Biomacromolecule Assembly Structure Amplified Fluorescence Anisotropy And Its Analytical Applications

Fluorescence anisotropy(FA)has been widely used in clinical diagnosis and biochemical analysis.When the temperature and viscosity of the solution are constant,the FA value is directly related to the rotation motion and mass or volume of the fluorescent labeled molecule.Because most molecules have small mass and volume,it is difficult to produce significant FA changes,so the sensitivity of detection needs to be improved.Up to now,various traditional FA amplifiers such as biomacromolecules and inorganic nanomaterials have been successfully used for biosensing.However,these materials have some disadvantages in enhancing FA signals.On the one hand,most reported inorganic nanomaterials possess broad and strong absorption within the visible light range,which overlaps with the emission of commonly used fluorescent probe and quench the fluorescence of fluorophore by the means of fluorescence resonance energy transfer(FRET),leading to the obtained FA value be greater than 0.4.Thus,one can confidently infer the presence of scattered light in addition to fluorescence.On the other hand,biomocules as FA amplifier can overcome the shortcoming of fluorescence quenching,but the limited mass and volume of a molecule to amplify FA will limit the detection sensitivity.Herein,we utilized the assembly structures of biomacromolecules without fluorescence quenching effect to amplify FA.By combining with nucleic acid signal amplification strategy,a series of biomolecules were analyzed and detected sensitively.The main contains are listed as follows:(1)In order to overcome the sensitivity reduction caused by limited FA amplification ability of one biomacromolecules,an universal detection strategy with high sensitivity and accuracy was established by using protein assembly structures induced by a DNA circuit(catalyzed hairpin assembly,CHA)to enhance FA.In this method,the aptamer of the target is fixed on the surface of the magnetic beads(MBs)and hybridized with the catalyst,and the catalyst is released from the MBs surface when the target was added.After magnetic separation,the free catalyst can trigger CHA process(CHA system is consists of two hairpin modified with biotin,one of which was modified with fluorescent group)to produce the biotin and fluorephore modified double stranded structure.In the presence of streptavidin(SA),the bingding of SA and biotin on the assembled hairpin structure caused multiple SA to aggregate,resulting in an increased FA value because of the large mass(volume)of the aggregation of SA.In the absence of target,each probe can only combine with one SA,which will result in a low FA value.This method was applied for the detection of miRNA-145,ATP and staphylococcal enterotoxin B(SEB),and it has been applied to detect SEB directly in complex samples.In addition,other targets could be detected by replacing the corresponding sequence of aptamer.This method has the following advantages: on one hand,because the protein cannot quench the fluorescence,the accuracy of the detection was greatly improved;on the other hand,the assembly structure of SA has excellent ablility to enhance FA because of the large mass(volume),which has dramatically improved the detection sensitivity.(2)In the above method,when target was absent,the probe was connected to SA,resulting in a relatively high blank FA value.In order to reduce the background value,we constructed an accurate and sensitive method for mi RNA-21 detection by using DNA dendrimer to amplify the FA signal.In this design,the DNA dendrimer with large mass was synthesized through the hybridization chain reaction and the affinity between biotin and SA.The probe DNA(pDNA)hybridized with linker DNA and was fixed on the DNA dendrimer indirectly.The rotation of fluorophore was limited by the high-mass DNA dendrimer,resulting in an increased FA value.After the addition of miRNA-21,the miRNA-21 trigered the CHA process,and the assembled hairpin structure can form a complex with the pDNA.Then the pDNA was released from the DNA dendrimer,resulting in a decreased FA value.Therefore,the miRNA-21 was detected by the reduced FA signal with high accuracy and sensitivity.There is a good linear relationship between mi RNA-21 and the decreased FA value in the concentration range from 1 nM to 19 nM and detection limit was found to be 52 pM.By taking advantage of large molecular mass of DNA dendrimer and the characteristics of without fluorescence quenching effect,the accuracy of detection was greatly improved.In addition,the single-stranded pDNA has a small FA value,which greatly reduces the background value.(3)In the above two methods,the detection accuracy was improved by employ the assembled biomacromolecules without fluorescence quenching as FA amplifiers.However,the precision of detection was limited due to the uncontrollable size and number of connected probes of the FA amplifier.In order to solve the above problems,we constructed a strategy for enhancing FA based on two-dimensional DNA nanosheets with controllable size and without fluorecense quenching ablility.In this design,pDNA was indirectly immobilized on DNA nanosheets surface by the handle DNA(hDNA)extended from DNA nanosheets,which leads to an increased FA signal due to the increase of molecular weight and the limitation of the rotation movement of the fluorescent group on pDNA.After the addition of the target,the pDNA was released from the DNSs surface,resulting in a decreased FA value.As a result,the target could be detected by the dramatically decreased FA.We have applied this new method in the detection of single-strand DNA(ssDNA),ATP and thrombin successfully.This new strategy has the following advantages: on the one hand,the detection accuracy was guaranted because the fluorescence of dye-labeled pDNA cannot be quenched by DNA nanosheets;on the other hand,the precision of detection was improved due to the controllable size of DNA nanosheets and the controllable number of pDNA linked to the DNA nanosheets.In conclusion,we have designed a novel FA method by using the assembly structures of biomacromolecules(protein assembly,DNA self-assembly)as new FA amplification platforms.These methods have been successfully applied to the detection of a series of biological molecules,such as nucleic acids,proteins and ATP.The sensitivity was improved because of the large mass and volume of the biomacromolecule assembly structures.The accuracy of detection was also improved because the biomacromolecules assembly structures don't have fluorescence quenching effect.As we have successfully applied these new methods in the detection of a series biomolecules,they are universal sensing platforms and can be applied to detect other targets by adapting the corresponding recognition sequence.