Fluorescence Methods For Chloramphenicol And Tetracycline Detection Based On Aptasensorsand Hemin

Due to the bacteriostatic and bactericidal effects of antibiotics,they are often illegally used in bee breeding and may caused long-term and low-dose intake of antibiotics for consumers,which is harmful to health.Therefore,based on the specific recognition of aptamer and target,fluorescence detection methods for chloramphenicol?CAP?and tetracycline?TC?were established respectively,and applied to the detection of CAP and TC in honey.The main research results were described as follows:Section 1,a method for the aptamer-based determination of CAP was developed by exploiting the peroxidase mimicking activity of hemin.The method includes two hemin-modified DNA termed P1 and P2.P1,which was modified at its 5?end with one hemin monomer,contains the CAP-binding sequence.The hybridization between P1 and P2 brings the two hemin monomers in close proximity,resulting in the formation of a hemin dimer with low peroxidase mimicking activity.The duplex structure was dehybridized in the presence of CAP.The formed hemin monomer featured a strong peroxidase mimicking activity and catalyzed the conversion of non-fluorescent tyramine into fluorescent tyramine by hydrogen peroxide.Fluorescence?with excitation/emission maxima at 320 and 410 nm,respectively?increased linearly in the 0.1 ng mL^-1 to 10 ng mL^-1 CAP concentration range.The detection limit based on the 3?/k criterion reached 0.07 ng mL^-1.The proposed assay was successfully employed for CAP detection in?spiked?honey samples with recoveries of 94.3%¹17.2%.Given its high sensitivity and good stability,this method shows potential in providing a platform for antibiotic detection.Section 2,Construction of tetrahedral DNA nanostructure magnetic microsphere?MB?.Tetrahedral DNA nanostructures have high assembly efficiency,mechanical rigidity and easy modification of chemical groups.The density of aptamers can be adjusted on the surface of MB to improve the detection sensitivity.Compared with directly connecting the aptamer to the MB surface,the density and spatial orientation of the aptamer can be adjusted to avoid entanglement between the aptamers.Detection of target DNA in serum,the aptamer on tetrahedral DNA can quickly bind to target DNA,the binding time is shortened from 120 min to 10 min of traditional MB surface aptamer,and the detection range is from 0.5 nmol L^-1 to 100 nmol L^-1 widens to 0.01nmol L^-130 nmol L^-1,and is not affected by complex media.It shows that the tetrahedral DNA modified on the MB surface as an aptamer detection probe connected to the scaffold has the characteristics of fast and sensitive,and strong specificity and anti-interference ability.Section 3,Aptamer-modified magnetic beads?MBs?have been widely used in small molecules detection.However,unfavorable aptamer density leads to electrostatic repulsion between negatively charged aptamer and strong steric hindrance,leading to the decreases of the binding efficiency.Herein,we design a facile and sensitive aptamer for TC detection using DNA tetrahedron-functionalized MBs and hybridization chain reaction?HCR?method.The use of DNA tetrahedron ensures the aptamer in well controlled densities with improved target accessibility.Upon the addition of target TC,aptamer combined with TC and released from the MBs,and the complementary sequence of aptamer triggered the hybridization chain reaction?HCR?for signal amplification.By monitoring the increases of fluorescence intensity,TC concentrations were detected.Under the optimized conditions,the fluorescence intensity was linearly dependent on the TC concentrations in the range of 0⁵0 ng mL^-1.The detection limit based on 3?/k criterion was 0.035 ng mL^-1.Furthermore,the presented method was successfully employed for TC detection in honey samples with recovery rates of 94.85¹08.1%.