Study On Signal Enhanced Electrochemical Sensor For Quantitative Determination Of Small Molecules

In this thesis,the small molecules mainly refer to three kinds of antibiotics and asenosine triphosphate(ATP),which possess smaller relative molecular mass and biological activity.The determination of these small molecules plays an important role in pesticide safty dosage,food quanlity inspection,drug content determination,and cell biological activity research.Electrochemical sensor,based on the highly specific molecular recognition(antibody or aptamer)and the electrochemical technique,has become one of the predominant analytical teniques for target analyte due to its low cost,high sensitivity,high selectivity,fast,simple instrumentation and excellent compatibility with miniaturization technologies.For the successful development of an electrochemical sensor,the immobilized strategy of biomolecules or protein and the amplification of elelctrochemical signal should be two key issues.Importantly,they typically complement each other within.In this thesis,we report the proof-of-concept of several new electrochemical sensors for the determination of some small molecules by coupling with the nano immobilized technique,hydrogels immobilized technique,enzyme catalyzed amplification,nano signal amplification,molecular biological amplification,and target recycling amplification.The thesis consists of 6 chapters as follows:In the first chapter,a general introduction of small molecules(the definition)and the present detection method was made,and detailed introduction of several specific small molecules(molecular structure,pharmacological effects,application and clinical significant)and the present detection method was made.Then,the detection principle and categories of the electrochemical sensor was described in detail,and emphasis on the immobilized techniques and signal amplified techniques.In the meanwhile,a general introduction of multiplex electrochemical sensor was made.In addition,the purpose and signification of this thesis were summarized also.In the second chapter,we synthesized redox-active organosilica nanostructures with three-dimensional network(TRSiN)for the preparation of the electrochemical sensor,which were utilized for the determination of streptomycin(STR)residues in food with a competition-type format by using nanogold-decorated mesoporous silica nanoparticles(GMSNs)as bionanolabels.The assay was carried out by using differential pulse voltammetry measurement method.Under optiamal conditions,the electrochemical sensor exhibited a wide linear range of 0.05?50 ng mL-1 with a low detection limit of 5 pg mL-1 STR(at 3SB).Based on its three-dimensional network,the TRSiN possess large specific surface area and could capture a large number of biological molecules(anti-STR in this assay).Moreover,the as-prepared GMSNs with highly loading capacity and good biocompatibility possess the ability to load multiple horseradish peroxidase and biomolecules(antigen).They are helpful to the improvement of the electrochemical sensor properties.In addition,the methodology was evaluated for the analysis of spiked food sample,receiving a good consistency between the electrochemical assay and high-performance liquid chromatography for determination of STR.In the third chapter,a novel multiplexed electrochemical assay protocol in was designed for simultaneous monitoring antibiotics(tetracycline,TC;chloramphenicol,CAP).Based on the specific interaction between biotin-avdin,two kinds of metal sulfide nanoclusters(CdSNC,PbSNC in this assay)had been prepared to be as distinguishable signal tags.In a typical assay,the haptens(conjugates of TC and of CAP with bovine serum albumin)immobilized on glassy carbon electrode compete with the added target analytes for binding to the corresponding antibodies on the metal sulfide nanoclusters.Experimental results revealed that the multiplexed electrochemical sensor enabled the simultaneous monitoring of TC and CAP in a single run.The developed assay exhibited good electrochemical behaviors toward TC in the working range of 0.01?50 ng mL-1 with a low detection limit of 7.5 pg mL-1(at 3sB),and CAP in the working range 0.01?50 ng mL-1 with a low detection limit of 5.4 pg mL-1(at 3SB).In addition,the selectivity,stability and reproducibility of the assay were acceptable.In the fourth chapter,a new homogeneous electrochemical analysis model based on home-made electrochemical detection cell was proposed.Based on the principle of complementary based paring,an omega-like(?)DNA nanostructure consisted of two DNA strands(strand A,SA;strand B,SB)had been prepared.In the presence of target ATP,it triggered the ATP-based aptamer at the 5'-end of SA,resulting in the opening of the ? DNA nanostructures.The released aptamer at the 3'-end of SA could intercalate with the hemin,and form the DNAzyme.The formed DNAzyme could catalyze the substrate,resulting in the production of an electro-chemical signal.By monitoring the change in the signal,we could quantitatively determine the concentration of target ATP in the sample without the need for sample separation and multi-step washing.The dynamic concentration range spanned from 1.0 pM to 10 nM with a detection limit of 0.6 pM ATP.Importantly,the electrochemical assay was application for the analysis of spiked ewborn calf serum sample.In the fifth chapter,coupling with DNase I-catalyzed target recycling,a novel,reproducible magnetic control sensor analysis model was designed.A redox-active Au(III)-assisted core-shell iron oxide@poly-(o-phenylenediamine)nanostructure(MB@Au-POPD)was first designed as a sensing platform for ultrasensitive electrochemical detection of small molecules(ATP,used as a model here).Due to the presence of redox-active POPD,the MB@Au-POPD-modified electrode by external magnet exhibits strong current response.Upon addition of the aptamers,the single-stranded DNA can bind onto the MB@Au-POPD owing to the ?-? stacking interactions between the nucleobases and the POPD.The introduced aptamers act as an inert layer and coat the surface of MB@Au-POPD,thus hindering the electron transfer.In the presence of ATP,the analyte reacts with the aptamer,and disturbs the interaction between the aptamers and MB@Au-POPD.Such interaction enables the release of the aptamer from the MB@Au-POPD.Meanwhile,the released ATP/aptamer complex can be cleaved by DNase I,and the ATP molecule is delivered from the complex.The released ATP then re-attacks other aptamers on the MB@Au-POPD,and triggers another target recycling,resulting in the successive release of the aptamers from the MB@Au-POPD.Thereby,it could restore the redox behavior of the MB@Au-POPD,and cause the increase in the current.By monitoring the increase in the current,we can determine the concentration of the ATP.The electrochemical sensor was evaluated by square wave voltammetry.It was found that the peak currents had a good linear response to the ATP concentration in the range of 0.1 pM?5.0 mM with a detection limit of 0.05 pM.In the sixth chapter,a new flow-through electrochemical aptasensor was designed for ultrasensitive monitoring of ATP by coupling microvalve-programmable capillary column with CdS-functionalized DNA concatamer for signal amplification.Initially,a layer of primary DNA-conjugated polyacrylamide hydrogel was covalently linked onto the internal surface of capillary column.In the presence of target DNA aptamer,the immobilized primary DNA hybridized with partial bases of the aptamer.The excess aptamer fregment could trigger the formation of DNA concatamer between auxiliary DNA1 and CdS-labeled auxiliary DNA2.Upon ATP introduction,a specific ATP-aptamer reaction was excuated,thereby resulting in the release of CdS-functionalized DNA concatamer from the capillary.Subsenquent anodic stripping voltammetric detection of cadmium released under acidic conditions from the released CdS nanoparticles could be conducted in a homemade detection cell.Under optimal conditions,the dynamic concentration range spanned from 0.1 pM to 10 nM ATP with a detection limit of 0.06 pM ATP.