A Simple Yet Effective Preanalytical Strategy Enabling the Application of Aptamer-Conjugated Gold Nanoparticles for the Colorimetric Detection of Antibiotic Residues in Raw Milk

Abstract

The misuse of antibiotics in the cattle sector can lead to milk contamination, with concomitant effects on the dairy industry and human health. Biosensors can be applied in this field; however, the influence of the milk matrix on their activity has been poorly studied in light of the preanalytical process. Herein, aptamer-conjugated gold nanoparticles (nanoaptasensors) were investigated for the colorimetric detection in raw milk of four antibiotics used in cattle. The effect of milk components on the colorimetric response of the nanoaptasensors was analyzed by following the selective aggregation of the nanoparticles, using the absorption ratio A520/A720. A preanalytical strategy was developed to apply the nanoaptasensors to antibiotic-contaminated raw milk samples, which involves a clarification step with Carrez reagents followed by the removal of cations through dilution, chelation (EDTA) or precipitation (NaHCO3). The colorimetric signals were detected in spiked samples at concentrations of antibiotics as low as 0.25-fold the maximum residue limits (MRLs) for kanamycin (37.5 μg/L), oxytetracycline (25 μg/L), sulfadimethoxine (6.25 μg/L) and ampicillin (1 μg/L), according to European and Chilean legislation. Overall, we conclude that this methodology holds potential for the semiquantitative analysis of antibiotic residues in raw milk obtained directly from dairy farms.

A Novel Preanalytical Strategy Enabling Application of a Colorimetric Nanoaptasensor for On-Site Detection of AFB1 in Cattle Feed

“…Each NAS was characterized by the detection of AFB1 at 0, 0.25, 0.5, 1, and 2 times the concentration corresponding to the MRL in the European Union [ 22 ]. The OD ratio 620/520 nm is widely used to follow the aggregation of colorimetric sensors based on AuNPs [ 60 , 61 ], even though we showed in a previous work that this parameter is not necessarily the best way to perform analyte detection with NAS [ 39 ]. With this in mind, NAS aggregation was analyzed spectrophotometrically between 400 and 750 nm to select the reading parameters that maximize the signal associated with AFB1 detection.…”

“…In comparison to antibodies, aptamers have superior stability and can be produced for a wide range of low molecular weight, toxic, and nonimmunogenic compounds [ 38 ]. Thus, they offer huge advantages over protein-based antibodies due to their higher stability and their capability to recover their native conformation after denaturation by heat treatment [ 39 , 40 ]. The combination of gold nanoparticles (AuNPs) with aptamers (NAS; nano-aptasensor) is gaining considerable attention to investigate the detection of a variety of analytes in the field of food safety [ 41 ].…”

“…AuNP-based NASs enable colorimetric detection by following a red-to-purple blue shift of the absorption spectrum during the aggregation of the nanoparticles, providing a versatile, sensitive, selective, and easy-to-apply platform. We recently reported NAS systems for the colorimetric detection of antibiotic residues from raw milk samples, addressing both the preanalytical problem [ 39 ] and the application of a machine-learning algorithm to improve the sensitivity of the analytical process [ 43 ]. Aptasensors for AFB1 detection have been successfully reported in rice, peanuts, and wheat flour upon extraction using methanol/water in 80/20 and 60/40 mixtures [ 19 , 44 ].…”

Dual fluorescent aptasensor for simultanous and quantitative detection of sulfadimethoxine and oxytetracycin residues in animal-derived foods tissues based on mesoporous silica

“…These methods have the advantages of high sensitivity and specificity but can only be used to detect a single target. Therefore, Díaz-García et al created a gold-based colorimetric assay to simultaneously detect SDM and OTC residues in milk ( 20 ). However, due to the physical and chemical properties of gold nanoparticles, which are easily affected by the surrounding detection environment, this method has shortcomings such as poor reproducibility and inconsistent detection results.…”

Optical Sensors Technology and Applications

“…In this framework, the authors of Ref. [ 6 ] developed a simple but effective method for the colorimetric detection of antibiotic residues in raw milk by using aptamer-conjugated gold nanoparticles. In fact, the improper use of antibiotics in cattle breeding can cause milk contamination, which has negative effects on the dairy industry and human health.…”