The Research For Nucleic Acid Biosensor Based On Gold Nanoparticles-enzyme Signal Amplilcation System

Colorimetric sensor is a new type of visual detection system based on the different colors of gold nanoparticles accumulation and dispersion . The method can be combined with a variety of experimental techniques and it can detect a variety of substances. The gold nanoparticles have many advantages: fast and intuitive detection, low cost, small size easy for portability, etc. However, there are some shortages in these methods. For example, using nucleic acids modified gold nanoparticles to detect DNA directly is not very sensitive. Moreover, the current widespread using the bio-enzyyme signal amplification technology to improve the sensitivity of the colorimetric system always exist a lot of limitations and it can't meet all of the nucleic acid detection systems.Therefore, designing a sensitive, simple detection system for any nucleic acid sequenceis is one of the important issues.In this paper,we also use the enzyme signal amplifition assay combined with the modified gold nanoparticles to detect target DNA. Two detection methods for any single-stranded nucleic acid have been designed.The first method was developed by the strand displacement amplification technique and the colorimetric method of gold nanoparticles: when the target nucleic acid sequence exits,as the primer of the cross-linker to cause the strand displacement amplification reaction . And the product of the reaction can be complementary with the Linker sequences, preventing the accumulation of modified gold nanoparticles ; In contrast, when no target DNA exits, the Linker will lead to the accumulation of modified gold nanoparticles and a concomitant color change. This method is intuitive, simple, low cost, without any background, etc., and the limit of detection is 1 pM. The another method is used the restriction enzyme digestion cycle to amplify the signal: the system needs HincII enzyme, a hemiphosphorothioate-modified hairpin probe, cross-linker and nucleic acid-modified gold nanoparticles. Upon recognition and hybridization with the target DNA, the modified stem of the hairpin probe is opened, after which the opened probe anneals with the linker and cause the restriction enzyme digestion cycle amplification reaction. The Linker nicked constantly will not trigger two sets of oligonucleotide-modified gold nanoparticles aggregation.On the other hand,in the absence of a target,the very stable hairpin probe can not lead to the enzyme digestion cycle reaction and modified gold nanoparticles aggregation will occur. The design concept for the detection of target DNA is very easy,sensitive and selective.The colorimetric detection limit of 5 pM is 3 orders of magnitude more sensitive than that of a traditional three-component sandwich assay format.Because the Tm value for the normal and mutant target DNA hybriding with hairpin is different. So using this principle, the experimental method is also applicable for DNA mutation identification.Using the simulation test, this paper aims to show a more simple,intuitive and sensitive detection method for target DNA .This approach is expected to lay the foundation for single-stranded nucleic acids based on the colorimetric detection of modified gold nanoparticles.In addition, these new methods can be easily combined with the current conventional analytical techniques, and the test field can be extended to proteins, nucleic acid aptamers of small molecules and metal ions, etc.