Based On Signal Amplification Technology In Tumor Cells And Their Markers

This study uses a series of SERS signal amplification methods and fluorescence analysis method to design Nanoparticle-based Bio-barcode for SERS active detection. It includes cycle amplification activated by target and polymerization shear, cycle amplification based on triple helix molecular probes and rolling-circle replication, target-catalytic activated hairpin assembly reaction cascade amplification. These measures were used to detect lysozyme, microRNA. Due to high sensitivity and high selectivity,these methods could be easily used for monitoring tumor markers.The main contents and research results of this paper include the following aspects:1. A unique four-way helical junction molecule(FHJM) probe was ingeniously designed with simple method, this FHJM was fabricated including the target recognition region, the triggering region and the template region.In this work, a novel FHJM was tactfully manufactured, and introduced into DNA signal amplification as a unique SERS probe for the first time.Furthermore, a FHJM-SERS method was successfully established to detect lysozyme. The new FHJM-SERS strategy exhibited high sensitivity with the detection limit of 0.5 fM for lysozyme, and fine performance in real human serum assay. If various recognition units may be merged into the FHJM structure, the new platforms can be applied to analyze various targets with general applicability. The proposed method provides a high sensitive sensing platform, and exploits an exciting novel horizon for the detection technique.2. We firstly develop a new and highly sensitive method for miRNA detection and intracellular imaging based on the novel nucleic acids molecular aggregates(NAMAs) self-assembled on graphene oxide nanoplates(GONPs) and a functionalized THP containing the aptamer region for target recognition and the trigger DNA region for RCA was introduced into single cells to activate RCA reaction for the first time.Interestingly, NAMAs labeled with 6-carboxyfluorescein(FAM) was a complex of being hybridized by RCA products and FAM-DNA and it was a complex of dsDNA and ssDNA. NAMAs could partly self-assemble on GONPs, in the meantime, NAMAs-FAM could extend from GONPs, which led to the quenched FAM appearing renewedly. Significantly, NAMAs were successfully applied to low-abundance miRNA detection and visualizing in single cells.In contrast with conventional RCA, the self-assembled NAMAs could generate the agminated and prominent fluorescence-bright spots in single cancer cells, which may significantly distinguish between cancer cells and normal cells. Such original intracellular imaging technique for miRNAs based on NAMAs may promote the progress of early cancer detection, which will spur the development to use a new and effective technology for visual cell research.3. Orderly nucleic acid aggregates(ONAAs) self-assembled on mesoporous silica Nan particles(Mons) with positively charged aminopropyl groups(PC) were firstly developed. Interestingly, a novel electrostatic DNA self-assembly could realize hybridization chain reaction(HCR) on the surface of PCMSNs in single cells. Significantly, a non-destructive amplification strategy based on ONAAs-PCMSNs was successfully developed for miRNA detection and in situ imaging by the prominent and geminated fluorescence-bright spots in living cells. A novel ONAAs-PCMSNs method was employed for analyzing target miRNA in single cells. A key design was a new nucleic acid nanoassembly that could realize target-initiated HCR-PCMSN for signal amplification and molecules gathering by electrostatic self-assembly. First, two hairpin-structured DNA probes N1 and N2 were designed, N2 labeled with Rox and BHQ. The loop regions of N1 and N2 could be flexible enough to synergistically interact with the positively charged surface, enabling N1 and N2 probes to be steadily assembled on the aminopropyl-coated MSNs. Second, after N1 and N2 probes assembled on PCMSNs were incubated in living cells, a target miRNA initialized the hybridization with N1 and produced a single stranded tail in N1, which might dissociate or increase the mobility of N1 on the PCMSNs, promoting its hybridization with N2 and recovering a single-strand tail in N2 with the same sequence of target. Third, in this manner, a chain reaction was triggered for alternating hybridization between N1 and N2, producing a chain-like assembly of N1 and N2.16 The HCR product(products) had an orderly conformation, self-assembled on the surface of PCMSNs through electrostatic attraction, as be(the)ONAAs, which could result in a concomitant fluorescence signal change based on the fluorescence resonance energy transfer. Compared with the intensity of the fluorescence signal generated by the above system which graphene oxide nanoplates as carrier, the amplification efficiency improved significantly.