Abstract
Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.
Highlights
Infections with resistant pathogens represent one of the major health issues of concern in the 21st century, especially with nosocomial infections being among the most challenging tasks to handle for healthcare facilities
The concept consists of two major technical components: novel bovine serum albumin (BSA) hydrogel-based beads and specific aptamer molecules as binding entities to functionalize a material with an affinity towards P. aeruginosa cells as a proof of concept for pathogen-capturing particles
ethyl-3-(3-dimethylaminopropyl) carbodiimide-hydrochloride (EDC) first reacts with a carboxyl group to form an amine-reactive O-acylisourea intermediate that quickly reacts with an amino group to form an amide bond and releases a non-toxic isourea byproduct which is removed by washing the hydrogel after fabrication [21,22]
Summary
Infections with resistant pathogens represent one of the major health issues of concern in the 21st century, especially with nosocomial infections being among the most challenging tasks to handle for healthcare facilities. The high mortality from blood infections is partly due to the inability to rapidly detect and identify the causative pathogen and treat patients with appropriate antibiotics in the early stages of infection [6]. Amplification-based molecular diagnosis methods such as polymerase chain reaction (PCR) can reduce the assay time to hours This methodology is often limited and not sensitive enough to detect the low concentrations of bacteria, needing additional steps such as initial enrichment [8]. The recently introduced variation of the SELEX process is based on monitoring the successive selection rounds by fluorescence measurements of the developing libraries This FLuCell-SELEX was directed against P. aeruginosa and generated polyclonal aptamer libraries which were better suited to reliably bind carbapenem-resistant P. aeruginosa than the best individual aptamers identified by deep sequencing of the library and bioinformatic analyses [17]. BSA as a natural component of blood circulating in the bloodstream is an ideal candidate for manufacturing surfaces free of interactions with blood cells and has been used as a coating for medical devices [20]
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