Rapid Immunoassay For Microcystin-LR Determination Based On Carbon Nanomaterials

Microcystins-LR (MC-LR) was the most toxic cyanotoxins with highest frequency, causing serious problems during cyanobacterial bloom. MC-LR can induce liver damage and even cancer by long-term and low-dose intake, posing great threat to human health. Therefore, it is of great importance for ultrasensitive and selective determination of MC-LR in trace level, which was meaningful for environmental water pollution control and ecological safety insurance. In comparion with traditional methods, biosensing methods have been applied for MC-LR determination due to their tremendous advantages, including simplicity, rapid response, high sensitivty and high selectivity. In this thesis, ultrasensitive recognize interface was developed for the fabrication of photoelectrochemical (PEC) immunoassay, electrochemical immunoassay, and colorimetric aptasensor by coupling with several signal amplification strategies based on functional nanomaterials. Linearity range, limit of detection (LOD), and sensitivity of these approaches were investigated. Furthermore, the signal amplification mechanisms based on functional nanomaterials were discussed. Contents and.results are as follows:(1) Highly oriented silicon nanowires (SiNWs) arrays were prepared by chemical etching method, and then incorporated with graphene quantum dot (GQDs) to form GQDs/SiNWs nanohybrid electrode. The transient photocurrent responses of GQDs/SiNWs were2.2times and6.4times as much as that of SiNWs and GQDs, respectively. Ab/GQDs/SiNWs electrode was fabricated by immobilization of Ab, and the specific recognition of MC-LR by Ab affected the electron transfer properties of the immunoassay system, leading to the photocurrent decrease. In the linear range of0.1μg/L-10μg/L, the photocurrent response was proportional to the MC-LR concentration with a LOD of55ng/L (3σ/κ). This method could meet the provision guideline value of drinking water (1μg/L) provided by WHO and China. The results of real water analysis by this approach were accordant with that obtained by liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS), and its spiked recoveries were97.8%-117.2%.(2) CdS/graphene (Gr) nanocomposites were prepared by solvothermal method, in which Gr was used to promote the charge separation and electron transfer, leading to the obvious enhancement of photocurrent response. By optimizing the reaction parameters, the optimal amount of graphene oxide (GO) was found to be8%for the synthesis of CdS/Gr, which can obtaine maximum photocurrent response. The maximum photocurrent was13.6times as much as that of CdS. The decrease of photocurrent response was obtained due to the increase of the electrical conductivity of elelctrode by the formation of immunocomplex of MC-LR and Ab. On the basis of this principle, this approach displayed a linear range of0.1μg/L-25 μg/L for the quantitative detection of MC-LR with a LOD of21ng/L (3σ/κ). The spiked recoveries for real water analysis were98.1%-106.7%.(3) Gr and CNSs were prepared by hydrothermal reduction method. CNSs served as a vector for loading multiple HRP. Meanwhile, electroconductivity of the electrode was improved by Gr modification. Dual electrochemical signal amplification was achieved by multi-labeled HRP and Gr modification. The electrochemical current response obtained by the proposed electrochemical immunoassay was5.1times higher than that one without employing CNSs and Gr. This approach provided a linear range of0.05μg/L-15μg/L with a LOD of16ng/L (3σ/κ). The proposed method provided precise and reliable results for the analysis of MC-LR in reservoir water, tap water and river water, and its spiked recoveries were88.0%-107.8%.94.6%-103.0%,99.2%-103.4%, respectively.(4) G-quadruplex DNA (G4) was selected as signal molecule, and carboxylated CNTs were used as a vector to improve the amounts of G4by forming G4/CNT/MC-LR biolabels. Meanwhile, CNTs was further applied for Ab immobilization on electrode, which acted as nanowires to promote the electrons transfer. Therefore, the electric-catalytic of H2O2by G4was accelerated, which can significantly enhance the current response. In the linear range from0.01μg/L-7μg/L, the current response was proportional to the MC-LR concentration with a LOD of2.31ng/L (3σ/κ). The results of real water analysis by this approach were in accordant with that obtained by LC-MS/MS, and the spiked recoveries were91.6%-105.2%.(5) Au nanoparticles (Au NPs)/Gr nanocomposite with peroxidase mimic activity was prepared by hydrothermal method, and the peroxidase-like activity of Au NPs/Gr was investigated. Furthermore, a new colorimetric aptasensor was explored for the label-free, rapid and sensitive detection of MC-LR based on the peroxidase mimic. Colorimetric signals generated by the catalytic Au NPs/Gr could be used to qualify the amounts of MC-LR with a liner range of0.01μg/L-1.0μg/L, and LOD was7.14ng/L.As a result, the PEC immunoassay and electrochemical immunoassay were developed based on carbon nanomaterials. In PEC immunoassays, charge separation and electron transfer have significantly improved using GQDs and graphene, leading to the photocurrent enhancements. In electrochemical immunoassys, electron transfer of enzyme-catalytic reaction was increased by graphene and CNTs, and more HRP were imported using CNSs and CNTs as vectors, leding to current enhancements. Ultra-sensitive and selective determination of MC-LR was realized coupled to specific discrimination of MC-R by Ab.