The Study On Novel Fluorescent Biosensing Method Based On Polydopamine Nanomaterials And Enzymatic Cyclic Amplification Strategy

Nucleic acids,biological enzymes,proteins,and small biological molecules are involved in many important biological processes.The analysis and detection of biological molecules has received more and more attention.Optical biosensors are widely used for the detection of biomolecules due to their excellent properties such as high sensitivity,low detection limit,good specificity and high accuracy.Polydopamine is the material of choice for biosensing applications due to its simple synthesis method,unique optical properties,adsorption properties,adhesion properties,and good biocompatibility and biodegradability.Combining the advantages of polydopamine and nuclease-assisted target circular amplification strategies,a series of new fluorescence sensing methods have been constructed for the detection of biomolecules.The specific content includes the following three aspects:1.A new fluorescence sensing method based on polydopamine nanotubes(PDANTs)and ? exonuclease(? exo)was constructed to detect T4 polynucleotide kinase(T4 PNK).PDANTs can quickly adsorb ss DNA and quench its fluorescence,but have less affinity for ds DNA.When T4 PNK is introduced into the system,T4 PNK phosphorylates the 5?-terminus of ds DNA to produce a 5?-phosphoryl end product.? exo hydrolyzes the 5?-phosphoryl end product of ds DNA and releases ss DNA.The energy resonance transfer(FRET)effect occurs between the labeled fluorophore and PDANTs on ss DNA,and the fluorescence intensity decreases.Based on the above strategy,this system successfully established a highly sensitive fluorescence sensing method for the detection of T4 PNK and its inhibitors.The detection limit of this method is 0.01 U/m L,and the linear range is 0.01 to 50 U/m L.At the same time,this method has achieved the detection of T4 PNK in complex systems,indicating that this method has great potential in biomedical and drug screening.2.A circular amplification strategy based on polydopamine nanotubes(PDANTs)and exonuclease I(Exo I)was constructed to detect cytochrome C(cyt C).PDANTs can rapidly adsorb aptamers of cyt C and quench their fluorescence.When the target cyt C is introduced into the system,cyt C can specifically bind to its aptamer adsorbed on the surface of PDANTs,and the fluorescence signal is enhanced.Exo ? is a single-stranded exonuclease that cleaves the aptamer of cyt C off the surface of PDANTs,releases cyt C into the next cycle,and the fluorescence signal is stronger.Based on the above strategy,this system successfully established a highly sensitive fluorescence sensing method for detecting cyt C.The detection limit of this method is 0.003 ?M,and the linear range is 0.01-100 ?M.This method enables the detection of cyt C in complex systems,indicating that the method can be used to monitor cell apoptosis to study certain cell-level diseases.3.Construction of a circular amplification platform based on polydopamine nanotubes(PDANTs)and DNase I assisted target detection to detect micro RNAs.PDANTs can rapidly adsorb DNA probes and quench their fluorescence.When the target micro RNA is introduced into the system,the micro RNA can quickly bind to the complementary sequence adsorbed on the surface of the polydopamine nanotubes to form an RNA-DNA double-stranded structure,which is detached from the nanotube surface,resulting in fluorescence recovery.The DNase I only acts to cleave the DNA strand in the RNA-DNA double-stranded structure,but has no activity on the RNA strand.The released micro RNA continues to bind to the complementary sequences adsorbed on the surface of PDANTs to implement a signal amplification strategy.Based on the above strategy,the system has successfully established a highly sensitive method for detecting micro RNAs with a detection limit as low as 0.01 n M.This method has the advantages of simple and fast,high sensitivity and low detection limit.In addition,the experimental method can be used to analyze micro RNA samples directly in serum,indicating that the method has great potential for clinical diagnosis.