Construction And Application Of Protein-mediated Fluorescence Resonance Energy Transfer(FRET) Probe

At present,there are two mainstream methods for constructing fluorescence resonance energy transfer（FRET）probes.One is traditional organic methods;Another method is synthesis using nanomaterials as the medium.But these methods have some problems more or less,such as complex organic synthesis and biocompatibility issues.Serum albumin is the most abundant protein in the plasma of living organisms,and its numerous structural domains can spontaneously bind with organic small molecule fluorescent dyes.In this paper,we aim to develop a novel synthesis method for the simple construction of FRET probes with good biocompatibility.Using bovine serum albumin（BSA）as the model protein and 7-（Diethylamino）coumarin-3 carboxylic acid（COU）,fluorescein（FL）and（2’,7’-dichlorodihydrofluorescein）DCFH₂ as raw materials,FRET probes Probe A and Probe B were constructed through spontaneous binding with BSA,which not only perfectly avoided the complex organic synthesis,but also endowed the probes with good biocompatibility,and successfully realized the micro detection of hydroxyl radical（·OH）.The specific research contents are as follows:1.To ensure the successful construction of FRET probes,we investigated the binding ability between COU,FL,DCFH₂,2’,7’-dichlorofluorescein（DCF）and BSA.The quenching process between COU,FL,DCFH₂,DCF and BSA was determined as a static quenching process by fluorescence spectroscopy;the binding constants and the number of binding sites between COU,FL,DCFH₂,DCF and BSA were calculated using a double logarithmic equation,the binding constants were all greater than 10⁴,and the number of binding sites was approximately 1;the main type of interaction force between COU,FL,DCFH₂,DCF and BSA was determined through the van der Hoff isotherm formula to be hydrophobic interaction;through drug competition experiments,it was determined that COU was mainly bound to the binding SiteⅡof subdomainⅢA of BSA,and both DCFH₂ and DCF were bound to the binding SiteⅠof subdomainⅡA of BSA.Molecular docking experiments further verified the results of drug competition experiments were correct.2.FRET probes Probe A and Probe B were constructed using COU as energy donor,FL and DCFH₂ as energy receptors,respectively.Among them,with the addition of·OH,the detection mode of Probe A is that the FRET process undergoes a change from presence to absence.Probe A showed a rapid response to·OH,and the response was complete within 6 min.The detection mode of Probe B is that the FRET process undergoes a change from scratch with the addition of·OH.The fluorescence signal of Probe B exhibits a good linear relationship with the concentration of·OH,and the limit of detection（LOD）is 5×10^-8 mol/L.Finally,the advantages of Probe B compared to the intensity fluorescent probe DCFH₂ were explored.The LOD of Probe B is lower than that of the intensity fluorescent probe DCFH₂,reflecting the ultra high sensitivity of Probe B,which can be used for precise detection of trace·OH;the interference of environmental factors（such as changes in fluorescence spectrometer parameters and probe concentration）does not affect the ability of Probe B to detect·OH,indicating that Probe B has excellent anti-interference ability.