Novel Biosensing Methods Research Based On Gold Nanomaterials

Optical biological sensing technology is usually means that optical signal was produced in the object to be measured and the recognition unit combination process, the determinand and identify the object under test unit combination process of optical signal, through the transducer signal conversion and amplification processing, directly into a readable data, and qualitative or quantitative analysis of the object under test can be read directly by the final output data or conversion. Optical sensing technology without biological separation, can be in situ determination of real-time live is easy to operate, so more and more get the attention of people. Nanomaterials, referring in three-dimensional space in at least one dimension in the nanometer scale (1-100nm), or by the scale of the material as the basic structural unit of nanoscale materials with special effects. Because of its special structure, nanomaterials often have special properties, including optical properties, thermal conductivity, electrical conductivity, magnetism and so on. The nanoparticles materials, between the atom clusters and macroscopic object boundary, it has a quantum size effect, small size effect, surface effect, quantum tunnel effect, dielectric confined effect, etc. Nanomaterials have been widely used in electronics, chemical industry, light industry, textile, military, medical and other fields. By combining both nano materials and optical biological sensing technology, applicationing in the small molecular protein, clinical diagnosis, drug screening and target therapy is a very important and urgent. To sum up, put the protein, phospholipids signal inositol, protein post-translational modifications enzymes, toxins and other major diseases, such as human tumor nuclear important biological functions related biomarkers as the research object, development a series of new biological sensing method whit simple and quick, low cost, high flux, wide linear range, good stability, and high sensitivity, high selectivity. The mainly points are as follows:(1) Enzyme mediated phosphoinositide signaling plays important regulatory roles in diverse cellular processes and has close implication in human diseases. Phosphoinositide signaling system, regulated by reversible phosphorylation of the inositol headgroup by phosphoinositide kinases and phosphatases, plays important roles in diverse fundamental cellular processes. In chapter2, we have developed a novel enzyme-activated AuNPs assembly strategy as a homogeneous colorimetric biosensor for activity detection of phosphoinositide kinases and phosphatases. This strategy utilizes a biomimetic mechanism of phosphoinositide signaling, in which AuNP supported phospholipid membranes was constructed to mimic the cellular membrane substrate, and AuNP modified binding PH domain was designed for specific recognition of the phosphorylated phosphoinositides. The biomimetic strategy enables efficient enzymatic reactions of the substrate and highly selective detection of the target enzyme with minimized interference. The developed biosensor strategy allows convenient, rapid, sensitive visual detection of phosphoinositide enzymes with a pM detection limit and a four-decade wide dynamic range. The biosensor is also demonstrated for the utility for enzyme assay in real cell lysate samples. Moreover, the biosensor is configured into a homogeneous format that increases the potential for high-throughput analysis. In view of these advantages, the developed strategy might create a general biosensor platform for visual detection of phosphoinositide signaling with high sensitivity and selectivity in biomedical research and clinical diagnostics.(2) Vibrio cholerae causes cholera, cholera can cause infections caused by severe diarrhea is widespread and ancient deadly infectious diseases, the cholera toxin (CT) is the external toxin produced by the bacterium vibrio cholera, so, monitoring the cholera toxin (CT) content on the cholera prevention and control work has very important significance. Human c-reactive protein (CRP) is the index factors of inflammation related diseases in the human body, these diseases include bacterial infection, or tissue damage and cardiovascular system diseases (including coronary heart disease), etc. For assessment of human c-reactive protein (CRP) has extraordinary significance. In chapter3, based on the previous chapter, improved the modification methods of phospholipids membrane on AuNPs, use with hydrophobic long chain alkyl NH3of cationic surfactant molecules (CTAB), Au-covalent NH3key role in the synthesis of modified directly on the surface of AuNPs. Glycosphingolipids N-end is inserted into the gold nanoparticle surface,forming a single molecular layer of glycosphingolipids. Using the Powerful combination between different scabbard glycolipids glycolipids and its receptor (such as the cholera toxin (CTB), human c-reactive protein (CRP), tetanus toxin and beta glycosidase, etc.), it can easily achieve the detection of toxins and proteinase accurate quantification of purpose.(3) Protein post-translational modifications (PTMs) play key roles in functional proteomics, via regulating the activity, localization, interactions with other biomolecules, and degradation of proteins. There is a grand need but a daunting challenge in the development of simple and label-free methods that allow rapid and real-time detection of PTM enzymes. In chapter4, we developed a simple, rapid method for highly fluorescent AuNCs synthesis with different peptide templates and discovered that PTM enzymes were able to exert chemical modifications on the peptide-templated AuNCs and quench the AuNCs’fluorescence. The peptide-templated synthesis and the enzyme-responsive property of AuNCs were successfully demonstrated using two PTM enzymes, HDAC1and PKA. It was found that the synthetic method produced highlyfluorescent Au8clusters with compact peptide coating. Enzymatic modification of the peptides could largely quench thefluorescence of the AuNCs, presumably because chemical modifications destroyed the protective peptide coating on AuNCs and induced O2-mediated quenching and oxidation of thefluorescent clusters. This enzyme-responsivefluorescent nanocluster beacon was shown to be highly sensitive and selective for label-free and real-time quantification of HDAC1and PKA with wide linear detection ranges. In virtue of the important biological roles of PTM enzymes and the advantages of the enzyme-responsive nanocluster beacon, this technology indeed created a useful label-free biosensor platform for the detection of PTM enzymes and their inhibitors, implying its great potential in proteomics, biomedical imaging, and clinical theranostics.(4) MDM2oncogene, is a clone of new genes associated with wide variety of tumor, basic physiological processes in the cell, its mutation andamplification and tumor (such as glioma, osteosarcoma, soft tissue sarcomaand breast cancer cell line) of metastasis. Gp120surface protein, is a glycoprotein virus outer, it for HIV entry into host cells caused by viral infection is very important. In chapter5, based on the previous chapter, proposed a method of biological sensor protein protection of biologically active molecules for protein analysis based on the study of small molecule. In theory, polypeptide by carboxypeptidase Y hydrolyzed into amino acid fragments, but, when the biological polypeptide chain and its target protein binding, theprotein prevents hydrolysis. Based on this principle, combined with the synthesis method of peptide-template AuNCs, this paper presents a new label-free biosensing platform method for rapid, sensitive detection MDM2and gp120. Also, the new method provides a new ultra sensitive biosensing platform for the study of small molecules and protein binding of specific interaction.