Studies on Cyclodextrin Inclusion Complex by Fluorescence Anisotropy

An equation for determining the equilibrium association constant (KA) of cyclodextrin inclusion complex with fluorescence anisotropy is derived and used to determine KA of pyrene-B-cyclodextrin inclusion complex. The existing forms of cyclodextrin inclusion complex in solution, the interaction type of host with guest, and the possibility of application of B-cyclodextrin in the analysis of metal ions using naphthalene derivative as a ligand are discussed based on the equation derived along with the curve of fluorescence anisotropy versus cyclodextrin concentration of guest/cyclodextrin system.

Octopus-like DNA nanostructure coupled with graphene oxide enhanced fluorescence anisotropy for hepatitis B virus DNA detection

Fluorescence Anisotropy(FA)is an effective biochemical detection method based on molecular rotations.Graphene oxide(GO)has been extensively used as an FA amplifier.However,the enhancement of FA by GO alone is limited and the strong scattering of GO will easily make the measurement of FA inaccurate.In order to address these problems,an octopus-like DNA nanostructure(ODN)was designed and coupled with GO to enhance the FA together in this work.By mimicking the multi-clawed structure of the octopus,the ODN can be adsorbed on GO tightly,which not only could improve the sensitivity because of the double FA enhancement abilities of GO and ODN,but also could improve the specificity due to the decrease of the nonspecific interaction in complex samples.Furthermore,ODN could maintain a certain distance between the fluorophore and GO to reduce the fluorescence quenching efficiency of GO,which could improve the accuracy.This method has been applied for the detection of hepatitis B virus DNA(HBV-DNA)in a range of 1-50 nmol/L and the limit of detection(LOD)was 330 pmol/L.In addition,the proposed method has been successfully utilized to detect HBV-DNA in human serum,indicating that this method has a great practical application prospect.

A simple fluorescence anisotropy assay for detection of bisphenol A using fluorescently labeled aptamer

Bisphenol A(BPA)is one of the environmental endocrine disruptors(EDCs),and BPA contamination in environment can cause high risks to human health.Rapid determination of BPA on sites is in high demand in environmental analysis.Taking advantage of aptamers as affinity ligands and fluorescence anisotropy(FA)analysis,we developed a simple and rapid FA assay for BPA by employing a single tetramethylrhodamine(TMR)labeled short 35-mer DNA aptamer against BPA.The assay is based on the BPA-binding induced conformation change of TMR-labeled aptamer and alteration of interaction between TMR and guanine bases,resulting in change of FA signals.We screened the FA change of aptamer probes having TMR label on a specific site of the aptamer upon BPA addition.The aptamer with a TMR label on the 22nd T base showed large FA-decreasing response to BPA and maintained good binding affinity to BPA.By using this TMR-labeled aptamer,we achieved FA detection of BPA with a detection limit of 0.5μmol/L under the optimized conditions.This assay was selective towards BPA and enabled the detection of BPA spiked in tap water sample,showing the potential applications on water samples.

Aptamer structure switch fluorescence anisotropy assay for aflatoxin B1 using tetramethylrhodamine-guanine interaction to enhance signal change

Fluorescence anisotropy(FA)assay in homogenous solution is simple,sensitive and reproducible.Here,we reported an aptamer structure switch FA assay for detection of aflatoxin B1(AFB1),one of the most toxic mycotoxins,by using tetra methylrhodamine(TMR)-labeled aptamer probe and its complementary DNA(cDNA)with tandem G bases extension,to meet the demand in sensitive and selective detection of AFB1,The hybridization of aptamer and cDNA drew TMR close to the repeated guanine(G)bases,and a high FA value was induced due to TMR-G inte raction and re stricted local rotation of TMR.In the presence of AFB1,aptamer bound to AFB1 instead of the cDNA due to competition.Thus,the TMR-G interaction was eliminated,and FA value of TMR decreased.This assay enabled the detection of AFB1 with detection limit of 125 pmol/L and dynamic range from 125 pmol/L to 31.2 nmol/L.

Super-resolution fluorescence polarization microscopy

Fluorescence polarization is related to the dipole orientation of chromophores,making fuores-cence polarization microscopy possible to_reveal structures and functions of tagged cellularorganelles and biological macromolecules.Several recent super resolution techniques have beenapplied to fluorescence polarization microscopy,achieving dipole measurement at nanoscale.In this review,we summarize both difraction limited and super resolution fluorescence polari-zation microscopy techniques,as well as their applications in biological imaging.

Conformational change of E.coli sulfurtransferase YgaP upon SCN- in intact native membrane revealed by fluorescence lifetime and anisotropy

Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN-remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7-HC to study the conformational change of YgaP upon SCN-binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN-. SMA is a good material to reflect the in situ conformation changes of protein than micelles.