| Adenosine(AD)plays an important role in human physiological activity.For example,AD can regulate myocardial oxygen consumption,blood flow,and the release of brain neurotransmitters such as dopamine through the AD receptor.In addition,it can help regulate smooth muscle contraction,neurotransmission,renal hemodynamics and release of chymosin.Under normal physiological conditions,trace amounts of AD are released into the extracellular space to exert an important pathophysiological effect.However,when the oxygen supply balance is disturbed,a large amount of AD is released into the body fluid/blood.Therefore,it is very necessary to detect AD for clinical diagnosis.Carbon dots(CDs)are spherical particles with size less than 10 nm.Nowadays,CDs have attracted much attention due to its good physical and chemical properties such as good water solubility,good biocompatibility,low toxicity,excellent photostability and strong fluorescence emission.And because of its excellent properties,CDs are often used for sensing.Aptamers,which are selected by the systematic evolution ofligands by exponential enrichment(SELEX),are special nucleicacid sequences.Aptamers provide multiple advantages for use as sensors,such as relatively easy synthesis,good stability,excellent affinity,and compatibility withdiverse modifications.Thus,based on aptamerlabeled CDs have many advantages,such as good stability,high sensitivity and selectivity.At present,it has not been reported to adapt the AD aptamer modified CDs to detect AD.In this paper,CDs were modified with AD aptamer to form CDs-aptamer.Then CDs-aptamer were used as fluorescence energy donor and nano-graph ite(NG)was used as energy acceptor to constructthe sensing platform for the purpose of high selective and sensitive detection of trace amounts of AD in biological samples.And the probe could be successfully applied to the detection of trace AD in urine samples.Summary is as follows:1.CDs-aptamer/NG sensing platformFirst,the activated carboxy groups on the surface of CDs interacted with AD aptamer modified with amino group to form CDs-aptamer complex.Then NG was introduced into CDs-aptamer solution to quench the fluorescence of CDs-aptamer by Fluorescence resonance energy transfer(FRET)between CDs-aptamer and NG becauseof close proximity between aptamer with NG caused by the,?-? interaction between aptamer and NG.When AD was added into the CDs-aptamer/NG system,AD could combine with AD aptamer specifically to form double-stranded DNA(dsDNA)structure.Therefore,CDs-aptamer detached from NG and the process of FRET was blocked.Eventually the fluorescence of CDs-aptamer was recovered.Based on the linear relationship between the degree of fluorescence recovery and the natural logarithmic value of the concentration of AD,quantitative detection of AD concentration in urine sample was achieved.The ratio of reactants,temperature,time and other experimental conditions for the synthesis of CDs-aptamer complexes and the application of the sensing platform were optimized.Finally,the structure and morphology of the fluorescence sensing platform were characterized by Fourier transform infrared spectroscopy,agarose gel electrophoresis and high resolution transmission electron microscopy to prove that the successful construction of CDs-aptamer/NG sensing platform.2.Application of CDs-aptamer/NG sensing platformBased on the fluorescence sensing platform of CDs-aptamer/NG system,we established a high sensitive and selective method for the determination of AD in urine.The interference test showed that the CDs-aptamer/NG sensing platform had good selectivity.The results demonstrated that the relative change of the fluorescence intensity of the constructed sensing platform was in good linear relationship with the natural logarithm of the AD concentration in the range of 2-50 nM under the optimum test conditions.The regression equation could be expressed as(F-F0)/F0=0.10477Ln[AD]+0.00646(AD represented the concertration of adenosine,nM),with a correlation coefficient of 0.9925 and the limit of detection for AD was 0.63 nM based on 3?/?(? is the standard deviation of ten blank measurement of CDs-aptamer/NG,and k is the slope of the regression line).We applied the as-established method to detect the amount of AD in the urine of the laboratory volunteer(80-fold dilution after filtration).The results showed that the concentration of AD was 226.4 nM,the spiked recoveries of AD were between 97.5 and 107.3%,and the relative standard deviations were less than 8.3%(n=3).The experimental results indicated that the off-on fluorescence sensing platform could meet the requirement of detection of AD in complex biological samples.The establishment of the proposed fluorescent sensing platform is green and convenient,and it can be applied to the rapid determination of AD in complex biological samples just after filtration and dilution. |
PDF Full Text Request