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Development of New Methods for Microcystins Detectionbased on Gold Nanomaterial

Author: ZhuYingYue
Tutor: HuaYuFei
School: Jiangnan University
Course: Cereals, Oils and Vegetable Protein Engineering
Keywords: microcystins detection gold nanomaterials controlled assembly SERS G-quadruplex DNAzymes
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Type: PhD thesis
Year: 2012
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Abstract


Due to the unique physical and chemical properties of gold nanomaterial, the goldnanomaterials are widely applied for various analytical and biological fields. This dissertationdiscuess with the issues of developing series novel, rapid and sensitive toxin detection methoscombine the gold nanomaterials with the traditional and advanced detection methos.Firstly, MC-LR detection was successfully achieved with controllable assembly of goldnanorods (GNRs), which are modified either on the sides or ends, using covalent or electrostaticroutes of protein attachment. The side-by-side and end-to-end assembly of the gold NR wererealized using complementary Ab and antigen. Both sensitivity and detection ranges using UV-visand DLS are markedly better for the end-to-end motif. The controllable immunoassembly methodsfollowing the described approach can be applied to a large variety of environmental toxins bysimple modification of the preparation procedure.Secondly, gold nanorods (GNRs) probes were prepared by modification of GNRs withantibody (Ab) and antigen (Ag, MC-LR-OVA) respective. Using the GNR-Ab and GNR-Ag as theprobes, GNRs were assembled into nanorod chains (end-to-end) through the bio-recognition ofantibody and antigen. The SERS signal of the probe molecule modified to the end of the NRscould be enhanced due to the hot spot between the NRs. The SERS signal could be tuned bychanging the degree of the assembly structure of the NRs. With increased MC-LR concentration inthe solution, the toxin molecules competed with the MC-LR-OVA antigen immobilized on theNRs. We characterized the changes in the degree of nanorod chains by TEM UV-vis and SERS.The limit of detection of MC-LR was0.01ng/mL, and the time necessary for the analysis usingour method (about35min) was decreased by approximately5-fold as compared with traditionalELISA.Thirdly, we demonstrate the application of versatile G-quadruplex-hemin DNAzymes in animmunoassay for detecting a toxin. Taking advantage of the high peroxidase activity ofG-quadruplex hemin complexes and the enhancement effect of gold nanoparticles (AuNPs), themethod showed simple, high sensitive and selectivity detection of target toxin residues in watersamples. The coated antigen, microcystin-LR (MC-LR)-ovalbumin (OVA), when coated on a plate,competed for MC-LR antibody with added target analyte to form antibody-antigen immunecomplexes. Subsequently, the immune complex reacted with G-quadruplex-labeled secondaryantibodies for colorimetric detection of MC-LR. This assay specifically determined MC-LR in thelinear range of0.1-10ng/mL, with a limit of detection (LOD) for MC-LR of0.05ng/mL. Theresults indicated that the novel immunoassay is an alternative to traditional plate-basedimmunoassays.Lastly, a novel transfection vector was designed to deliver the antisense oligodeoxynucleotideinto the cancer cells (HL-60). Antisense oligodeoxynucleotides (ASODNs) were conjugated to thegold nanoparticles (GNPs) through a chemical reaction. The conjugates were then bound to thesingle-walled carbon nanotubes (SWNTs). This vector system can effectively for inducedapoptosis of the cancer cells. The gold nanoparticles helped to improve both transfection andRaman signal. This vector system can effectively for induced apoptosis of the cancer cells, andthus has the potential to be used as a cancer therapy.

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