Extraction and detection of pathogens using carbohydrate-functionalized biosensors
Inventors
Alocilja, Evangelyn C. • Cloutier, Barbara Christine • Anderson, Michael J.
Assignees
Michigan State University MSU • US Army Medical Research and Development Command
Publication Number
US-12332238-B2
Publication Date
2025-06-17
Expiration Date
2032-08-29
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Abstract
The disclosure relates to the extraction and detection of pathogens using carbohydrate-functionalized biosensors. Immobilized carbohydrate moieties on the biosensor provide a means for non-specific binding of a plurality of target analytes. When a sample containing the target analyte is applied or otherwise transported to the biosensor detection surface, non-specific binding interactions between the carbohydrate moiety and the analyte immobilize/retain the analyte at the detection surface. The carbohydrate moiety is a stable binding pair member that allows on-sensor rinsing of a sample to enhance detection of an analyte in the sample. Specific analyte identification can be achieved with an analyte probe having a detection moiety and a binding pair member specific to the target analyte of interest.
Core Innovation
The invention relates to the extraction and detection of pathogens using carbohydrate-functionalized biosensors. The biosensor detection surface is immobilized with carbohydrate moieties that provide a means for non-specific binding of a plurality of target analytes such as bacteria. This non-specific binding permits immobilization and retention of target analytes on the biosensor surface when a sample containing the analyte is applied or transported to the biosensor. The carbohydrate moiety is a stable binding pair member, enabling on-sensor rinsing to enhance analyte detection by removing non-target interferences without substantial loss of the analyte.
The biosensor is combined with an analyte probe comprising a specific binding pair member and a detection moiety specific to the target analyte of interest. The analyte probe specifically binds the target analyte, forming an analyte conjugate that can be immobilized on the carbohydrate-functionalized biosensor by non-specific binding of the carbohydrate moiety to the analyte. Detection of the analyte is achieved by detecting the probe's detection moiety when immobilized on the biosensor. The detection moiety can include conductive polymers bound to magnetic nanoparticles, allowing both magnetic separation and electrical detection (conductimetry or electrochemistry).
The problem being solved is the need for rapid, accurate, and selective detection of pathogens, which traditionally require time-consuming manual processes with incubation times of 24 to 48 hours. Existing methods such as immunomagnetic separation and biosensor devices have limitations including specificity, shelf stability, and inability for multiplex detection. The invention addresses these by using a carbohydrate-functionalized biosensor with stable immobilization of carbohydrate moieties that non-specifically bind multiple bacterial analytes, combined with analyte probes specific to individual target analytes. This enables multiplexed pathogen detection with improved shelf stability and rapid assay kinetics.
Claims Coverage
The patent claims cover one independent kit claim focused on a biosensor kit for binding a target analyte, and several dependent claims refining aspects of the kit. The main inventive features relate to the analyte probe, the biosensor with carbohydrate-functionalization, the specific carbohydrate moieties used, and the detection moieties incorporating conductive polymers and magnetic nanoparticles.
Kit comprising analyte probe and carbohydrate-functionalized biosensor
The kit includes an analyte probe with a specific binding pair member capable of specifically binding to a target analyte, and a detection moiety bound to it, alongside a biosensor having a non-specific binding pair member immobilized on the biosensor's working electrode. This non-specific binding pair comprises a carbohydrate moiety capable of non-specific binding to the target analyte, with conductivity-enhancing nanoparticles also immobilized on the working electrode.
Carbohydrate moiety comprising mannose moiety
The carbohydrate moiety immobilized on the biosensor working electrode can comprise a mannose moiety, which is capable of non-specific binding to a plurality of bacteria.
Stable covalent immobilization of non-specific binding pair member
The carbohydrate non-specific binding pair member is immobilized on the biosensor working electrode via a stable covalent bond, enhancing shelf stability and robustness.
Target analyte comprising bacterial genera and strains
The target analyte can be a bacterium selected from genera including Escherichia, Bacillus, Staphylococcus, Klebsiella, Shigella, Pseudomona, Vibrio, Enterobacter and their respective species and strains, where the specific binding pair member specifically binds the bacterium and the carbohydrate moiety non-specifically binds a plurality of bacteria from this group.
Multiplexing through plurality of analyte probes
The kit may comprise a plurality of analyte probes, each with a specific binding pair member for a different target analyte, with the carbohydrate moiety on the biosensor capable of non-specific binding to each of these different analytes, enabling multiplexed detection.
Detection moiety with conductive polymer shell and magnetic nanoparticle core
The detection moiety of the analyte probe can be a conductive polymer shell bound to a magnetic nanoparticle core, with the specific binding pair member bound to the conductive polymer shell, facilitating magnetic separation and electrical detection.
Separate magnetic nanoparticle capture composition
The kit can further comprise a magnetic nanoparticle capture composition distinct from the conductive polymer nanoparticle detection moiety. This capture composition includes magnetic nanoparticles bound to an additional specific binding pair member capable of specifically binding the target analyte, improving analyte capture and concentration.
Biosensor as screen-printed carbon electrode with conductive nanoparticles
The biosensor can be a screen-printed carbon electrode (SPCE) with the non-specific binding carbohydrate moiety and conductivity-enhancing nanoparticles such as gold nanoparticles immobilized on its working electrode.
Carbohydrate moiety composition ranges
The carbohydrate moiety can be selected from monosaccharides, glycosides thereof, or combinations including glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, rhamnose, N-acetylneuraminic acid, glucuronic acid, galacturonic acid, arabinofuranose acid, and xylose moieties.
Conductive polymer types
The conductive polymer in detection moieties or shells includes polyanilines, polypyrroles, polythiophenes, their derivatives, combinations, blends with other polymers, or copolymers of the monomers, preferably without metallic form metals.
The claims define a kit comprising a carbohydrate-functionalized biosensor for non-specific binding of target analytes on its working electrode, combined with analyte probes containing specific binding pair members and conductive polymer-based detection moieties often combined with magnetic nanoparticles. The carbohydrate moiety, e.g., mannose, is immobilized via stable covalent bonds, ensuring shelf-stability and multiplexing capabilities. The biosensor platform frequently employs screen-printed carbon electrodes enhanced with conductive nanoparticles, while the detection moieties enable magnetic separation and electrical detection of target bacteria from various genera and strains.
Stated Advantages
Ability to perform dual functions of magnetic concentration and biosensor transduction.
Faster assay kinetics owing to compositions being in suspension and near target analytes.
Increased surface area facilitating biological events.
Minimized matrix interference through improved separation and washing steps.
Magnetic manipulation capabilities using permanent magnets or electromagnets.
Avoidance of complex pre-enrichment, purification, or pre-treatment steps.
Design of inexpensive, sensitive, highly specific, and rapid detection devices for diverse targets using different biological modifications.
Use of both electrical and magnetic properties of BEAM nanoparticles for rapid detection.
Documented Applications
Rapid, accurate, and selective detection of pathogens including various bacteria in samples such as food, water, biological fluids (e.g., milk), environmental and industrial samples.
Multiplexed detection of different target analytes in samples using biosensors functionalized with the same carbohydrate moiety.
Food and water safety surveillance, clinical diagnostics, and food defense applications to detect food-borne pathogens like Escherichia coli O157:H7, Bacillus cereus, Bacillus anthracis, and others.
Immunomagnetic separation and concentration of pathogens from complex sample matrices followed by conductimetric or electrochemical detection on biosensors.
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