A New One‐Pot Fluorescence Derivatization Strategy for Highly Sensitive MicroRNA Analysis

Abstract

MicroRNAs (miRNAs)m odulate the expression of over 30 %o fm ammalian genes during development and apoptosis, and abnormale xpression of miRNAs may lead to arange of humanp athologies. Therefore, analysis of miRNAs is valuable for disease diagnostics. In this work, an ovel onepot fluorescence derivatization strategy wasd eveloped for miRNA analysis. The mechanism of the derivatization reaction was explored by using instrumental methods, including liquid chromatography,f luorescence spectroscopy,a nd mass spectrometry.H ighly fluorescent N 6 -ethenoadenine (e-adenine) was formed and detached from the miRNA sequence through the reaction of adenine in nucleic acids with 2-chloroacetaldehyde (CAA) at 100 8C. This is the first experimental evidence that the cooperation of formed e-adenine and water-mediatedh ydrogen-bond interaction between the protona tt he 2'-a nd the oxyanion at 3'-positions stabilized the oxocarbenium significantly,w hich makes the depurinationa nd derivatization of miRNA highly effective. Based on this derivatization strategy,afacile and sensitive highperformancel iquid chromatography method was developed for quantitative assay of miRNAs. In combination with magnetic solid-phase extraction( MSPE), the HPLCm ethod was shown to be useful for the determination of microRNAsa t sub-picomolar level in serum samples.[a] Dr.

An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids

“…This reaction was carried out for pre-column derivatization of adenyl purines in several HPLC methods with uorescence detection. [27][28][29][30][31] In this work, we studied ESI-MS detection of etheno-derivatives. It's worth mentioning that the derivatization was performed at an elevated temperature and in a signicant excess of the derivatizing reagent, CAA (the mole ratio of CAA to the test compound was >500 in all cases) to ensure a good tagging yield.…”