Rapid, in-plant veterinary drug screening in animal carcasses using electrochemical aptamer-based sensors

The main objective of the proposed research is to develop an electrochemical aptamer-based (E-AB) sensor that can achieve simultaneous, in-plant detection of veterinary drugs in animal carcasses to increase the accuracy, efficiency, and cost-effectiveness of veterinary drug screening, and thereby improve food safety and protect consumers. This project will 1) improve the efficiency of chemical residue detection in meat products and 2) establish novel scientific techniques and methods for the streamlined development of E-AB sensors that can broadly detect numerous agriculturally-relevant substances in foodstuffs. As a demonstration, this project will focus on the veterinary drugs associated with the most residue violations - the beta-lactam antibiotics and the anti-inflammatory drug flunixin - within kidney tissue from cattle and swine. The development of these E-AB sensors entails the accomplishment of four aims:Isolation of a DNA-based affinity element (aptamer) specific for flunixin.Isolation of an aptamer that binds cross-reactively to beta-lactam antibiotics.Fabrication of E-AB sensors using the isolated aptamers.Validation of the E-AB sensors in bovine and porcine kidney juice.The animal agriculture industry is a multibillion-dollar market. Veterinary drugs are an integral part of modern animal husbandry and provide numerous agricultural and economic benefits. However, the misuse of such drugs leads to harmful quantities of residues in animal tissues intended for human consumption, and thus poses a great threat to public health and safety. As such, strict legal limitations are set for veterinary drug residues in animal carcasses to minimize human exposure. The enforcement of chemical residue limits requires close monitoring, and in-plant screening is vital for achieving this goal. However, the only currently available in-plant screening assay for antibiotics is vulnerable to both false-positives and -negatives, and there is no in-plant method available for monitoring of non-antibiotic residues in animal carcasses. As a solution, we propose to develop a set of scientific techniques and methods that will allow for the streamlined development of portable and cost-effective E-AB sensors that can detect chemical residues in a rapid, specific, and sensitive manner. As a demonstration, we will develop E-AB sensors for the simultaneous detection of beta-lactam antibiotics and flunixin, drugs that are commonly used in animal agriculture and frequently associated with chemical residue violations. We will first isolate two high-affinity aptamers--one specific for flunixin, and another cross-reactive for beta-lactam antibiotics--through a well-established process of systematic evolution of ligands by exponential enrichment (SELEX). We will subsequently utilize these aptamers to fabricate E-AB sensors and validate the performance of these sensors in bovine and porcine kidney juice. The key deliverable of this project will be E-AB sensors that enable rapid, sensitive, specific, robust, facile, and quantitative detection of beta-lactams and flunixin in animal kidneys. Such sensors can be used by both farmers and USDA inspection program personnel to ensure that the levels of veterinary drug residues in animal carcasses are compliant with federal tolerance levels, thereby improving food safety. More broadly, the techniques and methods developed in this proposal can further be used for the streamlined development of E-AB sensors for many other analytes of interest to the USDA.