Method for detecting enzymatic reaction product
Inventors
TABATA, Kazuhito • MINAGAWA, Yoshihiro • Noji, Hiroyuki
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Publication Number
US-12173348-B2
Publication Date
2024-12-24
Expiration Date
Abstract
Provided is a technique that can be used to detect a pathogenic microorganism such as influenza virus with high sensitivity, and specifically, a method for detecting a reaction product, including reacting an enzyme with a substance serving as a substrate for a reaction involving the enzyme in a hydrophilic solvent that is in interfacial contact with a hydrophobic solvent, wherein the reaction product is produced directly and released from the substrate, and wherein the hydrophilic solvent contains at least one of a buffering substance or trimethylamine oxide (TMAO).
Core Innovation
A method is provided for detecting a reaction product by reacting an enzyme with a substrate in a hydrophilic solvent that is in interfacial contact with a hydrophobic solvent. The reaction product is produced directly and released from the substrate, and the released reaction product is retained in the hydrophilic phase to enable sensitive optical detection.
The hydrophilic solvent contains at least one of a buffering substance or trimethylamine oxide (TMAO), and the concentration of the buffering substance in the hydrophilic solvent is at least 500 mM. In disclosed embodiments, the hydrophilic solvent pH is set above the pKa of the reaction product to support retention in the hydrophilic phase, and buffering substance and/or TMAO suppress migration of the reaction product into the hydrophobic phase.
The disclosed approach further includes enzyme-substrate pairs for pathogen detection, including influenza virus with neuraminidase using 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (4 MU-NANA) to produce 4-methylumbiferone (4 MU) for optical detection. A droplet-array workflow is described using a microfabricated array with hydrophobic-walled receptacles and an enclosing hydrophobic solvent to form picoliter-to-nanoliter droplets that accumulate product in minimal volume for fluorescence, absorbance, or optical-rotation detection.
The documented use cases include quantification and influenza subtype determination (influenza A/B), and digital quantitative determination.
Claims Coverage
The document provides two independent claims covering a reaction-product detection method based on enzyme-substrate conversion at a hydrophilic/hydrophobic solvent interface with retention of the produced product in the hydrophilic phase via buffering substance and/or TMAO. The independent claims include 1 inventive-feature set centered on interfacial enzymatic release with a quantitative buffering threshold, and a second inventive-feature set emphasizing suppression of migration with added buffering substance or TMAO under the same interfacial conditions and concentration threshold.
Interfacial enzyme reaction with direct product release
Reacting an enzyme with a substance serving as a substrate for a reaction involving the enzyme in a hydrophilic solvent that is in interfacial contact with a hydrophobic solvent, wherein the reaction product is produced directly and released from the substrate.
Retention by buffering substance or TMAO with quantitative threshold
The hydrophilic solvent contains at least one of a buffering substance or trimethylamine oxide (TMAO), and wherein the concentration of the buffering substance in the hydrophilic solvent is at least 500 mM.
Migration suppression by adding buffering substance or TMAO
Adding at least one of a buffering substance or trimethylamine oxide (TMAO) to the hydrophilic solvent to suppress migration of the reaction product to the hydrophobic solvent from the hydrophilic solvent, wherein the concentration of the buffering substance in the hydrophilic solvent is at least 500 mM.
Across the independent claims, the inventive coverage is directed to interfacial enzymatic conversion in hydrophilic/hydrophobic solvent contact with direct release of the reaction product, combined with a hydrophilic-phase retention strategy using buffering substance and/or TMAO and an explicit buffering concentration threshold of at least 500 mM.
Stated Advantages
The released reaction product is retained in the hydrophilic phase to enable sensitive optical detection.
Buffering substance and/or TMAO suppress migration of the reaction product into the hydrophobic phase.
A droplet-array workflow accumulates product in minimal volume for fluorescence, absorbance, or optical-rotation detection.
Documented Applications
Pathogen detection, including influenza virus detection.
Influenza subtype determination (influenza A/B).
Quantification and digital quantitative determination.
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