Study On Electrochemical Biosensor Based On Multiple Amplifying Techniques For The Detection Of Biomakers

Electrochemical biosensors were developed by integrating electrochemical analytical methods and biosensors. With the merits of high sensitivity, good selectivity, rapid response, low cost, the electrochemical biosensor has been extensively used in biological analysis, clinical detection, environmental monitoring and so on. In order to improve the property of the electrochemical biosensor, multiple amplifying techniques have been employed to construct biosensor. Among them, nanomaterials show board application prospects, owing to their characteristics of large specific surface area, good electric conductivity and electrochemical catalysis activity. Therefore, the article uses various functional nanomaterials as nanocarriers and focuses on the combination of nanomaterials with appropriate amplifying methods（enzyme catalysis amplification and hybridization chain reaction amplification） to develop a series of sensitive electrochemical biosensors for the detection of thrombin（TB） and matrix metalloproteinase 2（MMP-2）.Part 1 A sensitive electrochemical aptasensor based on palladium nanoparticles-functional graphene-molybdenum disulfide flower-like nanocomposites and enzymatic signal amplificationIn this study, poly（diallyldimethylammonium chloride） functional graphene/molybdenum disulfide nanocomposites（PDDA-G-Mo S₂） were prepared and further immobilized with Pd NPs. With flower-like structure, large surface area and excellent conductivity, Pd NPs/PDDA-G-Mo S₂ was used as nanocarriers and functionalized by hemin/G-quadruplex, glucose oxidase（GOD）, and toluidine blue（Tb） as redox probes. GOD could effectively catalyze the oxidation of glucose, coupling with the reduction of the dissolved oxygen to H₂O₂. Then, both Pd NPs and hemin/G-quadruplex acting as hydrogen peroxide mimicking enzyme could further catalyze the reduction of H₂O₂, resulting in significant electrochemical signal amplification. The proposed aptasensor showed high sensitivity with a wide dynamic linear range of 0.0001 to 40 nmol·L-1 and a relatively low detection limit of 0.062 pmol·L-1 for TB determination.Part 2 An amplified electrochemical aptasensor for thrombin detection based on pseudobienzymic Fe₃O₄-Au nanocomposites and electroactive hemin/G-quadruplex as signal enhancersAn electrochemical aptasensor for TB detection was constructed based on hemin/G-quadruplex as signal label and Fe₃O₄-Au nanocomposites with glucose oxidase（GOx-） and peroxide-mimicking enzyme activity as signal enhancers. Due to the large surface area and good biocompatibility, Fe₃O₄-Au nanocomposites were employed to immobilize amount of electroactive hemin/G-quadruplex. Based on the GOx-mimicking enzyme activity, Au nanoparticles（Au NPs） on the surface of Fe₃O₄-Au effectively catalyzed the oxidization of glucose in the presence of dissolved O2, accompanying with the produce of H₂O₂. Both Fe₃O₄ cores of Fe₃O₄-Au and hemin/G-quadruplex with hydrogen peroxide mimicking enzyme activity could catalyze the reduction of the generated H₂O₂, which promoted the electron transfer of hemin and amplified the electrochemical signal. The proposed electrochemical aptasensor showed a wide dynamic linear range of 0.1 pmol·L-1-20 nmol·L-1 with a lower detection limit of 0.013 pmol·L-1. Part 3 A ‘signal on-off’ electrochemical peptide biosensor for matrix metalloproteinase 2 based on target induced cleavage of peptideIn this work, a ‘signal on-off’ electrochemical peptide biosensor was developed for the determination of MMP-2 on the basis of target induced cleavage of a specific peptide using nanomaterials and HCR as amplification strategies. The prepared S1-p Pt NPs-P1 bioconjugates were employed as nanoprobes, where the biotion-labeled peptide（P1） was used as a cleavage-sensing element, offering the capability of ‘on-off’ electrochemical signalling for the target MMP-2. S1-p Pt NPs-P1 was immobilized on the electrode surface through the conjunction of biotin-streptavidin. Then, HCR was triggered to embed the electroactive thionine（Thi）. p Pt NPs could effectively catalyzed the decomposition of H₂O₂, resulting in the electrochemical signal of Thi enhanced significantly（‘signal on’ state）. Upon cleavage with MMP-2, p Pt NPs and Thi left from the electrode surface, leading to observably decreased electrochemical signal of Thi（‘signal off’ state）. Owing to the combination of nanomaterials amplification with HCR, the proposed ‘signal on-off’ electrochemical peptide biosensor exhibited good sensitivity and satiability with a detection limit of 0.32 pg·m L-1 and wide linear range: 1 pg·m L-1-10 ng·m L-1.