Carbonic Anhydrase Activity Detection Based On Anti-Fouling DNA Elecrochemical Sensor

The water body are severely polluted leading to damage to the ecosystem by rapid development of industrialisation and urbanisation.The amount of planktonic algae in water due to eutrophication of water body has increased dramatically.The frequently occurring abnormal growth of algae poses a serious threat to both the ecological balance and human health.Real-time monitoring of algae growth plays an important role in early warning of water blooms and water quality monitoring.Carbonic anhydrase is an important biological catalyst for algae growth,and its activity can be used as an indicator to predict the growth trend of algae.In this thesis,based on the catalytic hydration reaction to carbonic anhydrase,an anti-fouling electrochemical biosensor was constructed for sensitive detection of carbonic anhydrase activity by quantitative conversion of i-motif induced by H⁺.（1）Construction of DNA anti-fouling electrochemical sensing interfaces and study of their anti-fouling properties:In complex samples,the detection of carbonic anhydrase activity is interfered by its coexisting substances,and the electrode interface is easily contaminated,which affects the detection results.Therefore,the anti-fouling of electrode surface is very important for electrochemical detection of enzyme activity.DNA is a hydrophilic polymer with excellent biocompatibility.In this study,single-stranded DNA（ss DNA）was used as an anti-fouling coating to investigate the anti-fouling performance of anti-fouling biosensing interfaces prepared by different base types and different amounts of adenine.The adsorption capacity of different bases on the gold surface was explored by DFT calculation.The effect of DNA chain length on anti-fouling ability was investigated by DNA surface coverage test.An anti-fouling electrochemical sensing interface was constructed to study the anti-fouling mechanism and anti-fouling ability of ss DNA.Based on this,an electrochemical sensor was constructed for the quantitative detection of protein and DNA in serum using ss DNA as an anti-fouling coating.（2）Carbonic anhydrase catalyzed H⁺-induced quantitative conversion of i-motif:In order to realize the H⁺-induced i-motif quantitative conversion catalyzed by carbonic anhydrase,the conversion strategy of enzyme-catalyzed reaction and single-stranded DNA was constructed.The Double-stranded DNA（dsDNA）is designed as a capture probe,where the C-rich base sequence was the H⁺capture strand and labelled with a fluorescent bursting group,and its complementary sequence labels the fluorescent molecule as the output end of the fluorescent signal.The fluorescence signal was weak or absent when the probe was a double-stranded structure.Through the carbonic anhydrase catalytic reaction,the structure of the DNA capture probe is changed,the i-motif structure is generated,and the ss DNA is released to obtain a strong fluorescence signal.The capture of H⁺catalyzed by carbonic anhydrase by DNA single strand reduces the interference to the detection of coexisting interferents to a certain extent and improves the accuracy of detection.（3）H⁺-induced i-motif quantitative conversion to construct electrochemical biosensors for sensitive detection of carbonic anhydrase activity:In order to further improve the sensitivity of carbonic anhydrase activity detection,a highly sensitive electrochemical biosensor was constructed by designing H⁺DNA nano-capture probe combinied with DNA Mg²⁺cleavage-assisted DNA cyclic amplification strategy.When carbonic anhydrase catalyzes the production of H⁺,H⁺induces conformational changes in the capture probe,releasing part of the hybridized DNA double strands,which are further added to the electrode surface.The dsDNA is hybridized with the methylene blue-labeled hairpin DNA immobilized on the electeode surface to form a Mg²⁺shear site.In the presence of Mg²⁺,the methylene blue-labeled hairpin DNA is cleaved,and the methylene blue is released from the electrode surface to obtain a reduced electrochemical signal.At the same time,dsDNA is released,which will further react with other hairpin DNA to achieve DNA circulation.Among them,the number of methylene blue-labeled DNA strands released by the hairpin dsDNA strand is proportional to the amount of H⁺generated by carbonic anhydrase.The greater the carbonic anhydrase activity,the significant decrease in the electrochemical signal.According to this mechanism,the electrochemical sensitive detection of carbonic anhydrase activity was realized.