Design and GC-MS Validation of a Highly Sensitive and Portable Colorimetric Biosensor for the Detection and Quantitation of Amphenicol Residues in Seafoo

In recent years, antibiotic resistance has emerged as a potent threat to human health. Antibiotic resistant bacteria result in 2 million infections, 23,000 deaths, and $20 billion in healthcare related costs in the United States annually. The consumption of meat from livestock reared on antibiotics represents one possible route of exposure of antibiotic resistant infection for humans. Seafood from aquaculture is of particular concern due to the use of prophylactic antibiotic treatment. This treatment is motivated by the proximity of the fish to one another, high demand for seafood, and aquaculture ponds providing bacterial breeding grounds. The United States government strictly regulates antibiotic use, but many foreign countries lack strict monitoring of livestock treatment, and 90 percent of seafood consumed in the US is imported. The Food and Drug Administration (FDA) typically screens seafood imports with liquid chromatography or gas chromatography coupled to mass spectrometry (LC-MS/MS or GC-MS) due to the high sensitivity and accuracy of these methods. However, chromatographic detection methods are time consuming, require technical expertise and machinery, and utilize large quantities of organic solvents. Thus, development of an ultrasensitive biosensor that outperforms LC-MS/MS and GC-MS in terms of sample time, convenience, and environmental footprint without sacrificing sensitivity and accuracy is vital to efforts in reducing the occurrence of dangerous antibiotic exposure.;The research presented in this document involved the design of a biosensor to detect chloramphenicol (CAP), a toxic antibiotic belonging to the amphenicol class, as well as the development of a GC-MS method to validate and calibrate biosensor measurements. CAP is frequently used in South American and Asian countries in aquaculture and linked to the promotion of antibiotic resistant bacteria. The GC-MS method used 5-alpha cholestane as an internal standard for amphenicol quantitation. Other antibiotics, including thiamphenicol (TAP), florfenicol (FF), were also detected using GC-MS. The biosensor incorporated the enzyme-linked immunosorbent assay (ELISA) method, which manipulates antibody/antigen pair affinities, onto a poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous polymer membrane structure prepared by electrospinning. PVA-co-PE membranes introduced several advantages, including portability with naked eye detection, functional sites for covalent bond formation, ultrahigh specific surface areas available for protein (antibody) immobilization, and hydrophilicity to repel interfering matrix molecules such as lipids. Detection occurred by competitive reaction between CAP and horseradish peroxidase-conjugated CAP (CAP-HRP) molecules with polyclonal CAP antibodies and subsequent application of a tetramethylbenzidine (TMB) substrate that induced a blue color. The color intensity correlated with the amount of immobilized CAP-HRP; thus, stronger color intensity indicated lower CAP concentration. It was possible to detect CAP at 0.2 parts per billion (ppb) with the naked eye, which is on the same order of magnitude as ELISA and LC-MS/MS sensitivity. Color signal was not uniform on the membranes, and sensitivity was not as drastically improved as expected relative to conventional ELISA; it was hypothesized that restriction of mass transport of proteins through the porous membrane structure caused these undesirable results. Protein diffusion studies were conducted by placing a nanofibrous membrane in side-by-side diffusion cells, where the donor and receptor cells contained protein and buffer solutions, respectively. The data indicated that smaller proteins penetrate the porous structure more quickly and that protein adsorption and electrostatic attraction both inhibit protein diffusion through the nanofibrous membrane.