Sulfamethoxazole (SMX) is an antibiotic that is used in treating a variety of bacterial infections. However, increased and inappropriate use of antibiotics in humans and animals is the main cause of antibiotic resistance. According to the World Health Organization antibiotic resistance is one of the biggest threats to global health and development today. Thus, it is necessary to continuously, sensitively, and accurately monitor the levels of antibiotics in biological samples and soil to protect the environment and public health. The goal of our lab is to develop a DNA aptamer-based fluorescent biosensor for detection of SMX using a method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). We are using a protocol for the isolation of aptamers by solution-phase selection. The method is based on attaching to agarose-streptavidin matrix a biotin-strand complementary to one of the PCR primers in the single-stranded DNA library and then attaching a library through these complementary interactions. In each round, we introduced a solution of the SMX to collect all the specific DNA that binds to it. In order to isolate high binding affinity aptamers, we lowered the concentration of the SMX throughout the multiple rounds. We completed 20 SELEX rounds and the SMX concentrations were decreased from 100 uM to 2.5 uM. After completing SELEX, we performed cloning by incorporating the purified PCR product directly into the plasmid vector. The plasmids were subsequently transformed into competent cells, and positive colonies containing recombinant plasmid DNA were screened via blue/white screening. We randomly picked 24 colonies and cultured them in LB media. Isolated plasmids from the cultured cells were sent for sequencing and we received 18 sequences and proceeded with sequence analysis. In order to design and develop the first DNA aptamer-based biosensor for the detection of the antibiotic Sulfamethoxazole, we determined the Kd for several aptamer candidates. These findings will help us to design and develop the first DNA aptamer-based biosensor for the detection of antibiotic sulfamethoxazole.

Development of DNA aptamer-based fluorescent biosensor for antibiotic sulfamethoxazole detection

ACS Fall 2020 Virtual Meeting

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