The use of low-cost immobilized DNA aptamers on cellulose filter to remove antibiotic residues from effluents

Effluents in general and hospital wastewater in particular can have high concentrations of antibiotics. When untreated, such residues have great potential to generate resistant bacterial strains in the environment. The main methods for removing antibiotic residues from effluents are based on chemical processes through the addition of compounds to react with or degrade antibiotics or physical methods that rely on expensive technologies. This project proposed the development of a cellulose filter obtained from agro-industrial residues with immobilized DNA aptamers that act as specific and high-affinity chemical probes for the absorption and retention of antibiotic molecules in effluents. The main methods for removing antibiotic residues from effluents are based on chemical processes and physical methods that depend on technology and expensive maintenance. The hypothesis is that after immobilization on cellulose filters, aptamers bind with high affinity to antibiotic molecules in effluents of different types, reducing the concentration of these molecules in the final effluent. The proposal uses a low-cost environmentally friendly technique. The filters are reusable and can be used in effluents without the risk of chemical contamination and are easily adapted to various types of environments such as domestic and hospital effluent treatment stations and small and medium agricultural properties. The technology produced is easily scalable and can be applied to many air and waterborne pathogens and has also been applied to the elimination of SARS-CoV-2 viral particles found in air and effluents.

The project's first step was to identify sequences and aptamers from the literature with the ability to interact with antibiotics with high affinity and test their removal power in an aqueous solution. The identification of new chemical probes used a systematic evolution of ligands by exponential enrichment (SELEX method), with the specific aim of detecting chemical probes that bind to beta-lactams, aminoglycosides, fluoroquinolones, and macrolides. The chemical probes identified were linked covalently to cellulose membranes. Binding efficiency was measured and characterized by spectrophotometry, calorimetry, and scanning electron microscopy. After validating the efficiency in removing beta-lactams, the functionalized membrane was tested in hospital and agricultural effluents from different communities in different Brazilian regions.

Four chemical probes bound to diverse classes of antibiotics were identified. In parallel, a new RNA aptamer was identified and used as an RNA guide for the CRISP-Cas9 system to reverse the resistance of gram-negative bacteria. The chemical probes, validated in the laboratory, removed antibiotics from aqueous solutions and synthetic effluent. The filter was found to be efficient in removing five classes of antibiotics at concentrations generally found in real effluents obtained from the Arrudas sewage plant in Belo Horizonte, Minas Gerais. The HPLC gold standard confirmed this effectiveness. The filter can undergo two regeneration processes, with estimated durability of 48 months. Antibiotic residues retained on the filter were efficiently and easily recovered as a concentrated solution that was either inactivated by small-scale oxidative processes or purified for reuse. A rapid colorimetric system for detecting antibiotics in effluents was also developed that can identify and quantify these contaminants in one minute, enabling assessment of the filters´ efficiency.

The idea is creative because unlike traditional effluent treatment methods, it uses low-cost probes, is environmentally safe, and provides a user-friendly system. The aptamers and cellulose membranes are biodegradable, and the membrane is produced from plant residues used in various industries. The filter was structured as a plug-in part that allows connecting different units to create a filter with the size customized to the target environment.

Considering that antimicrobial residues can select multidrug-resistant strains, removing these antibiotics from effluents is paramount for controlling AMR. The proposed technology in this project using cellulose immobilized aptamers shows high performance and low environmental impact.

The next steps include scaling up the production of filters to generate units for installation in hospitals and effluent treatment plants. In addition, other versions of the filter are also under development to remove other pathogens for the control of human and veterinary diseases, as well as filters to remove phytopathogens from reused agriculture water.