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Study On Electrochemical Biosensors Based On Nanomaterial And Loop Mediated Isothermal Amplification

Posted on:2017-02-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:S B XieFull Text:PDF
GTID:1108330509954490Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
The fast, simple and sensitive detection of biological molecular in clinical diagnosis, environmental monitoring, food analysis and pathogenic microorganisms research is one of the most attractive research areas in analytical chemistry. Electrochemical biosensor has been vigorously developed and studied for biological molecules detection due to its inherent advantages such as simplicity, low cost, high sensitivity and easy miniaturization. Concerning new methods and new technology in electrochemical biosensors construction, using various molecular recognition elements with good specificity, combining novel nanomaterials with excellent performance, introducing effective signal amplification strategies to improve the performance of electrochemical biosensors, has very important significance for a variety of biological molecules specific and sensitive detection. This thesis focus on various signal amplification strategies by combining with functional nanomaterials and loop mediated isothermal amplification to construct electrochemical biosensors with lower cost, stronger practicability and easier operation. The details are mainly as follows: 1 Manganese porphyrin-double strand DNA complex guided in-situ deposition of polyaniline for electrochemical thrombin detectionManganese porphyrin-double strand DNA(MnTMPyP-dsDNA) complex has been reported as an excellent mimicking enzyme of peroxidase, while to date, MnTMPyP-dsDNA-based catalyst for the polymerization of aniline have been rarely reported. In this work, we proposed an electrochemical strategy for sensitive detection of thrombin(TB) based on in suit generated polyaniline(PANI) as redox mediators by using MnTMPyP-dsDNA peroxidase-like artificial mimicking enzymes as powerful catalyst and template. The dense MnTMPyP-dsDNA catalyst units on the fabricated “sandwich-type” aptasensor would catalyze the oxidation of aniline to PANI and lead to a readily measurable “turn-on” electrochemical signal. For further amplified current signal, polydopamine(PDA) and gold nanoparticles functionalized Pd@Pt nanocages(Pd@Pt/PDA/Au) were used as label for enhancing the loading rate of biomolecular and providing a favorable microenvironment for electron transfer. Therefore, the MnTMPyP-dsDNA with the advantages of label-free, excellent catalytic performance, good stability, coupled with the remarkable amplification of the nanomaterials, the constructed aptasensor exhibited a good linear response toward TB over a wide range of 0.5 pmol/L~30 nmol/L with a detection limit of 0.14 pmol/L. 2. Multifunctional hemin@metal-organic framework and its application to construct an electrochemical aptasensor for thrombin detectionA new type of multifunctional metal-organic frameworks(MOFs) have been synthesized by encapsulating hemin into the nano-sized Fe-MIL-88 MOFs(hemin@MOFs) and first applied in an electrochemical aptasensor to detect thrombin(TB) with the aid of enzyme for signal amplification. The gold nanoparticles functionalized hemin@MOFs(Au/hemin@MOFs) have not only been simultaneously served as redox mediators and solid electrocatalyst, but also been utilized as an ideal loading platform to immobilize a large number of biomolecules. In this aptasensor, Au/hemin@MOFs conjugated with glucose oxidase(GOD) and thrombin binding aptamer(TBA II) were used as the secondary aptamer bioconjugates(Au/hemin@MOFs-TBA II-GOD bioconjugates), TB was sandwiched between Au/hemin@MOFs-TBA II-GOD bioconjugates and the amino-terminated TBA I which was self-assembled on the gold nanoparticles(AuNPs) modified electrode. The GOD could oxidize glucose into gluconic acid accompanying with the generation of H2O2. The generated H2O2 on the electrode surface was further electrocatalyzed by hemin@MOFs to amplify the electrochemical signal of hemin contained in hemin@MOFs. Therefore, the synthesized hemin@MOFs represented a new paradigm for multifunctional materials since it combined three different functions including catalyst, redox mediators and loading platform within a single material. With such ingenious design, a wide linear ranged from 0.0001 nmol/L to 30 nmol/L was acquired with a relatively low detection limit of 0.068 pmol/L for TB detection 3. An electrochemical peptide cleavage-based biosensor for prostate specific antigen