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-10739337-B2
Publication Date
2020-08-11
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 extraction and detection of pathogens using carbohydrate-functionalized biosensors. The biosensor includes immobilized carbohydrate moieties on a detection surface, which provide a means for non-specific binding of multiple target analytes. When a sample containing the target analyte is applied, the carbohydrate moiety non-specifically binds and retains the analyte on the biosensor surface, allowing for on-sensor rinsing to enhance detection sensitivity. Specific identification of the analyte is achieved with an analyte probe containing a detection moiety and a binding pair member that specifically binds the target analyte.
The disclosed method includes providing an analyte conjugate composed of the target analyte bound to a specific binding pair member and a detection moiety; providing a biosensor with a non-specific binding pair member comprising a carbohydrate moiety immobilized on the working electrode; immobilizing the analyte conjugate on the biosensor via non-specific binding between the carbohydrate moiety and the target analyte; and detecting the detection moiety. The carbohydrate moiety is stable, allowing rinsing to remove non-target interferences without loss of the target analyte. The analyte probe can be a biologically enhanced, electrically active magnetic (BEAM) nanoparticle composition that facilitates both separation and detection.
The problem being solved addresses limitations of conventional pathogen detection methods which require manual labor and incubation times of 24 to 48 hours. Existing immunomagnetic separation and biosensing techniques face challenges including lack of stability of specific binding moieties on biosensors, limited multiplexing capability due to specificity, and lengthy sample preparation. There is a need for rapid, accurate, selective, and field-portable detection of pathogens with shelf-stable biosensors capable of non-specific binding to multiple analytes while allowing specific analyte identification via analyte probes.
Claims Coverage
The claims cover one independent method claim that includes multiple inventive features relating to the detection of target analytes using carbohydrate-functionalized biosensors.
Use of carbohydrate moiety as non-specific binding pair member immobilized on biosensor electrode
The biosensor comprises a non-specific binding pair member immobilized on the working electrode, where the member includes a carbohydrate moiety capable of non-specific binding to the target analyte.
Immobilization of analyte conjugate via non-specific carbohydrate binding
The method immobilizes the analyte conjugate on the working electrode through non-specific binding interactions between the carbohydrate moiety and the target analyte of the analyte conjugate.
Detection moiety comprising conductive polymer bound to magnetic nanoparticle core
The detection moiety of the analyte conjugate includes a conductive polymer shell bound to a magnetic nanoparticle core, with the specific binding pair member bound to the conductive polymer shell.
Inclusion of conductivity enhancing nanoparticles on biosensor electrode
The biosensor's working electrode includes nanoparticles (e.g., gold nanoparticles) immobilized thereon for enhancing conductivity prior to immobilizing the analyte conjugate.
Multiplexed detection using same carbohydrate-functionalized biosensor
The method can be replicated for multiplexed detection of a plurality of different target analytes, all using biosensors comprising the same carbohydrate moiety or moieties.
Use of antibodies as specific binding pair members against bacteria
The specific binding pair members are antibodies capable of binding specifically to bacterial target analytes selected from various genera including Escherichia, Bacillus, Staphylococcus, among others; whereas the carbohydrate moiety non-specifically binds a plurality of bacteria from the group.
Biosensor embodied as screen-printed carbon electrode (SPCE)
The biosensor is embodied as a screen-printed carbon electrode with the carbohydrate moiety immobilized on its working electrode.
Optional washing of biosensor after analyte conjugate immobilization
The method includes an optional step of washing the working electrode after immobilizing the analyte conjugate and before detecting the detection moiety to remove non-bound probes and reduce false positives.
Conductimetric or electrochemical detection of conductive polymer detection moiety
Detection of the detection moiety in the analyte conjugate can be performed conductimetrically or electrochemically by detecting the conductive polymer moiety bound to the magnetic nanoparticle.
The inventive features combine the use of a carbohydrate moiety immobilized on a biosensor electrode as a stable, non-specific binding platform for multiple analytes, together with analyte probes comprising specific binding members and conductive polymer-magnetic nanoparticle-based detection moieties. The method supports multiplexed pathogen detection with enhanced conductivity through nanoparticles on the biosensor, realized particularly in screen-printed carbon electrodes.
Stated Advantages
Ability to perform dual function of magnetic concentration and biosensor transduction in pathogen detection.
Faster assay kinetics due to compositions being in suspension and proximity to target analytes.
Increased surface area for biological events to occur, improving detection.
Minimized matrix interference via efficient separation and washing steps.
Capability to design cheap, sensitive, highly specific, and rapid detection devices for diverse targets using biological modifications.
Shelf-stable biosensor platform allowing multiplexed detection of multiple pathogens with the same carbohydrate-functionalized surface.
Elimination of lengthy pre-enrichment steps required by standard detection methods.
Field portability and rapid detection with estimated detection limit of about 2 CFU/ml and total testing time around 2.5 hours including PCR confirmation.
Documented Applications
Rapid, accurate, and selective detection of various pathogens (e.g., bacterial strains such as Escherichia coli O157:H7, Bacillus cereus, Bacillus anthracis, Staphylococcus aureus, Klebsiella pneumonia, Shigella, Pseudomona aerugenosa, Vibrio fischeri, Enterobacter) in food, fluid milk, and broth samples.
Multiplexed pathogen detection using the same carbohydrate-functionalized biosensor to enable detection of multiple different analytes in a sample.
Field-portable biosensor platform for food safety, food defense, and clinical diagnostics to identify contamination rapidly without requiring lengthy culture enrichment steps.
Use in immunomagnetic separation processes for extraction, concentration, and detection of target analytes in complex sample matrices including food and environmental samples.
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