| Combined with DNA cycling amplification and chain growth amplification technology, this paper explored a novel and sensitive detecting methods for the determination of DNA and biological molecule based on colorimetric, chemiluminescence, Surface enhanced Raman spectroscopy (SERS). These methods we developed provides basic research information to the diagnosis of DNA and biological molecule. In this paper, three types of methods are summarized as follows:1. A novel and sensitive DNA detecting method based on DNA cycling amplification and hybridization chain reaction technology has been developed. First and foremost, combined with DNA cycling amplification and hybridization chain reaction technology, this DNA biosensor had a fast detecting rate and high detection sensitivity which can reach1.0×10-16M. Besides, the background signal can be reduced to a great extent with the introduction of magnetic nanoparticles. Finally, the horseradish peroxidase labeled streptavidin (HRP) was added, and bound with the DNA labeled by biotin which would form OPDA-H2O2-HRP system that can be detected by using a UV spectrophotometer. As a consequence, the measured content of DNA are proportional to the extent of the OPDA-H2O2-HRP absorbance within a certain range, it can be used to detect DNA content.2. On the basis of the DNA amplification technology, a highly sensitive and selective chemiluminescence detecting method for cocaine using proximity-dependent circle amplification (PDCA) as a new strategy has been developed. Here we described new probes which possess not only two recognition sequences of cocaine aptamer, but also a short complementary sequence which create a proximity probe pair with the additional hairpin probe. When the two aptamer (recognition) probes bound with the cocaine molecule simultaneously, the formation of the tripartite complex caused the opening of the hairpin probe. With the hybridization between the cocaine aptamer and the hairpin probe, the recognition site of nicking endonuclease was formed. When the nicking endonuclease and polymerase were added, the proximity-dependent circle amplification reaction was activated, while the displaced probe and cocaine became available for the circle amplification reaction. In order to improve the sensitivity of this sensor,a dual amplification system using nicking endonuclease and polymerase was designed. Overall, with two successive amplification steps and one magnetic separation procedure, this flexible aptameric sensor system exhibited a high sensitivity with the detection limit as low as1.0×10-9M which provides a great promise for the sensitive cocaine determination in law enforcement and clinical applications.3. Based on the above chemiluminescence aptameric sensor for cocaine, we explored a new method combined with the Surface enhanced Raman spectroscopy (SERS). Not only the double circle amplification technology was introduced, we also explored a new raman dye-conjugated bio-barcode. As the concentration of cocaine and the SERS signal has a certain relationship within a certain range, the detection limit of cocaine was5.0×10-10M at the best experimental conditions, and the result was also perfect in the recovery detection of human serum which puts a great promise for the application in biological and medical field. |
