Vitamin C In The Cell Design Synthesis And Dynamic Visualization Analysis Of Fluorescent Probes

Oxidizing and reducing substances are very important to maintain the normal process of lifeactivities. The reducing substances play a significant role in maintaining intracellular redoxbalance. In addition, they also have manymorephysiological functions, such as oxidationresistance, stress resistance, enhancing immunity and anticancer effect. Appropriate oxidizingsubstance isof great importance to keep the normal redox balance. They also maintain the normalimmune response in the body, participate in an important material synthesis and metabolism inthe cell and cell signal transduction.The concentration of oxidizing substances which is higherthan that of normal level will affect the physiological functions in the cell, leading to differentdegrees of damages on protein and nucleic acid. On the other hand, it also can make the changesof other active small molecules and enzymes, causing cell dysfunction and diseases. So thenormal organism should exist antioxidant defense system, mainly including: superoxidedismutase (SOD),Selenium glutathione peroxide enzyme (GSH-Px) and other protein substances;glutathione (GSH) and ascorbic acid (Vc) and other small molecular substances.At present, various approaches have been developed to monitor ascorbic acid anddehydroascorbicacid, such as spectrometry, liquid chromatography (HPLC), electrochemicalanalysis, mass spectrometry, fluorescence method, etc.However the method which can be used inbiological systems is seldom. Because there is always a problem in the measurement, forexample, fluorescence method: interference is aserious problem, nitric oxide and some reducedmaterials will cause interference to the detection of ascorbic acid; low sensitivity, ascorbic acidconcentration increasing to millimole level will have response to probe; operation is moretroublesome. Therefore, designing a kind of effective method for the detection of ascorbic acidis now the focus of medical research.We carried out the two aspects of investigation:First, we designed and synthesized a near-infrared(NIR) fluorescent probe fordehydroascorbic metabolites detection, arginine as specific center, tircarbocyanine as fluorescentdye. Based on the guanidine of arginine can specifically recognize the adjacent diketone, wehave designed the fluorescent probes for studying transformation processof ascorbic acid in thecell. Probe synthesis procedure is simple, mild reaction conditions, particularly in highproduction rate, and the product is very stable. Under simulated physiological conditions, we tested the optical properties of probe, the excitation and emission wavelength of680nm and749nm, respectively, stokes shift is69nm. Then we used the cell disruption liquid in chemicalsystem for analyzing the metabolites of dehydroascorbic. There is a good linear relationshipbetween the fluorescence intensity and the concentration of dehydroascorbic, the linear rangesfrom0to10μM. The linear equation is F=323.09+31.25[DHAA](μM), with a correlationcoefficient of0.9969. The probe shows high selectivity. There is no interference with othersubstances, such as: Cystine, Glucose, NO, H2O2, Dopamine, K+, Na+, ect.Fluorescence imaging by confocal microscopy show that the probe can be applied to theimaging of dehydroascorbic metabolites in the cell. Experiments show that the probe can bequickly and well monitor process of dehydroascorbic metabolic in cancer cells, in addition, ithas good low membrane permeability and biological toxicity. Then we use the probe, because theliver cancer cells in a kind of type oxidation environment, there are quite a few ofdehydroascorbic reduced to ascorbic acid, the majority of metabolism, and normal liver cells in areduced environment, most of the dehydroascorbic reduced to ascorbic acid, only a small numberof metabolism. So we use the probe real-time monitor dehydroascorbic transformation in theliver cancer cells and liver cells. Anyway, due to the redox state changes in normal cells andcancer cells, the dehydroascorbic transformation process is different in the cell, so our probeprovides a new tool for further study of dehydroascorbic transformation in the cell and signaltransduction.