Development Of Bioanalytical Methods Based On Copper Nanoparticles

Nano-materials have attracted wide attention because of their unique property. Copper nanoparticles have wide applications on sensors, catalysis for their special properties. In recent years, copper nanoparticle's bioanalytical application has been well developed. Immunoassay and DNA hybridization assay are very important in life science and medical science. Atomic absorption spectrometry?AAS? and inductively coupled plasma mass spectrometry?ICP-MS? have a wide range of applications in elements analysis. They can directly detect metal elements in nanoparticle probes. No special properties such as optical, electrical properties are required, thus the difficulty of the synthesis of the probes was significantly reduced. Besides, their high sensitivity made them intriguing in bioassays.In Chapter 1, we described the basic properties and the preparation of nanoparticles, the application and research status of copper oxide nanoparticles, AAS instrument, and ICP-MS instrument. The research status of immunoassay and label-free DNA detection are also introduced.In Chapter 2, we established a new immunoassay. Depending on the probes and detection methods, labeled immunoassay technology can be divided into radio immunoassay?RIA?, fluorimmunoassay?FIA?, enzyme immunoassay?EIA?, metal oimmunoassay?MIA?, luminescent immunoassay?LIA?, electrochemical immunoassay?ECIA?, etc. After copper oxide nanoparticle labeling, we studied the immunoassay based on atomic absorption spectrometric detection. First, we examined the preparation of nano copper oxide colloid. Then CuO NPs-antibody conjugates were prepared. The sandwich immunoassay was performed in a polystyrene 96-well high binding ELISA plate for the detection of human IgG. Copper ions were released into solution with diluted nitric acid before AAS detection. We optimized the concentration of blocking reagent?BSA? and the dilution radio of the bio-conjugate?CuO NPs-IgG antibody?. The results demonstrated a good linearity in the range of 10-9-10-5 g/mL human IgG. The detection limit was calculated to be 0.19 ng/mL human IgG.In Chapter 3, we established a novel label-free DNA detection method. DNA assays are usual y divided into label and label-free method. Label-free method has been developed rapidly in recent years for its simplicity. The double-strand DNA?dsDNA? can act as an efficient template for the formation of copper nanoparticles?Cu NPs?, whereas the single-strand DNA?ssDNA? and triplexes cannot support the formation of Cu NPs. DNA amplification techniques include polymerase chain reaction?PCR?, ligase chain reaction?LCR?, strand displacement amplification?SDA?, rolling circle amplification?RCA?, etc. However, a common and serious problem is that the activity of enzymes adopted in these techniques can be easily affected by various factor. So hybridization chain reaction?HCR? which is enzyme-free has become an attractive technique. In this study, we established a label-free and non-enzymatic assay for DNA based on ICP-MS detection. First, the magnetic microbeads are modified with capture DNA. Then capture DNA can hybrid with target DNA. HCR was carried out for dsDNA chain extension. Copper nanoparticles can be synthesized in the presence of dsDNA. Finally, copper ions were released into solution with diluted nitric acid and detected by ICP-MS. We optimized the concentration of Cu²⁺, sodium ascorbate, and hairpin probe H1/H2 and the reaction time of HCR. The results demonstrated a good linearity in the range of 1-100 nM target DNA. The detection limit was calculated to be 0.803 nM.