Novel Biosensing Methods Based On Target-controlled Assembly Of SERS Nanoparticles

Spectacular advances in nanofabrication techniques and deeper fundamental understanding of the phenomenon stimulated an exponential increase of surface enhanced Raman scattering (SERS) applications which nowadays have expanded across many fields of science. Compared to other optical-based analytical methods, SERS technique offers the advantages of extremely high sensitivity, rich spectral information, narrow vibrational line signatures, little or no sample pretreatment and so on. Considering some key points in the biomarker detection, especially protein, enzyme and toxin, a series of novel SERS biosensing strategies have been developed with high sensitivity, high specificity, speediness and simpleness in the thesis. The mainly points are as follows:(1) Dysregulation of DNA demethylases is involved in many important diseases such as cancers, imprinting-related diseases, and psychiatric disorders. In chapter2, we develop a novel concept for enzymatic control of plasmonic coupling as a SERS nanosensor for DNA demethylation. This nanosensor is constructed by decorating gold nanoparticles (AuNPs) with Raman reporters and hemimethylated DNA probes. Demethylation of DNA probes initiates a degradation reaction of the probes by methylation-sensitive endonuclease and single-strand selective exonuclease. This destabilizes AuNPs and mediates the aggregation of AuNPs, generating a strong plasmonic coupling SERS signal in response to DNA demethylation. To our knowledge, the work is the first time that enzymatic degradation of DNA substrate probes has been utilized to induce aggregation of AuNPs such that reproducible, sensitive SERS signals can be achieved from biological recognition events. This nanosensor has the advantages in its high signal-to-noise ratio, superb specificity, rapid, convenient, and reproducible detection with homogeneous, single-step operation. Thus, it may create a simple and useful platform for detecting DNA demethylation and related molecular diagnostics and drug screening.(2) Sensitive and selective detection of molecular biomarkers represents an essential approach in proteomics and clinical diagnostics. In chapter3, we report the development of a novel single-step, sensitive, multiplexed, homogeneous immunoassay platform based on our realization that substantial SERS signal enhancement can be achieved by controlled immunological assembly of SERS nanoparticles in response to protein targets. This platform, called as target-controlled assembly based SERS immunoassay (TCA-SERS immunoassay), uses gold spherical nanoparticles or nanorods co-decorated with antibody half-fragments, non-fluorescent Raman-active dyes and passivating proteins as the SERS nanoparticles. This nanoparticle can offer a facile approach to accomplish orientational immobilization of antibodies, minimized plasmonic coupling distance, multi-color Raman fingerprint coding, as well as excellent biocompatibility and stability. This platform is demonstrated for multiplexed quantification of three cytokines, IFNy, IL-2and TNFa, with large signal-to-noise ratio. The developed SERS immunoassay platform may create a simple but valuable tool for facilitating accurate validation and early detection of disease biomarkers as well as for point-of-care tests in clinical diagnostics.(3) Identification and analysis of cholera toxin (CT) have great potential as useful diagnostic tools for the prevention and control of cholera. In chapter4, we develop an extremely sensitive bioassay for CT, using GM1-incorporated liposome-SERS nanoparticle composite. CT binds to GM1-incorporated liposome-SERS nanoparticle composite and induces aggregation of the nanoparticles, generating a strong plasmonic coupling SERS signal. Compared to other methods, our biosensing strategy has the advantages in its high signal-to-noise ratio, superb specificity, and rapid, convenient, and reproducible detection with homogeneous, single-step operation. Thus, the developed strategy may provide a useful method for detecting CT and preventing and controlling of cholera.(4) Identification and analysis of histone demethylases (HDMs) have great potential as useful diagnostic and therapeutic tools for the related diseases, which represent an active area in epigenetic research, molecular diagnostics and drug screening. In chapter5, we develop a novel SERS-based biosensing strategy for sensitive detection of HDMs activity by direct observation of by-product formaldehyde. A thiolated reactive probe, is used for the SERS-based detection of formaldehyde, which allows a selective reaction with formaldehyde under mild conditions, and the product can self-assemble on peptide-stabilized AuNPs to deliver the SERS signal. To the best of our knowledge, the present work reports for the first time the use of formaldehyde-selective reactive probe for SERS-based detection of histone demethylases. This biosensor has the advantages in its high signal-to-noise ratio, superb specificity, rapid, convenient, and reproducible detection with homogeneous single-phase assay. Thus, the developed strategy may create a simple and useful platform for the characterization of HDMs and related researches into their biological functions.