Study On The Optical Aptasensors For The Highly Sensitive Detection Of Fluoroquinolones Residues

Thousands of antibiotics have been developed,which are widely used in medicine,animal husbandry and aquaculture,agricultural control and other fields.Due to the abuse of antibiotics,human health and ecological environment have been greatly affected.Ciprofloxacin（CIP）and ofloxacin（OFL）are typical fluoroquinolone antibiotics（FQs）.Because of their strong antibacterial properties,they have been widely used in medicine to prevent diseases caused by livestock and poultry diseases and various bacterial pathogens.FQs are resistant to degradation and highly absorbable in vivo.Extensive use of FQs will lead to incomplete metabolism in the body.FQs can also accumulate in the environment,especially in soil.More seriously,the overuse of antibiotics is accelerating the emergence of antimicrobial resistance,which makes microbial-induced infections difficult to treat and sometimes incurable,endangering human health.Although many optical aptasensors have been developed to detect FQs,they still have shortcomings.Therefore,it is of great significance to develop an analytical method with high sensitivity,strong specificity,simple operation and rapid detection in real time for the detection of antibiotic residues in the environment or food.In this study,FQs was used as the target,and the sensitivity and specificity of the optical aptasensors were studied.Three types of optical aptasensors were developed to detect FQs by fluorescence resonance energy transfer（FRET）,photoinduced electron transfer（PET）,and amplification of gold nanoparticles（AuNPs）.The main research contents and conclusions are as follows:（1）A fluorescent aptasensor based on FRET was constructed to detect OFL with high sensitivity.In this study,the Cy3 modified OFL aptamer（Cy3-Apt）and the complementary strand modified by the BHQ2（BHQ2-cDNA）form Cy3-Apt/BHQ2-cDNA complexes,and FRET occurs between Cy3 and BHQ2,contributing to the fluorescence quenching of Cy3.In the presence of the target OFL,OFL preferentially binds specifically to Cy3-Apt to separate BHQ2-cDNA from Cy3-Apt/BHQ2-cDNA and recovery fluorescence of Cy3.In addition,OFL can induced the fluorecence enhancement of Cy3.Under the optimal experimental conditions,the detection limits of the method for standard solution and fresh milk were 0.20 nmol/L(7.2×10^-2μg/L)and 20.57 nmol/L（7.4μg/L）,the limits of quantitation were 254.11 nmol/L and 90 nmol/Land the linear ranges were 0.01-0.50μmol/L and 0.005-0.10μmol/L,respectively.The recoveries of the FRET aptasensor were obtained between 91.39%and118.73%in real water and fresh milk samples.（2）Based on PET and split aptamer（SPA）a quencher-free fluorescence aptasensor was constructed to detect CIP with high sensitivity.The original intact CIP aptamer was broken into two fragments SPA 1 and SPA 2,which could specifically recognize the target CIP.Among them,one of SPA segment SPA 1 was modified with fluorophore FAM,namely FAM-SPA 1.In this study,SPA and FAM were employed as the recognition and singnaling element,respectively,and the target substance CIP played role as the quencher.In the presence of CIP,SPA specifically recognizes and combines with it to form a sandwich structure of"aptamer/target/aptamer（FAM-SPA1/CIP/SPA2）".Due to PET phenomenon occurring between CIP and FAM,the fluorescence quenching phenomenon of FAM is promoted.Therefore,a novel SPA biosensor is constructed for CIP detection.Experimental results show that the method has strong selectivity and high sensitivity to CIP,the detection limit was as low as 5.0 nmol/L（1.6μg/L）,the limit of quantitation is 32.5nmol/L,and the linear range was 0.0-200.0 nmol/L;the spiked recoveries in actual water samples were layed between 88.21%and 119.18%.（3）Based on the amplification growth of AuNPs by gold enhancememnt and its LSPR peak shift,a simple,fast and label-freecolorimetric aptasensor was developed for CIP analysis.In the absence of CIP,the AuNPs are highly dispersed by the aptamer due to the electrostatic interaction between the aptamer and AuNPs.After gold enhancement,the dispersed AuNPs are grown to be larger particles,and the color of solution could change from light pink to red-wine color.In the presence of CIP,aptamer preferentially binded specifically to CIP and detached from AuNPs surface.It is because AuNPs lost the protection of aptamer and occouredaggregated CIP induced aggragation.However,the color was still almost indistinguishable from the AuNPs solution without CIP.In the addition of gold enhancing solution,the aggregated AuNPs were used as seeds to grow into the larger size.It was observed that the color of solution turned to dark blue and the LSPR peak of AuNPs was a red-shift than the control.The biosensor can not only recognize the difference of CIP concentration with naked eyes,but also improved its detection sensitivity through LSPR peak-shift phenomenon.The experimental results show that the detection limit of CIP was29.64 nmol/L（9.8μg/L）,the limit of quantitation was 799.82 nmol/L,and the linear range was0.01-1.0μmol/L.The spiked recoveries in actual water samples were 86.67%-109.99%.In summary,three optical aptasensors were successfully constructed in this study and applied to the detection of residual FQs in actual water and milk.The FRET-based fluorescence aptasensor was constructedto detect OFL based on fluorescence recovery and enhancement of Cy3.Furthermore,the quencher-free fluorescence apsensor was developed for the detection of CIP based SPA and PET phenomena between CIP and FAM.In addition,the colorimetric LSPR aptasensors was constructed to detect CIP based onsignal amplification by the sensitizer.The optical aptasensors could be applied for fast,highly sensitive and selective detection of the small molecules in water,food and medical environment.