Methods, microfluidic devices, and systems for detection of an active enzymatic agent
Inventors
Sommer, Gregory J. • Hatch, Anson V. • Singh, Anup K. • Wang, Ying-Chih
Assignees
National Technology and Engineering Solutions of Sandia LLC • Sandia National Laboratories
Publication Number
US-8871496-B1
Publication Date
2014-10-28
Expiration Date
2029-08-20
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Abstract
Embodiments of the present invention provide methods, microfluidic devices, and systems for the detection of an active target agent in a fluid sample. A substrate molecule is used that contains a sequence which may cleave in the presence of an active target agent. A SNAP25 sequence is described, for example, that may be cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety. The substrate molecule is exposed to the sample, and resulting reaction products separated using electrophoretic separation. The elution time of the reporter moiety may be utilized to identify the presence or absence of the active target agent.
Core Innovation
The invention provides methods, microfluidic devices, and systems for the detection of an active target enzymatic agent in a fluid sample using a substrate molecule that contains a sequence cleavable by the active agent. A specific example uses a SNAP25 sequence that is cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety which enables detection of cleavage by the active enzymatic agent. The assay exposes the substrate molecule to the sample, and electrophoretically separates the resulting reaction products. The elution time of the reporter moiety after separation indicates whether the active enzymatic agent is present.
The background problem being addressed is that Botulinum Neurotoxin (BoNT), a highly potent toxin, requires rapid, sensitive, and specific detection of its active form for effective therapeutic intervention. Existing assays such as the mouse bioassay, while sensitive, are time-consuming and require large animal populations. Immunoassays detect presence of toxin proteins but not enzymatic activity and cannot differentiate between active and inactive forms. There is a need for rapid, highly sensitive assays that specifically detect enzymatic activity, preferably with serotype specificity, and that can be integrated into automated systems.
The innovation further includes the use of microfluidic devices integrating assay steps such as metering, mixing, preconcentration, rinsing, separation, and detection on small volumes with high sensitivity and speed. The substrate molecule, sample, and reaction products may be concentrated near size exclusion membranes within the microfluidic channels, facilitating enzymatic cleavage followed by separation under denaturing conditions to produce distinct elution times for cleaved versus intact substrate molecules. The system can include electrodes to electrophoretically control transport of molecules and reagents such as denaturants, enabling automation and efficient assay workflows.
Claims Coverage
The patent includes one independent claim describing a microfluidic device to detect an active enzymatic agent, and several dependent claims that add specific features and embodiments to this device.
Microfluidic device configuration with multiple channels and membranes
A microfluidic device comprising a substrate defining first, second, and third channels, with first and second membranes positioned in the first and second channels respectively, where the first membrane blocks substrate molecules including a reporter moiety and cleavable sequence, and the third channel transports reaction products between the membranes.
Electrophoretic concentration and transport of substrate molecules and reaction products
Using pairs of electrodes positioned to generate electrophoretic transport across the first and second membranes, allowing concentration of substrate molecules and reaction products, facilitating enzymatic cleavage and subsequent separation within the device.
Inclusion of a separation channel with separation gel for electrophoretic separation
A separation channel including a separation gel coupled to the second channel is configured to separate reaction products, where elution time of the reporter moiety to a detection region indicates presence or absence of active enzymatic agent.
Use of denaturing agent delivery channel and electrophoretic transport
A fourth channel coupled to the third channel delivers a denaturing agent near the second membrane, with electrodes transporting the denaturant electrophoretically toward the second membrane and away from the first membrane, enabling separation under denaturing conditions separately from enzymatic cleavage.
Specific substrate sequences and enzymatic targets
The substrate molecule sequence can include SNAP25 or VAMP-2 sequences configured to be cleaved by active enzymatic agents such as Botulinum Neurotoxin or Tetanus Neurotoxin, with the reaction products including a cleaved portion containing the reporter moiety, which is smaller than the unconnected portion.
Reporter moiety properties and membrane pore size selection
The reporter moiety comprises a fluorescent tag, and the first membrane has a pore size selected to exclude the substrate molecule, assisting in concentration and separation steps within the microfluidic device.
The independent claim covers a microfluidic device integrating sample and substrate concentration, enzymatic cleavage, denaturant exposure, electrophoretic separation, and detection of cleavage via elution times of reporter moieties, with inventive features including membrane placement, electrophoretic transport, substrate selection, and system integration to detect active enzymatic agents such as Botulinum Neurotoxin.
Stated Advantages
Faster assays with reduced time compared to conventional methods.
Smaller sample and reagent volumes leading to efficiency.
Higher sensitivity enabling detection of active enzymatic agents at low concentrations.
Better reproducibility and potential for automation reducing labor and costs.
Portability of the microfluidic device and system for widespread diagnostic use.
Documented Applications
Detection of active Botulinum Neurotoxin in fluid samples for rapid diagnosis of toxin presence.
Detection of other enzymatic agents such as Tetanus Neurotoxin using cleavable sequences like VAMP-2.
Serotype-specific activity assays useful for effective diagnostics and therapeutic intervention of neurotoxin exposure.
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