The Construction And Application Of Fluorescent Probes Based On Aptamer

Aptamer is a piece of DNA or RNA sequence through an in-vitro selection process,it can be binding to the target with high affinity and specificity and which is widely used in the field of biosensors.Nucleic acid dye SYBR Green I is a dye having a green excitation wavelength bound to dsDNA double helix groove region.The change of the fluorescent signal can be used to quantitatively detect the target.Exonuclease III can catalyze the stepwise removal of mononucleotides of double-stranded DNA from 3'to 5',and that can improve the sensitivity of the sensor.In recent years,gold nanoparticles(AuNPs)which is widely used in biological,chemical and other fields because of its good optical properties has become one of the hot spots of scientists.In this paper,we mainly use the spectral characteristics of gold nanoparticles and nucleic acid dye to construct different biosensors to detect tetracycline,hepatitis B virus DNA and adenosine,which mainly include the following three parts:(1)A label-free fluorescent strategy for tetracycline detection based on aptamer.Based on tetracycline aptamer and signal transduction probe(STP),a triple-helix molecular switch(THMS)was constructed.After adding the tetracycline,the structure of THMS was destroyed,and aptamer specifically binds to tetracycline,releasing signal transduction probe.Then adding SG,the fluorescence intensity decreased.The decrease in fluorescence intensity of SG is proportional to the concentration of tetracycline,and the linear range is 1.1-23 nM,the detection limit is 130 pM.This method can be applied to the determination of tetracycline in milk samples,the recovery values were in the range of 92.5-96.1%.(2)A novel label-free exonuclease III-aided amplification assay for Hepatitis B Virus DNA based on nucleic acid dye SYBR Green I.Design two hairpin DNA with a protruding 3'-terminus.In the absence of HBV,ssDNA successively open the two hairpin DNA,and lead to the formation of DNA duplex.After the duplex is digested by Exo III,some single-stranded DNA is released.The system presents a weak fluorescence signal after adding the SG.In the presence of HBV,ssDNA hybridizes with HBV to form a partly complementary double-stranded DNA rather thanhybridizes with the hairpin DNA,so that the two hairpin DNA can keep its structures to resist the digestion of Exo III.The signal of SG significantly increases.The results showed that,for the change of fluorescence intensity of SG and the concentration of HBV,the linear range is from 0.1 to 10 nM,the detection limit of HBV was determined to be 59 pM.It is simple in design,fast in operation and possesses high sensitivity and selectivity,and it can be used very well in serum samples for the detection of HBV,the recovery values were in the range of 93.0-98.0%.(3)Label-free sensor for adenosine detection based on aptamer and gold nanoparticles.In the absence of adenosine,aptamer was adsorbed onto the surface of AuNPs through electrostatic attraction and helps to enhance the AuNPs-stability against salt-induced aggregation,the color of the solution was still red.Followed by the intercalation of SG into the aptamer and adsorbed on the gold nanoparticles,and the aptamer may keep the fluorescent dye SG and AuNPs in close proximity,and the emission of SG and absorption of AuNPs overlaps very well,thus the efficient fluorescence resonance energy transfer(FRET)occurs,so that the fluorescence of SG was quenched by AuNPs.In the presence of adenosine,aptamer specifically binds to adenosine,and the salt easily induced the AuNPs aggregated,the color of the solution changed from red to blue.After adding SG,SG molecules can intercalate into the formed aptamer-adenosine complex,the fluorescence of the system gradually recovered.Accordingly,we can use the colorimetric,UV-Vis and fluorescence methods for the detection of adenosine.Among them,the sensitivity of the fluorescence method is the highest.And the fluorescence method was linear in the concentration range of 5.5-110 nM with 0.8 nM as the limit of detection.This method can be applied to the determination of adenosine in serum samples,the recovery values were in the range of 92.0-103.4%.