Graphene Oxide Amplified Fluorescence Anisotropy For Biomolecules Detection

In recent years,the fluorescence anisotropy(FA)has been widely applied in biochemical analysis and molecular disease diagnosis.FA is established based on changes in rotational motion of fluorescently labelled molecules,which depends on molecular volume and local movement of fluorescent molecule.However,traditional FA assays showed relatively low sensitivity for molecule detection,because widespread molecular masses are too small to produce detectable changes in FA value.Therefore,a general strategy that involves a design for mass amplification would be a highly valued addition to this field.Until now,different nanomaterials have been used as the efficient FA amplifiers.Especially,graphene oxide(GO)has already been demonstrated as an excellent FA amplifier because of its unique interactions with DNA and structure containing two-dimensional(2D)nanosheet with slower rotation rate than that of zero-dimensional(0D)spherical nanoparticles with the same surface area.However,there are some drawbacks associated with GO being used as FA enhancer.Firstly,the fluorescence of dye-labeled probe DNA(pDNA)on GO is found to be quenched strongly by GO,thereby substantially limiting the detection accuracy.Secondly,nucleic acid probes are not easily released from GO surface through target competition,thereby limiting the detection sensitivity.In this paper,we developed a novel GO amplified FA strategy for the detection of biomolecules.The main contents are listed as follows:(1)A novel and versatile GO amplified fluorescence anisotropy strategy with improved accuracy and sensitivity is explored.The probe DNA was first indirectly immobilized on GO through a capture DNA to get high FA.Upon addition of target,probe DNA was released from GO surface,resulting in obviously decreased FA.We successfully applied this new strategy in selective detecting of ssDNA,adenosine and thrombin.In this method,the introduction of capture DNA decreased the fluorescence quenching effect of GO,thus improving the accuracy of the FA detection.In addition,this design promoted the release of fluorophore-labelled pDNA from GO surface,thereby providing excellent detection sensitivity.(2)An exonuclease III(Exo III)-assisted GO amplified FA assay is constructed for ricin B-chain detection by using aptamer as a recognition element.This method is simple,quick and cost-effective compared with immunoassay.The aptamer was hybridized with a blocker sequence and linked onto the surface of magnetic beads(MBs).Upon the addition of ricin B-chain,blocker was released from the surface of MBs and hybridized with the dye-modified probe DNA on the surface of GO through the toehold-mediated strand exchange reaction.The formed blocker–probe DNA duplex triggered the Exo III-assisted cyclic signal amplification by repeating the hybridization and digestion of probe DNA,liberating the fluorophore with several nucleotides(low FA value).Thus,ricin B-chain could be sensitively detected by the significantly decreased FA.It existed a good linear relationship between the decrased FA value and ricin B-chain in the concentration range of 1.0 μg/mL – 13.3 μg/m L.The estimated detection limit was as low as 400 ng/m L,which is approximately comparable with the previous reported immunoassays and is 22.5 times lower than that without Exo III.This method improved the sensitivity of FA assay and it could be generalized to any kind of target detection based on the use of an appropriate aptamer.(3)We developed a new target-catalyzed hairpin assembly(CHA),an enzyme-free DNA circuit,assisted GO amplified FA strategy for microRNA-21(miRNA-21)detection.This method accomplishes the target recycling to improve the detection sensitivity and selectivity.In this method,the probe DNA was first indirectly immobilized on GO to get high FA.In the presence of miRNA-21,the CHA was initiated and plenty of H1–H2 duplexes were produced continuously.The obtained H1–H2 duplex could trigger the toehold-mediated strand exchange reaction to form a H1-H2-probe DNA complex which detached the dye-modified probe DNA from the GO surface,leading to a decreased FA.By monitoring the decrease of FA,miRNA-21 could be detected in the range of 0.1?16 nM.The limit of detection was 47 pM,which was 279 times lower than that without CHA.In addition,the selectivity of this method has also been enhanced greatly compared with that without CHA.The current strategy avoids the use of any kind of enzyme or sophisticated equipment,making it more stable and cost-effective.In addition,the proposed strategy has been used to examined the expression of miRNA-21 in cancer cell,expecting to provide a sensitive and selective platform for miRNA detection and clinical diagnosis.In conclusion,we developed a novel GO amplified FA strategy with improved accuracy and sensitivity for the detection of a panel of biomolecules.Meanwhile,by introducing the cyclic signal amplification strategy,the sensitivity and selectivity have been further improved.We believe that the proposed strategy has great potential to be used as a routine tool for biomolecules analysis in biomedicine and molecular disease diagnosis.Moreover,this approach may also be extended to other target molecules detection by simply switching the corresponding probe.