Studies On New Fluorescent Biosensing Methods Based On The Strategy Of Exonuclease-Assisted Signal Amplification

Developing highly sensitive novel biosensing methods and technologies is of great significance in the fields of biomedical research,clinical diagnosis,food safety and environmental monitoring.Nuclease assisted signal amplification technology provides an important route for designing sensitive biosensing methods.In addition,the development of nanomaterial and aptamer facilities the construction of molecule recognition and signal transformation in the bionsening methods.Combination of the advantages of exonuclease-assisted signal amplification,nanomaterials and aptamer,three sensitive,selective,simple and fast biosensing platforms have been developed,and used for the detection of adenosine deaminase,streptavidin and nucleic acids,respectively.The main contents of this work can be concluded as follows:Part one:Combining with grapheme oxide(GO)and exonuclease ?(Exo ?)-assisted signal amplification,a sensitive fluorescent aptasensor has been developed for the detection of adenosine deaminase(ADA)activity and its inhibitor.This work was based on the superior fluorescence quenching ability of GO and the difference of adsorption ability between nucleic acids with different structure,as well as based on the high digestion efficiency.In the present design,the adenosine(AD)specific aptamer(FAM-apt)with a fluorescein(FAM)fluorophore labeled at 3' terminus is first adsorbed onto the GO surface,and resulting a fluorescence quenching.Upon the addition of AD,a FAM-apt/AD complex was formed and released form the GO surface.After the deamination of AD by ADA,AD was converted into inosine,which could not be recognized by FAM-apt.Thus,the FAM-apt was then adsorbed onto the GO surface again.And the GO could protect the adsorbed FAM-apt form the digestion by Exo ?.On the contrary,in the absence of ADA or after the addition of inhibitor,the formed FAM-apt/AD complex was free in the solution.And the aptamer in the formed complex could be digested by Exo ?,liberating the FAM fluorophores and AD.The released AD would then bind with another FAM-apt which adsorption on the GO surface,and trigger the next digestion.After the cycling recognition and digestion process,numerous FAM fluorophores were released,the fluorescence signal was enhanced greatly.In the concentration range from 0.002 U/mL to 0.25 U/mL,there is a good linear relationship between the fluorescence intensity and the logarithmic concentration of ADA.This method is sensitive with a detection limit of 0.00092 U/mL.The proposed method was also applied for the detection of the ADA inhibitor.Part two:Based on the fluorescence quenching ability of self-assembly aggregates of perylene derivative induced by nucleic acid,and coupled with terminal protection of small-molecule-linked DNA and Exo ?-assisted signal amplification,a highly sensitive fluorescence detection method for protein was developed.Streptavidin was used as the model analyte.The streptavidin-biotin interaction will protect the biotin-modified DNA from digestion by Exo I.Based on the terminal protection,protein assay can be converted into a DNA detection method which is easier to design the signal transformation and amplification.Using the protein-protected single strand DNA to hybridize with a FAM-fluorophore-labeled signal probe,forming a double strand structure.This structure triggered the Exo ?-assisted signal amplification process,liberating numerous FAM fluorophores finally.The intact signal probe would adsorb the positive-charged perylene derivative through the electrostatic interaction,and the perylene derivative was then aggregated.The formed self-assembly aggregates could quench the fluorescence of FAM that labeled at the end of signal probes.However,the released FAM fluorophores could not adsorb the perylene derivative,resulting a high fluorescence signal.The introduction of Exo ?-assisted signal amplification,the sensitivity of the proposed method was improved with a low detection limit of 1.48 ng/mL.In addition,this method has a good selectivity,and a good analytical performance in diluted serum was also archived.Part three:Based on the strand displacement reaction and T7 exonuclease(T7 Exo)-assisted double cycling signal amplification,a simple,fast,and sensitive homogeneous fluorescent sensing system was reported for detecting nucleic acids.Two single strand DNA probes(P1 and P2)were hybridized to form a duplex structure.The addition of target nucleic acids(tDNA)would trigger the strand displacement reaction to produce a tDNA/P1 hybrid,releasing P2 probe.The released P2 probe could open the molecular beacon probes(MB probes),resulting in a fluorescence.After the introduction of T7 Exo into the analysis system,T7 Exo would digest the P1 probes in the tDNA/P1 hybrids and MB probes in the P2/MB hybrids,liberating tDNA and P2 probes.The released tDNA and P2 probes would then induce the cycle "hybridization-digestion" process.Finally,large amounts of free fluorophores were produced which were far away from the quenchers in MB probes,and the fluorescence were then further enhanced.The results indicated that in the concentration range of 1.0×10-10?1.0×10-8 mol/L,a good linear relationship between the fluorescence intensity and the target concentration was achieved.The detection limit of this method is about 5.612×10-11 mol/L.In addition,this method also worked well in complex diluted serum samples.