Establishment Of A Platform For Quantitative Detection Of Ciprofloxacin In Fish By Smartphone Based Quantum Dot Microsphere Test Strip

With the continuous development of economy and the improvement of people’s living standards,the variety and quantity of grains,meat,fish,milk and related processed non-staple food are also constantly abundant.Veterinary drugs play an important role in ensuring the supply of animal derived food.As a new,efficient and broad-spectrum synthetic antibacterial drug,ciprofloxacin has many functions,such as accelerating the growth and development of animals,improving High animal derived products,reduced animal incidence rate and mortality rate have been widely applied in aquaculture.However,the excessive abuse of these drugs often leads to the excess of their residues in aquatic products,causing potential harm to human health.China,North America,the European Union and other countries have stipulated ciprofloxacin as a banned drug for aquatic products,which shall not be detected.It can be seen that the residue of ciprofloxacin in aquatic products has become a must.At present,the common methods for ciprofloxacin detection include high performance liquid chromatography(HPLC),liquid chromatography-mass spectrometry(LC-MS),enzyme-linked immunosorbent assay(ELISA),etc.Both HPLC and LC-MS need expensive instruments.Sample pre-processing is very tedious and requires professionals who are proficient in complex operations.Detection cost is expensive and time-consuming.Although the operation of ELISA is a little simpler than that based on chromatographic instrument,it is still only suitable for laboratory operation,which limits its application in field rapid detection.Colloidal gold strip has the advantages of rapid detection,low cost and convenient operation,but there are still some defects,such as low detection sensitivity,obvious matrix effect and large background interference.In this study,a new method of detecting ciprofloxacin by quantum dot microsphere test strip was established by combining with a hand-held fluorescence detector based on smart phone.The hardware system design of the hand-held fluorescence detector can take photos of the test strip,and automatically process and analyze the acquired images.The results show that the accuracy of the test results is high and the processing speed is fast.The mobile application designed by its software system can be used in any Android smartphone,and its algorithm can accurately and quantitatively analyze the experimental results,effectively avoiding the error of direct observation by human eyes and the disadvantage of non quantitative detection.After the optimization of the conditions,it was determined that the optimal pH of the QD microspheres coupling antibody was 6.0 and the optimal antibody amount was 6 μg;the sample pad treatment solution was 0.01M pH 8.0 PBS containing 1.0%BSA,0.5%Tween-20 and 0.1%NaN3;the spraying concentration of the T-line antigen was 0.4 mg/mL;the spraying concentration of the C-line goat anti-mouse antibody was 1.0 mg/mL;the volume of the QD microspheres fluoroquinolone monoclonal antibody probe was 3 μL;incubation time was 3 min;reaction chromatography time was 10 min.The establishment of quantitative standard curve,the determination of detection limit,and the evaluation of accuracy,specificity,repeatability and stability of the established method were carried out.The results showed that the range of quantitative detection of ciprofloxacin was 0.1 ng/mL～100 ng/mL,the linear regression equation was y=-0.29017logx+0.61396,and the minimum detection limit was 0.05 ng/mL;the concentration measured by the test strip is highly consistent with the HPLC method,with good accuracy;chloramphenicol,tetracycline,sulfamethazine and penicillin have basically no cross reaction with the test strip,and the cross reaction rates with enrofloxacin and dapfloxacin are respectively 75.2%and 64.6%,but the cross reaction of fluoroquinolones with other homologous antibiotics is very low,with good specificity;the coefficient of variation is less than 15%,which shows that the batch difference of the test strip is small,with good repeatability;the concentration measured around the test strip preserved at 25℃and 37℃ is basically the same as the standard concentration added,with good temperature stability;for The results show that the recovery rate is 76.88%～102.34%and the coefficient of variation is 6.09%～10.48%,which shows that the quantum dot microsphere strip has good accuracy and precision in the detection of fish samples.At the same time;it has high accuracy,up to 98%;good specificity,up to 100%;and good sensitivity,up to 97%.It shows that its ability to detect ciprofloxacin and its detection standard curve are not affected by matrix effect,which is also suitable for the detection of actual fish samples.