Design, Synthesis And Analytical Application Of NADH Fluorescent Probes

Reduced nicotinamide adenine dinucleotide(NADH)and its oxidized form(NAD~+)are ubiquitous in all living systems and are essential coenzymes for more than 300 oxidoreductase.NADH is involved in most redox reactions,such as glycolysis,tricarboxylic acid cycle and mitochondrial respiration,and plays an important role in energy metabolism and electron transport in the respiratory chain.The traditional methods for detecting NADH were established based more on the autofluorescence of intracellular NADH,resulting in low sensitivity,weak selectivity,and the damage of biological sample due to UV radiation.Subsequently,electrochemical analysis,enzymatic cycling assay,high performance liquid chromatography and capillary electrophoresis were developed.Although these methods can be better used to detect NADH,most of them are only carried out in vitro and are difficult to apply in living cells and in vivo.Thus,to design and synthesize a fluorescent probe which can be applied in living cells and in vivo with high sensitivity and specificity to detect NADH is of great significance for studying on the NADH-related physiological and pathological processes.This paper reviewed the physicochemical properties,physiological functions and common detection methods of NADH,and the research status and development trend of detection of NADH based on fluorescent probes(genetically encoded probes,nanoprobes and small molecule probes).This paper mainly includes the following work:1.Near-infraed(NIR)fluorescent probe(DCI-MQ)was designed and synthesized by using dicyanoisophorone as a near-infrared chromophore and quinolinium as a reduction site of NADH.After the probe reacted with NADH,the maximum absorption wavelength shifted to 568 nm and the strong fluorescence was emitted at 660 nm due to the presence of a strong intramolecular charge transfer(ICT).We measured the response of DCI-MQ to NADH under simulated physiological conditions(10 mM PBS,pH=7.4).The enhanced fluorescence intensity of the probe with NADH showed a good linear relationship to the concentration of NADH in the range from 0to1?M.The regression equation was F=22.12+134.27[NADH],with a linear coefficient of0.9915.The limit of detection(LOD)was calculated as low as 12 nM.In addition,we successfully applied the probe to detect and image NADH in HepG2 cells and tumor-bearing mice.2.To improve sensitivity and selectivity,we used tricyanofuran(TCF)as chromophore to synthesize another fluorescent probe(TCF-MQ)for detecting NADH.After the probe reacted with NADH,the maximum absorption wavelength shifted to 582 nm and the strong fluorescence emitted at 610 nm.The enhanced fluorescence intensity of the probe with NADH showed a good linear relationship to the concentration of NADH in the range from 0 to 3?M.The regression equation was F=83.22+238.42[NADH],with a linear coefficient of 0.9819.The limit of detection was calculated to be 6 nM,which improves the detection sensitivity.