Preparation Of Novel Biosensors Based On Hybridization Chain Reaction Coupled With Gold Nanoparticles

Gold nanoparticles?AuNPs?have been widely used in the preparation of biosensors that used for the detection of nucleic acid,protein,metal ions and small molecules due to their unique photoelectric properties including high specific surface area,good biocompatibility,good conductivity,good dispersibility in solution,good fluorescence quenching and so on.In addition,hybridization chain reaction can be triggered by an initiator and hybridized between two stable complementary DNA strands to form long double-stranded DNA complex.As a kind of signal amplification technology,which can be carried out without the enzyme in the room temperature.Thus,it is widely used also in biochemical analysis.Combing hybridization chain reaction with novel gold nanoparticles,biosensors were prepared that can be used for the detection of double-stranded DNA?dsDNA?,metal ions and protein,and the strategies realized the sensitive and selective detection of targets,is expected to become a new biological diagnostic tool.The main contents of these researches were summarized as follow:Part one:Introdution.The concept and types of gold nanoparticles.The applications of gold nanoparticles and hybridization chain reactions in biosensor preparation.The contents and significances of this research were briefly summarized.Part two:Preparation of novel biosensors based on hybridization chain reaction coupled with gold nanoparticles.1.Double-stranded DNA detection was realized based on the hybridization chain reaction coupled with positively charged gold nanoparticles?+?AuNPs.Firstly,capture probe was immobilized onto the electrode surface.Upon addition of dsDNA,sandwiched DNA complex could formed between capture probe and detection probe.Then,another exposed part of the detection probe could open two alternating ferrocene?Fc?modified DNA hairpins and triggered the HCR to form a double-helix.Meantime,?+?AuNPs were electrostatically adsorbed onto the negatively charged surface of such double-helix to amplify electrochemical signal.Then,dsDNA could be recognized indirectly in the range from 15 pmol/L to 1.0 nmol/L by monitoring the electrochemical signal change of Fc,and the detection limit for dsDNA was 2.6 pmol/L.2.A sensitive sensor for Hg²⁺detection was reported by using DNA-based hybridization chain reaction coupled with positively charged Ag@Au core-shell nanoparticles??+?Ag@Au CSNPs?amplification.For constructing such a sensor,graphene-nafion?Gr-Nf?film was introduced to enlarge the electrode surface.Then gold nanoparticles?AuNPs?were electrodeposited on the modified electrode to provide a rough and stable surface for the immobilization of capture probe.Subsequently,capture probe would hybridize with the detection probe that modified onto the surface of AuNPs based on T-Hg²⁺-T coordination chemistry in the presence of Hg²⁺.After that,the carried detection probe then opened two ferrocene?Fc?modified hairpin DNA in sequence and propagated the happen of HCR for the formation of extended dsDNA polymers.Furthermore,?+?Ag@Au CSNPs were adsorbed onto such dsDNA polymers surface to amplify the electrochemical signal.Based on detectig the electrochemical signal change of Fc along with the change of Hg²⁺concentration,Hg²⁺could be detected in the range of 10 pmol/L-2.5 nmol/L,with a detection limit of 3.5 pmol/L.Meantime,the assay had good recovery in the detection of Hg²⁺concentration from water samples.3.A sensitive sensor for glucose detection was reported by using gold-DNA complex amplification.Firstly,the stem-loop structure of thiolated molecular beacon?MB?that immobilized onto gold electrode surface was opened based on the hybridization of it with the detection probe,and then another exposed part of it opened two ferrocene?Fc?modified DNA hairpins,which directly induced the happen of hybridization chain reaction?HCR?and the formation of extended dsDNA polymers.Meantime,?+?AuNPs were electrostatically adsorbed onto the negatively charged dsDNA polymers to amplify electrochemical signal.However,if the detection probe was incubated with the mixture of glucose,glucose oxidase and Fe²⁺firstly,it would be cleaved into DNA fragments and could not open the stem-loop structure of MB,accordingly resulted in no HCR happen and?+?AuNPs adsorption,which directly induced the decrease of electrochemical signal.Thus,glucose detection could be realized with a linear range of 50 pmol/L-2.0 nmol/L based on monitoring the Fc signal change.The detection limit for glucose detection was 21 pmol/L.4.Based on the combination of HCR and the fluorescence quenching of AuNPs,an ultrasensitive and enzyme-free biosensor was developed for the detection of AGR2.Two fluorophore labeled hairpin probes were firstly designed.The AGR2 aptamer was used as an initiator for HCR event,which could hybridize with the fragment“a”of HP₁and opened the hairpin structure of it.The newly exposed sticky section of HP₁ could then hybridize with the fragment of HP₂ to open its hairpin structure and expose a sticky end for the following hybridization with the fragment of the next HP₁.According to this principle,HCR happened between HP₁ and HP₂ to form a nicked DNA duplex,which could not adsorb onto the surface of AuNPs,and thus a strong fluorescence signal appeared due to the label of HP₁ and HP₂ with fluorophore.However,in the presence of AGR2,the AGR2 aptamer would specifically recognize with it and thus could not trigger the happen of HCR.Then,the sticky tails in HP₁ and HP₂ would adsorb onto the surface of AuNPs,resulting in the efficient quenching of the fluorescence signal due to the close proximity of the fluorophore to AuNPs surface.Thus,the quantitative detection of AGR2 could be realized indirectly based on monitoring the decrease of the fluorescence signal.The linear range for AGR2 detection was 5.0 pmol/L to 1.0 nmol/L,with a detection limit of 2.65 pmol/L?...