Preparation And Study Of Electrochemical Biosensor Based On Nucleic Acid Aptamer

In this paper,four new electrochemical biosensors were fabricated based on aptamers recognition and hybridization chain reaction?HCR?used as signal amplification method.We successfully achieved high sensitivity,high selectivity biosensors for the detection of Hg²⁺,8-hydroxydeoxyguanine?8-OH-dG?,adenosine triphosphate?ATP?and protein kinase A?PKA?.Mainly includes the four aspects as follows:1.A colorimetric assay for Hg²⁺detection based on Hg²⁺-induced hybridization chain reactionsIn this work,four different sequences of DNA were introduced:cDNA,pDNA and two biotin-labeled hairpin hybrid DNA strands H1,H2.In the presence of Hg²⁺,thymine?T?-rich probe DNA?pDNA?hybridized with cDNA via T-Hg²⁺-T base pairs.Then the HCRs were realized using pDNA as an initiator and two biotin-labeled hairpin DNAs?H1 and H2?as fuel strands.Finally,numerous avidin-labeled horseradish peroxidase?HRP?enzymes were immobilized on long nicked dsDNA strands,which can catalyze the H₂O₂-mediated oxidation of 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate?TMB?to cause a dramatic color change.Under the best experimental conditions,the absorbance of TMB showed a good linear relationship with the logarithm of Hg²⁺concentrations from1 fM to 1 pM,the detection limit was 0.33 fM.This method shows good selectivity and high sensitivity,which provides a potential for the practical detection of Hg²⁺in environmental.2.An electrochemical aptasensor for the detection of 8-hydroxy-2?-deoxyguanosine based on ferrocenescence?Fc?enhanced ABEI electrochemical luminescenceIn this work,A aptasensor was prepared for the determination of 8-OH-dG based on ferrocenescence enhanced ABEI electrochemical luminescence.The aptamer of 8-OH-dG is partially complementary paired with the capture DNA immobilized on the gold electrode,and the other part is complementary paired with the pDNA of the terminally modified Fc.Due to the introduction of Fc,the electrochemical luminescence signal of ABEI will be significantly enhanced.However,in the presence of 8-OH-dG,the conformation of Apt changed from single-stranded structure to tight G-quadruplex structure,and the number of Fc introduced on the surface of the electrode decreases,the electrochemi luminescence signal of ABEI also decrease.Under the optimal experimental conditions,the logarithm of ABEI luminescence intensity was linear with the logarithm of 8-OH-dG concentration in the range from 1nM to 100?M with the detection limit of 0.33nM.This method is applied to the detection of 8-OH-dG in human urine with satisfactory results,so this method has potential application value in clinical detection.3.Anelectrochemicalaptasensorforthehighlysensitivedetectionof8-hydroxy-2?-deoxyguanosine based on the hybridization chain reactionIn this work,a highly sensitive and selective aptasensor was developed for the determination of 8-hydroxy-2?-deoxyguanosine?8-OH-dG?based on the hybridization chain reaction?HCR?signal amplification.It was observed that the aptamer of 8-OH-dG could hybridize with the capture DNA immobilized on the gold electrode and the hybridized portion may induce hybridization of the hybridization strands H1 and H2 to achieve linear growth of the double-stranded DNA.Then the electroactive species?[Ru?NH₃?₆]Cl₃,RuHex?intercalated into the dsDNA grooves to generate the amplified signal.However,in the presence of 8-OH-dG,the aptamer containing G-rich nucleic acid sequences would be induced to form a G-quadruplex structure,which made it impossible to continue the HCR,so the detection signal will significantly decrease.Under the best experimental conditions,the peak current of RuHex was linear with the logarithm of8-OH-dG concentration in the range from 10 pM to 100?M with the detection limit of 2.5pM.The experimental results show that the prepared aptasensor not only showed high sensitivity for the detection of 8-OH-dG,but also exhibited good selectivity against to the uric acid,an important interferent in the urine sample.4.An electrochemical aptasensor for the detection of PKA based on the hybridization chain reactionIn this work,we developed a highly sensitive and highly selective electrochemical sensor based on HCR signal amplification for the detection ofprotein kinase?PKA?.In the presence of ATP,the aptamers bind to ATP-specific interactions which made it impossible to continue the HCR.So the detection signal will significantly decrease.In the presence of PKA,PKA converts ATP to ADP and increased MB loading onto DNA duplexes,can indirect measurement of PKA The linear range of PKA was from 4 U/L to4×10⁵ U/L with a detection limit of 1.33 U/L.The method shows good selectivity and high sensitivity,importantly,we have been used the method to the determination of PKA in HeLa cell lysate,which shows that the method has great potential value in clinical detection.