Colorimetric Detection of Tetracyclines in Fresh Milk Based on Aptamer-Hybridization Chain Reaction

A colorimetric method for the detection of multi-residues of tetracyclines (TCs) was proposed by using broad-spectrum aptamer against TCs as the recognition element, and hybridization chain reaction (HCR) as signal amplification strategy. The detection conditions were optimized and methodological studies were carried out. The 40 nmol·L^-1 biotinylated detection probe (bio-DP) solution was added to enzyme plate coated with avidin, and the mixture was incubated at room temperature. The enzyme plate was blocked with bovine serum albumin (BSA) solution for 1 h. The flesh milk sample diluent was added, and the mixture was incubated at room temperature for 20 min. When the TCs were present in the sample, TCs were captured by the aptamer sequence of bio-DP, and the hairpin structure would be opened. Then 200 nmol·L^-1 biotinylated hairpin DNA1 (bio-H1) solution and 200 nmol·L^-1 biotinylated hairpin DNA2 (bio-H2) solution were added, and the HCR was performed at room temperature for 40 min to form double stranded DNA (dsDNA) nanowires with multiple repeating units. Horseradish peroxidase (HRP) labeled streptavidin (SA-HRP) was added, and the mixture was incubated to label dsDNA at room temperature. The chromogenic agent containing 3,3', 5,5'-tetramethylbenzidine (TMB) was added, and catalyzed to format blue reactive organisms by HRP. The coloration reaction was terminated after 5-8 min, and the absorbance of the above system was measured at 450 nm by an enzyme-labelling measuring instrument. As found by the results, the bio-DP had high specificity for tetracycline, oxytetracycline, aureomycin and doxycycline, and had no cross reactivity with antibiotics such as kanamycin, gentamicin, ampicillin, tylosin, sulfadiazine and enrofloxacin. Linear relationships between values of the mass concentration sum and the absorbance sum of tetracycline, oxytetracycline, aureomycin, and doxycycline were kept in the range of 0.8-100 μg·L^-1, with detection limits (3s/k) of 0.18 μg·L^-1. Test for recovery was made by the standard addition method on actual fresh milk samples, giving recoveries in the range of 86.4%-106%, and RSDs (n=3) of the determined values ranged from 2.5% to 7.1%. The proposed method was applied to the analysis of fresh milk samples, and there was no significant difference (P>0.05) between the detection results of the proposed method and the national standard method of GB 31658.6-2021, and the detected amount of TCs detected did not exceed the standard (GB 31650-2019). Compared with other methods reported in the literatures, the above method had advantages of simplicity, speed, low detection limit, and high accuracy, and was suitable for the rapid detection of multi-residues of TCs in food.