Study On The Colorimetric Sensing Technology For Food Safety Based On G-quadruplex DNAzyme

Biosensing technology has been widely concerned by researchers because of its advantages such as simple operation,rapid detection,and good specificity.In recent years,food safety issues have frequently erupted,arousing widespread concern among people from all walks of life.In this thesis,we developed the colorimetric sensing technology for food safety via nucleic acid base complementary pairing,Hg²⁺-mediated formation of thymine-Hg²⁺-thymine(T-Hg²⁺-T)base and tetracycline-aptamer-mediated specific recognition,The main researches are summarized as follows:1.Based on catalyzed hairpin assembly(CHA)strategy,a colorimetric detection method for the transgenic promoter CaMV35S was successfully developed.One end of the two DNA probes is designed to complement the target nucleic acid molecule,and the sequence at the other end can trigger the CHA reaction.When the target DNA is present,the unhybridized part of the DNA probe approaches,triggers the CHA reaction and releases a G-rich sequence blocked by H2 to form a G-quadruplex DNAzyme,which catalyzes the color development of the chromogenic substrate TMB mediated by H₂O₂.It can form a stable yellow solution after termination by H₂SO₄.It was found that a linear range of 1-15 nM and a limit of detection of 0.66 nM for this method,which can specifically distinguish single-base mismatched DNA strands.In addition,for the detection of different target nucleic acids,only partial sequences of auxiliary probes need to be simply replaced according to the sequence of the target nucleic acids,which greatly improves the versatility of DNA probes.2.Based onMg²⁺-dependent DNAzyme double-cycle strategy,a Hg²⁺colorimetric detection method was successfully developed,which successfully realized the detection of Hg²⁺in milk.When Hg²⁺is present,the auxiliary probe P and the hairpin probe H0form a T-Hg²⁺-T structure under the action of Hg²⁺,releasing the DNAzyme enclosed in the H0 loop,and at the same time,the uncomplementary part of P and H0 forms a cleavage With the participation of Mg²⁺,two DNAzyme simultaneously catalyze the cleavage of the hairpin probe H1 that modifies ribonucleic acid(r A),releasing a large number of G-rich sequences to form G-quadruplex DNAzyme.The G-quadruplex DNAzyme can catalyze the color development of the chromogenic substrate TMB mediated by H₂O₂,and can form a stable yellow solution after being terminated by H₂SO₄.The T-Hg²⁺-T structure makes this detection method highly specific,and the dual-cycle strategy shows higher sensitivity.It was found that a linear range of 0.05-20nM and a limit of detection of 0.04 nM for this method.The actual sample test results show that this method has potential practical value.3.Based onMg²⁺-dependent DNAzyme and catalytic hairpin assembly(CHA)strategy,a TC colorimetric detection method was successfully developed,which successfully realized the detection of TC in milk.When TC is present,it binds to the nucleic acid aptamer and destroys the hybrid complex between the nucleic acid aptamer and DNA1.The released DNA1 combines with DNA2 in the solution to form Mg²⁺-dependent DNAzyme,which catalyzes the cleavage and modification of ribose.The H0of nucleic acid(r A)produces a large amount of short-stranded DNA,which further initiates the CHA cycle,releases a large number of G-rich sequences to forms a G-quadruplex DNAzyme.The G-quadruplex DNAzyme can catalyze the color development of the chromogenic substrate TMB mediated by H₂O₂,and can form a stable yellow solution after being terminated by H₂SO₄.This method shows the characteristics of high sensitivity and strong selectivity,and the detection limit as low as 0.89 p M.This method has been successfully used for the detection of TC in milk samples and has great practical value.