| Protein and nucleic acid have been regarded as the key biomarkers in cancer diagnosis and treatment. But, in the early stages of the disease, the content of some small molecules are often very low to be diagnosed. Thus, highly sensitive detection of protein and nucleic acid are of great importance in the early diagnosis of cancers and major diseases. In this study, with the technology of bio-barcode and surface enhanced Raman scattering, we have realized the rapid and sensitive method to detect PDGF-BB, miRNA-203 and ATP by primer self-generation polymerase strand displacement amplification, symmetric simultaneous signal amplification and asymmetric simultaneous signal amplification assay method. This paper mainly includes three aspects as following:1. A simple and novel one-two-three signal amplification surface-enhanced Raman scattering(SERS) method for the detection of protein is fabricated by using label-free aptamer and dual-primer self-generation. According to the unique structure of the aptamer, a hairpin DNA was fabricated after the hybridization of target molecule and aptamer, then the cooperator DNA automatically formed another hairpin DNA strand. This two kinds of hairpin assisted exponential polymerase strand displacement amplification reaction based on hairpin DNA-self generation and released a mount of triggers to initiate the next amplification, finally, numbers of bio-barcode were immobilized on the gold substrate to enhance the signal again, the limit of detection was 0.42 pM. This method is simply operation with good selectivity and sensitivity which opens an exciting new avenue for protein detection.2. As the important biological molecules for the cancer diagnosis, we have synthesized a bifunctional biological molecule Raman probes to realize a new symmetric signal amplification strategy for the simultaneous detection for miRNA-203 and ATP by hybridization chain reaction amplification and bio-barcode technology. This method used magnetic bead(MB) as a kind of carrier with the immobilization of two target aptamer and cooperator, when the two target moleculesoccured specific identification, two kinds of triggers were released into the system to initiate the HCR and immobilize the bio-barcodes onto the MBs. The detection of ATP is 5.6 × 10-9 M and the detection of miRNA-203 is 1.2 × 10-14 M. This two kinds of molecules detection type assay has good selectivity and sensitivity, provides a new platform for detecting multi-component.3. Simultaneous detection of cancer biomarkers holds great promise for the early diagnosis of different cancers. However, in the presence of high concentration biomarkers, the signals of lower-expression biomarkers are overlapped. Existing techniques are not suitable for simultaneously detecting multiple biomarkers at concentrations with significantly different orders of magnitude. Here, we propose an asymmetric signal amplification method for simultaneously detecting multiple biomarkers with significantly different levels. Using the bifunctional probe, a linear amplification mode responds to high-concentration markers and quadratic amplification mode responds to low-concentration markers. With the Combined bio-barcode probe and hybridization chain reaction(HCR) amplification method, the detection limits of miRNA-203 and ATP via surface-enhanced Raman scattering(SERS) detection are 0.15 fM and 20 nM on, respectively, with a breakthrough of detection concentration difference over 11 orders magnitude. Furthermore, successful determination of miRNA-203 and ATP in cancer cells supports the practicability of the assay. This methodology promises to open an exciting new avenue for the detection of various types of biomolecules. |
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