Exo Ⅲ-mediated Multiple Cycle Amplified DNA Walker Strategy For Ciprofloxacin Detection

Fluoroquinolones（FQs）are fully chemically synthesized broad-spectrum antibiotics that are commonly used for their lower price,longer half-life,and stable performance than comparably effective antibacterial agents.In the 2019 outbreak of COVID,FQs were utilized extensively as the most used antibiotics in clinical anti-infection.Among them,ciprofloxacin（CIP）is one of the most common used in FQs.As a universal drug for both humans and animals,CIP can also treat animal diseases as well as added to livestock feed.The residues of such antibiotics in processed animal food may lead to accumulative toxicity,which is one of the reasons why it is considered as an antibiotic that requires priority monitoring.So it’s important to achieve CIP trace detection.Sensor is a detection device that monitors information about the measured object,and converts the information into an optical or other form of signal according to the certain pattern,fluorescent sensors and electrochemical sensors are two of the most common detection techniques.The principle of fluorescence sensors is based on the fact that the object to be measured can absorb visible or ultraviolet light leading to electron transition at the energy level,then release energy in the form of radiative transition back to the ground state.Electrochemical sensors,on the other hand,are technologies based on reactions that generate or consume electrons,converting the chemical energy of the analyte to be measured into electrical energy for further detection.Both sensors have the advantages of low cost,high sensitivity,simple and reliable principles in most cases,thus gaining great interest in the fields of pharmaceutical analysis,environmental monitoring and food safety evaluation.DNA Walker,a highly efficient detection tool based on the field of biosensing that has been rapidly developed in recent years,is a DNA nano-device that can be driven by a variety of dynamics,similar to the movement of natural kinesin.With the development of biological and nanotechnology,the coupling of aptamer-based sensors and DNA Walker is of great importance for the detection of antibiotic residues in humans and the environment.In this thesis,we combine fluorescent sensor,electrochemical sensor,DNA Walker,nanotechnology and drug analysis to develop a complete research work in terms of substrate material preparation,construction of fluorescent and electrochemical sensing platform and its detection of ciprofloxacin in real human serum samples.The main aspects include the following:1.Based on the high selectivity of the ciprofloxacin aptamer,catalytic hairpin cycling reaction（CHC）was accomplished to achieve specific ciprofloxaxin capture.Using polydiallyldimethylammonium chloride（PDDA）as a modifier,Au NPs prepared by sodium citrate reduction were loaded on carboxylated Fe₃O₄ magnetic spheres by electrostatic adsorption,and the synthesis of three-dimensional spherical bioprobe was completed by chemical bonding of sulfhydryl groups at the ends of the aptamer and Au.Based on the large number of sites provided by the enriched Au NPs for the aptamer,DNA walking machine（DWM）was triggered by Exo Ⅲ,the constructed aptamer device showed a good linear response in the range of 1 n M-10 n M with a detection limit of 0.30 n M.2.Based on the duplex amplification of the fluorescence sensor,a hairpin probe containing a segment of hemin aptamer sequence was modified onto the gold electrode surface through Au-S bond.The signal strand obtained from the above fluorescence reaction was introduced to the electrode surface,then the hairpin probe on the electrode surface opened the hairpin structure and bound to the signal strand to form a duplex structure.Under the assistance of Exo Ⅲ,the formed double strands were dissipated while the signal strand was released again,so that it could participate in the new double strand formation-dissipation cycle,thus triggering the electrochemical signal cycle amplified reaction（ESCA）,finally a large number of hemin aptamers were left on the electrode surface.After introduce of hemin,a deoxyribonuclease（DNAzyme）:G-quadruplex/hemin complex was formed.The strong electrical signal obtained by reduction of hydrogen peroxide（H₂O₂）leaded to the construction of an electrochemical sensor with further signal amplification.Under optimal conditions,the two-step sensor eventually showed impressive linearity for the concentration values of ciprofloxacin in the range of 25 p M-20 n M,with a detection limit 1.08 p M,while enabling sensitive and rapid detection of ciprofloxacin in real serum samples.