Study On The New Aptasensing Methods Based On Biological Signal Amplification Techniques

With the rapid development of automated aptamer-screening techniques,the combination of the highly specific biorecognition of aptamers with different sensitive signal transduction strategies to construct various biosensors with excellent performance has attracted considerable attentions in the field of analytical chemistry.Compared with antibodies,aptamer possesses unique advantages such as smaller molecular weight,more easiness in chemical synthesis,better stability and higher specificity in biorecognition.Thus the construction of biosensors based on aptamer has better performance superiority and application potentials over the conventional immunosensors.More importantly,the property of aptamer as an oligonucleotide make its much feasible for the combination with various nucleic acid and nuclease-based biological signal amplification techniques to develop various biological signal amplification strategies and thus enhance the analytical sensitivity of the biosensors greatly.Therefore,based on the requirement of highly selective and accurate detection of low-abundant analytes like antibiotic molecules and microRNA biomarkers in complicated matrices,in this thesis I combine the highly specific biorecognition of aptamers and various biological signal amplification techniques to carry out three studies on the development of new ultrasensitive electrochemical or colorimetric biosensing methods,which are described in details as follows:1.Ultrasensitive electrochemical biosensing of kanamycin based on the signal amplification with enzyme-nanoprobeBased on the signal transduction with a horseradish peroxidase（HRP）-functionalized gold nanoprobe,this work successfully develops a new electrochemical aptasensing method for kanamycin（Kana）detection.The aptasensor was constructed through the hybridization of biotinylated Kana-aptamer at its complementary oligonucleotide strand-modified electrode,followed by intercalating methylene blue（MB）into the formed double-stranded DNA（dsDNA）.Then,the streptavidin and high-content HRP functionalized gold nanoparticle probes were bound onto the aptasensor.As the MB-mediated HRP-catalytic reaction could produce sensitive electrochemical signal and the aptamer-biorecognition toward Kana could cause quantitative decrease of MB intercalation and nanoprobe capture,convenient electrochemical signal transduction was achieved.Both the enzymatic reaction and nanoprobe signal amplification greatly enhance the electrochemical signal,offering ultrahigh sensitivity of the method.Under optimal conditions,this method showed a wide linear range over four-order of magnitude with a low detection limit of 0.88 pg mL^-1.2.Ultrasensitive homogeneous colorimetric biosensing of kanamycin based on the DNAzyme-catalytic reaction and target recycling for signal amplificationBased on the target-induced DNAzyme liberation and exonuclease III（Exo III）-assisted target recycling,this work successfully develops a homogenous colorimetric bioassay method for the ultrasensitive determination of kanamycin（Kana）antibiotic.TheDNAhybridizationbetweenKana-aptamerandthe peroxidase-mimicking DNAzyme sequence contained mononucleotide well inhibited the activity of the DNAzyme.The specific binding between Kana and its aptamer led to the liberation of the DNAzyme strand from DNA duplexes for colorimetric signal transduction.One adjacent-adenine extension of the DNAzyme sequence not only enhanced the catalytic activity of the DNAzyme remarkably but also ensured its easy activity inhibition and liberation.Meanwhile,the formation of the hairpin aptamer-Kana biocomposite enabled the Exo III-assisted target recycling for signal amplification and sensitivity improvement.Under optimal conditions,this method shows a wide linear range of five-order magnitude and a very low detection limit of0.045 pg mL^-1 for Kana assay.3.Exonuclease-assisted target recycling for ultrasensitive electrochemical biosensing of microRNA at vertically aligned carbon nanotubesBy combination of the one-step biorecognition reaction at a vertically aligned nanostructure-based biosensor with the T7 exonuclease（Exo）-assisted target recycling,this work successfully develops a novel electrochemical bioassay method for miRNA-21 detection.The vertically aligned nano interface is constructed through the covalent attachment of terminal carboxylated single-walled carbon nanotube（SWCNT）at an aryldiazonium salt-modified electrode,which enables the noncovalent adsorption of ferrocene（Fc）-labeled single-stranded signal DNA to obtain the biosensor.Upon its incubation with a target miRNA-21 solution,the DNA/RNA hybridized duplexes will form and release from the electrode surface,leading to corresponding electrochemical signal decrease of the biosensor.Moreover,this biorecognition reaction can also trigger the T7 Exo-assisted target recycling to achieve the great signal amplification.Together with the highly efficient biorecognition and excellent electron transfer promotion at the vertically aligned SWCNTs,this miRNA-21biosensor exhibits an ultrahigh sensitivity.Under the optimum conditions,this method can realize the convenient and sensitive detection of miRNA-21 in a wide linear range from 10 fM to 100 pM,and the detection limit is estimated to be 3.5 fM.