detection via host-guest interaction between ferrocene and β-cyclodextrinIn this work, we have developed a “signal-on” peptide cleavage-based assay for sensitive and specific detection of prostate specific antigen(PSA) by coupling the host-guest interaction between ferrocene(Fc) and β-cyclodextrin(β-CD). A specific peptide sequence with redox tag labeling(Fc-peptide), which could be recognized and cleaved by PSA, was assembled on Fe3O4@Au magnetic beads first. In the presence of target, PSA specifically cleaved the Fc-peptide, and the redox tag Fc with a fragment of peptide was released from the Fe3O4@Au surface. Since β-CD molecules immobilized on the electrode could efficiently capture the released Fc-labeled peptide fragment via the host-guest interaction, the Fc tags was concentrated on the electrode surface and subsequently yield highly sensitive electrochemical signal. Moreover, the adopted CNTs-PAMAM nanohybrids as a sensor platform can not only enhance the immobilization amount of β-CD for capturing the released Fc but also facilitate electron transfer from Fc to the electrode. By combining the host-guest interaction with the peptide cleavage events, this work provided a simple method for detecting of PSA, and with such design, our sensor showed a good linear relationship in the range of 0.001~30 ng/mL with a detection limit of 0.78 pg/mL for PSA detection. 4 Using a ubiquitous pH meter combined with loop mediated isothermal amplification method for facile and sensitive detection of Nosema bombycis genomic DNA PTP1Loop mediated isothermal amplification(LAMP) is emerging as a rapid diagnostic tool for early detection and identification of microbial diseases in resent years. Current technologies for LAMP-amplified products identification are mainly based on electrophoresis of amplicons, turbidity of magnesium pyrophosphate, and resistance of metal ion as well as electrochemical of an electroactive reagent, while very few of them are widely accepted to the public for quantitative LAMP-amplified products because of either the requirement of laboratory-based instrument or lack of quantitative detection. Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during the LAMP amplification procedure using a pH meter. And the genomic DNA of Nosema bombycis(N. bombycis) were amplified and detected by employing this LAMP-pH meter platform for the first time. The key conception of our method was that detecting the change in pH as the nucleic acid polymerization reaction procedure in LAMP and converting the chemical signals(H+) into a reliable readout of the nucleic acid amplification by using pH meter. Using this platform, as few as 0.5 pg/μL N. bombycis genomic DNA were quantitatively detected. In addition, the method was simple and cost-effective and may be of particular benefit to the farmer in underdeveloped countries. 5. Tracing phosphate ions generated during loop mediated isothermal amplification for electrochemical detection of Nosema bombycis genomic DNA PTP1Traditionally, amplified DNA detection in a loop mediated isothermal amplification(LAMP) was carried out in a complicated gel electrophoresis or expensive fluorescence-based methods. Here, instead of direct detection that relies on amplified DNA, the indirect detection based on tracing phosphate ions(Pi) generated during LAMP by using an electrochemical method has been proposed for sensitive nucleic acid detection. Pyrophosphate(PPi) as the byproducts of nucleic acid polymerization reaction in LAMP was hydrolyzed into Pi by the pre-addition of thermostable inorganic pyrophosphatase(PPase). Thus, the total amount of Pi in the LAMP amplified sample was proportional to the amount of starting DNA templates. The obtain Pi could then react with acidic molybdate to form the molybdophosphate precipitates on the of electrode surface, which serve as redox mediators to give a readily measurable electrochemical signal. We further demonstrate the efficiency of this strategy for sensitive and accurate quantification of Nosema bombycis genomic DNA PTP1. The electrochemical method presented here allows the quantitative analysis for target genomic DNA with a detection limit of 17 fg/μL. Thus, we supposed that the novel method proposed in this work with superior sensitivity and specificity, as well as the simple feature can be easily established for quantitative analysis of many other kinds of nucleic acid in assistance of LAMP for amplification.
Keywords/Search Tags:Electrochemical biosensor, Nanomaterials, Loop mediated isothermal amplification, Signal amplification
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