respectively. Therefore, it is reasonable to expect that the proposed mass-spectrometric method can be a competitive alternative for the label-free and multiplexed detection of multiple miRNAs in clinical diagnosis.

Magnetic-enhanced fluorescence sensing of tumor miRNA by combination of MNPs@PDA with duplex specific nuclease

“…Therefore, detection of disease-specific miRNA in vivo can effectively diagnose diseases and develop personalized treatment schemes. Electrochemistry, 1 surface-enhanced Raman scattering, 2 mass spectrometry, 3 microchip electrophoresis, 4 chemiluminescence imaging array, 5 surface plasmon resonance 6 have been developed for the detection of miRNA. These assays have utilized the different strategies and each of them has respective advantages.…”

Enhanced electrochemiluminescence of Ru(bpy)32+‐doped silica nanoparticles by chitosan/Nafion shell@carbon nanotube core‐modified electrode

“…In addition, as listed in Table 1, the sensitivity of the proposed biosensor was comparable with, or better than, that of other previously reported approaches for detection of microRNA. [ 21–26 ]…”

Highly Sensitive and Specific Mass Spectrometric Platform for miRNA Detection Based on the Multiple-Metal-Nanoparticle Tagging Strategy

“…Compared with spectroscopic-technique-based biosensors, mass spectrometry has the advantage of excellent specificity. However, its detection limit for direct detection of miRNA is generally in the nanomolar level. , One of the main reasons for the relatively low sensitivity of mass spectrometry for miRNAs is the low ionization efficiency of miRNAs. , Various methods including organic mass tags, metal element labeling, and isothermal amplification are used to increase the ionization efficiency and enhance the sensitivity for miRNA detection. − Among them, metal nanoparticles are quite promising mass tags offering high detection sensitivity because of the presence of many thousands of metal atoms in a single metal nanoparticle of a 10 nm diameter. − Although the sensitivity problem of mass spectrometry for miRNA detection is effectively solved by mass tagging techniques, its specificity is lost since the mass spectral signals come from the mass tags instead of miRNAs themselves, and the discrimination between the target miRNAs from nontarget ones with SNPs is based on the differences in their mass spectral intensities. Mass spectrometric methods possessing both high sensitivity and specificity are highly desired for the detection of miRNAs.…”