Detection Of Saxitoxin By SERS Aptamer Sensor Based On Enzyme Cycle Amplification Technology

Shellfish toxins in food are produced by some toxic algae in the ocean and accumulate in marine shellfish,clams,and snails through the food chain,which can lead to food poisoning when people ingest toxic seafood.Food poisoning caused by the consumption of toxic seafood is a frequent occurrence in China and abroad and has serious health consequences.There are many different types of shellfish toxins,among which the most widespread and toxic is the Saxitoxin（STX）,which is a paralytic shellfish toxin.In this paper,two Surface-enhanced Raman spectroscopy（SERS）aptamer sensors with signal amplification have been developed for the sensitive and rapid detection of shellfish toxins.The main studies in this paper are as follows:In chapter 2,the core-shell structure was used as the carrier of Raman signal molecules and combined with s DNA to form a Raman signal probe.The hairpin aptamer（Hp DNA）of STX is loaded on the magnetic bead.When STX was presented,Hp DNA will be opened.Then,under the action of primer and DNase,rolling ring amplification would occur to produce long ss DNA containing repetitive sequence.Then,Raman signal probe would complement and pair with it to enhance the intensity of Raman signal and realize the sensitive detection of STX.The linear range for the detection of STX is 2.0×10^-10mol·L^-1～5.0×10^-4mol L^-1,wide linear range,ΔI=10704.8 lg C+7153.21（C is the concentration of STX,ΔI=I–I₀）,linear correlation coefficient R²=0.998,detection limit 1.2×10^-11mol·L^-1。In order to further realize the sensitive detection of STX,the third chapter introduced hybrid chain reaction（HCR）and chain displacement amplification（SDA）two signal amplification method,gap endonuclease（Nb.Bbv CI）and Klenow fragment polymerase（KFP）,the presence of the system target and target aptamer complementary chain double cycle amplification,the cycle 2 used the chain displacement amplification method.The presence of magnetic nanoparticles Fe₃O₄@Au makes the reaction easier to proceed,and can enhances the Raman signal.Finally,four Hp DNA were introduced,one of which modified the Raman signaling molecule Rox,which formed the branched HCR,which greatly enhanced the Raman signal intensity and improved the sensitivity of the sensor.In order to further realize the ultra-sensitive detection of STX,two signal amplification methods,hybrid chain reaction（HCR）and strand displacement amplification（SDA）,were introduced in chapter 3.The existence of nick endonuclease（Nb.Bbv CI）and Klenow Fragment polymerase（KFP）makes the system undergo double-cycle amplification of target and target aptamer complementary chain.The presence of gold magnetic nanoparticles Fe₃O₄@Au makes the reaction easier to proceed,and can enhances the Raman signal.Finally,four Hp DNA were introduced,one of which modified the Raman signal molecule Rox.By opening the Hp DNA in turn,HCR was triggered,which greatly enhanced the Raman signal strength.At 5.0×10^-12mol·L^-1～1.0×10^-9mol·L^-1,SERS intensity increases linearly with the increase of STX concentration.The linear equation is:ΔI=8710.6 lg C～1272.1,R²=0.996,LOD is 1.15×10^-12mol·L^-1.This method has realized the super-sensitive detection of STX with good selectivity,and has broad application prospects in the detection of shellfish toxins.