| To achieve the food safety and targeted identification and detection of disease-related biomolecules is the primary prerequisite for food safety control and accurate diagnosis of disease.Due to easy synthesis,modification and highly specific affinity recognition function for the target,the nucleic acid aptamer can be used as an ideal carrier for biomolecular target recognition.What's more,the emergence of new nano-optical materials such as quantum dots and graphene oxide provides a new signal conversion substrate for the construction of different biometric sensing probes.The non-radiative fluorescence resonance energy transfer?FRET?mechanism based on the nanoscale distance change response between fluorescent substances provides an effective mode for biomolecular targeted recognition effect sensing and probe construction.However,how to effectively combine the advantages of nanomaterials with biomolecular interactivity is the key to develop highly sensitive nanobiosensor probes.Focusing on how to improve probe targeting and sensitivity to achieve accurate detection of biomolecules closely related to food safety and disease diagnosis and based on the FRET mechanism,this paper selected quantum dots and graphene oxide as energy donor and receptor pairs,and developed four nucleic acid aptamer-mediated FRET probes by virtue of selective recognition of nucleic acid aptamer to target molecule through the modification and assembly of energy donor and receptor pair and key mediator of nucleic acid aptamers as energy for distance regulation between donor and receptor pairs.The main contents are as follows:In view of the potential hazards of bacterial endotoxin?LPS?to food safety and human health and the practical demand for rapid detection of bacterial endotoxin,a FRET probe for simple and rapid detection of LPS was constructed by using lipopolysaccharide aptamers labeled with 6-carboxyl rhodamine as fluorescence target recognition probes and graphene oxide as fluorescence quenching agents based on the selective recognition of lipopolysaccharide aptamers and the fluorescence quenching properties of graphene oxide.Under the optimized experimental conditions,the probe has good detection performance for LPS.The linear range of the probe and detection limit was 25–1600 ng mL-1 and 15.7 ng mL-1.It can be used for the quantitative determination of lipopolysaccharide content in fruit juice,beer and other beverages,and has certain practical application performance.By virtue of the superior optical properties of quantum dots,specific recognition of target molecules by nucleic acid aptamers and fluorescence quenching properties of graphene oxide,an aptamer-mediated quantum dot/graphene oxide composite FRET probe was constructed aiming at the insufficiency of routine insulin detection method.Under the mediation by the aptamer,the insulin aptamer-modified quantum dots can be effectively adsorbed on the surface of graphene oxide,and quantum dot fluorescence will be quenched by graphene oxide.When insulin is present in the system,the high affinity recognition of aptamer and insulin will cause a change in the conformation of aptamer,preventing the effective binding of aptamer-modified quantum dots to graphene oxide and achieving the recovery of fluorescence.Under the optimal experimental conditions,the linear range of detection of insulin by the constructed probe was 4.3-206.4 nM,and the detection limit was2.74 nM.What's more,the constructed probe can be used for rapid calibration of insulin content in pharmaceutical preparations,and the detection process is simple and convenient.On the basis of achieving the detection of a single target by constructing FRET probes through quantum dots/graphene oxide,it is possible to construct a multi-component biomolecule synchronous detection probe based on the multi-color fluorescence properties of quantum dots and broad-spectrum fluorescence quenching performance of graphene oxide.In order to realize the simultaneous detection of three hepatitis virus nucleic acids of hepatitis A,hepatitis B and hepatitis C,three complementary nucleic acid ligands of hepatitis A,hepatitis B and hepatitis C were used as modifiers to respectively modify three kinds of CdSe/ZnS QDs quantum dots?emission wavelengths 525 nm,585 nm and 632 nm?with different homologous excitation and emission wavelengths,and finally,the three nucleic acid ligand-modified quantum dots were obtained and used as fluorescent energy donors.At the same time,by using graphene oxide as a fluorescent energy acceptor,the energy transfer between quantum dots and graphene oxide as well as the simultaneous detection of three hepatitis markers were achieved through the mediating regulation of specific recognition of complementarity by nucleic acid ligands and target nucleic acid sequences.The constructed probe provides a detection technique for the rapid diagnosis and typing of hepatitis,and also provides a reference for the simultaneous detection of multi-component biomolecules based on multi-color quantum dots.In order to improve the fluorescence quenching performance of graphene oxide,enhance the signal transduction efficiency of FRET probe,and further construct a highly sensitive FRET probe,a BHQ-2 modified graphene oxide composite fluorescent quencher?GO@BHQ-2?was prepared by selecting black hole fluorescent quencher BHQ-2 as a modifier and cross linking BHQ-2 to the surface of carboxylated graphene oxide by covalent condensation based on the effect of optical properties of energy acceptor on the detection performance of FRET probe and functionalization of the carboxylate of graphene oxide.As a dual energy receptor,BHQ-2 modified graphene oxide can effectively quench the fluorescence of quantum dots.Compared with pure graphene oxide,GO@BHQ-2 can increase the fluorescence quenching efficiency of quantum dots by 2.4 times,which can be used as a fluorescent super quencher for the construction of fluorescent probes.On this basis,a FRET probe for detecting ricin was constructed by using the quantum dots modified by the ricin?RTB?adapter as the fluorescent donor,GO@BHQ-2 as the fluorescent receptor and adapter as the mediator.The linear scope and detection limit of ricin detection was0.05-3.0?g mL-1 and 0.037 mL-1,respectively.The standard sample recovery experiment confirmed that the constructed probe can be used for RTB selective detection in actual samples,which not only provides a new technical method for the rapid detection of RTB,but also provides some ideas for the modification of graphene oxide.In summary,by using fluorescence energy resonance transfer as the probe construction principle,this paper constructed FRET probes of four biomolecules closely related to food safety and disease diagnosis such as lipopolysaccharide,insulin,hepatitis nucleic acid marker and ricin,by using aptamer functionalized quantum dots and graphene oxide as the energy donor-acceptor pairs based on the selective recognition performance of nucleic acid aptamer to a target molecule and its binding property to graphene oxide,under the regulation of fluorescence energy transfer between energy donor and receptor pairs through aptamer mediation.This not only provides a new method for the simple and convenient detection of four biomolecules mentioned above,but also provides a basis for the construction of target recognition fluorescent probes for other biomolecules based on functionalized quantum dots and modified graphene oxide. |
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