Development And Application Of Paper Chip-Based Quantitative Detection System For Tetracycline And Chloramphenicol

Antibiotics have been widely applied in disease prevention and treatment for a long time;however,the abuse of antibiotics is a problem that cannot be ignored.Especially antibiotic abuse in animal feeding inevitably leads to antibiotic residues in animal-origin foodstuffs such as honey,eggs,fish and milk.For instance,tetracycline(TET)residue causes abdominal pain,diarrhea,flatulence and vomiting;and chloramphenicol(CAP)residue causes fatal bone marrow depression and aplastic anemia which are harmful to human health.Therefore,TET maximum residue limit in milk and fish is 100 μg/kg,and CAP is prohibited in animal-origin foodstuffs set by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China.In order to detect antibiotics such as TET and CAP,gold standard methods including liquid chromatography-mass spectrometric and high-performance liquid chromatography are often used.Though they can detect antibiotics in extremely high sensitivity and accuracy,the required expensive setups,specialized personnel and extensive sample preparation illustrate that they can only be antibiotic detecting solutions in laboratory.Besides chromatography,electrochemical sensors were used for antibiotic detections with high sensitivity and fast speed;however,they still suffer from poor detecting stability.Different from collecting electrical signals,optical signals such as chemiluminescence and fluorescence signals can be used for antibiotic detections as well.Moreover,surface enhanced Raman scattering and surface plasmon resonance based methods were also adopted to further improve the detecting sensitivity.However,these optical sensors require expensive hardware such as UV-Vis spectrophotometer,Raman spectrometer or microscope and Biocore system,thus still not the proper solutions for on-site detections.In order to obtain sensitive and accurate TET and CAP detections especially in rapid on-site applications,we design a wax-printing paper chip based dual antibiotic detection relying on colorimetric indirect competitive ELISA.Paper chip can be fabricated only by wax printing and surface modification,making the cost of single detection rather cheap as～1 RMB;and it requires simple operations as sample adding,washing and coloration all finished in 10 min.Moreover,supporting smartphone application with functions of image recording,correction and analysis is designed for quantitative detections.Antibiotic antigen modified on the hydrophilic microwells can bond to introduced monoclonal antibody competing with antibiotic target:when there is no antibiotic target,monoclonal antibody bonds to antigen and is fixed on the paper chip surface;while when antibiotic target exists,monoclonal antibody bonds to antibiotic target and then is washed off.Target-free microwell tends to be dark blue,while target-existed microwell becomes white due to the reaction between colorimetric substrate and enzyme labeled secondary antibody bonded to monoclonal antibody fixed on the paper chip surface.The test to control intensity ratio in red channel(It/Ic)R is measured to quantify antibiotic concentration.Proved by using series standard solutions,paper chip reached extremely low limits of detection as 0.5 ng/mL and 0.05 ng/mL as well as wide effective linear detecting ranges as 1-103 ng/mL and 0.1-100 ng/mL in TET and CAP detections,respectively.Other antibiotics were also detected on our paper chip.It was found that the detection system had good anti-interference ability and could be used for the specific detection of tetracycline and chloramphenicol.The results showed that the coefficient of variation was 3.2%-3.8%for tetracycline and 2.2%-2.8%for chloramphenicol.The results of stability study showed that the paper chip can be stored at 4℃ for 90 days.In addition,by comparing the detection limit of this detection system with commercial ELISA kit and colloidal gold strip,it showed that the detection system of this experiment has excellent detection performance.Tetracycline and chloramphenicol in milk and fish samples were also detected by paper chip and HPLC respectively.According to the actual sample detection results,the detection limit of paper chip for tetracycline detection can reach 0.5 ng/mL,and chloramphenicol detection limit is 0.05 ng/mL,which is similar to the detection limit of standard samples,and has potential practical application value.