Preparation Of Novel Multifunctionalized Nano-Bioprobes And Their Applications In Bioassay

Highly sensitive, selective, and quantitative detection of nucleic acids associatedwith human diseases, proteins for host response and small molecules for profiling thehuman metabolome has played a vital role in the field of modern moleculardiagnostics and genomics research. There has been tremendous interest in thedevelopment of novel multifunctionalized nano-bioprobes for molecular detection,pathogen and live-cell sensing, tissues diagnosis and in vivo bioimaging. Recent years,functional nanomaterials have been actively explored to enhance the abilities ofbioanalytical systems for bioassay.Here, combining functionalized nanomaterials and DNA nanotechnology, weprepared rolling circle amplification (RCA)-based carbon nanotube probe, polyAnon-fluorescent nanotags (pA-nF-NTs)-based SERS probe and RCA-generatedmulti-fluorescence labeled DNA probe for highly sensitive and selective assay ofvarious bioactive molecules.1. Carbon nanotubes multifunctionalized by rolling circle amplification and theirapplication for highly sensitive detection of cancer markersWe have developed multifunctional MWCNTs by RCA which can be used as asignal amplification system. Long ssDNA is generated in situ on MWCNTs alongwith the loading of molecular labels. In this system, binding ability of proteins,hybridization and replication ability of DNAs and catalysis ability of enzymes, areintegrated on a single carbon nanotube. The system is then used for ultrasensitivedetection of cancer protein biomarkers in biological fluids, demonstrating that it is anavailable tool in molecular diagnosis for cancers.2. Gold Nanostructures Encoded by Non-fluorescent Small Molecules inPolyA-mediated Nanogaps as Universal SERS Nanotags for Recognizing VariousBioactive MoleculesWe have developed a universal SERS platform based on pA-nF-NTs nanotags for multiplex assay. Five different types of highly uniform and reproducible SERS activepA-nF-NTs with non-fluorescent small molecule Raman reporters were successfullysynthesized. The main problems such as high-cost and SERS spectra overlap havebeen solved satisfactorily by using polyA block and non-fluorescent small moleculeRaman reporter. It has been demonstrated that pA-nF-NTs SERS tags have excellentsensitivity and specificity for BRCA-1gene DNA assay. Moreover, highly specificand sensitive multiplex assay for virus gene DNA has been achieved with ourpA-nF-NTs. Even more important, we have successfully demonstrated thatpA-nF-NTs could be competent to multiplex assay for multiplex analytes includingnucleic acid, protein and small molecule, and showed high selectivity and sensitivity.To our knowledge, the universal multiplex detection system presented here is the frstto achieve simultaneous detction for all of the major classes of analytes with onemeasurement.3. Graphene oxide-based RCA generated multi-fluorescence labeled DNA probefor DNA recognitionA novel synthesis method for multi-fluorescence labeled DNA probe has beendeveloped via RCA. Combine multi-fluorescence labeled DNA probe with grapheneoxide, we further developed graphene oxide-based platform for DNA recognition withmulti-fluorescence labeled DNA probe. Both different lengths of DNA targets anddifferent concentrations of DNA targets were analyzed with this method successfully.