Enzymatic Assay for D-2-Hydroxyglutarate Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting (D)-2-hydroxyglutarate and (D)-2-hydroxyadipic acid are labor-intensive, costly, and not suitable for high-throughput analysis, particularly in diagnosing associated diseases and monitoring biotechnological production processes, due to limitations in distinguishing enantiomers and requiring time-consuming sample preparation.
Innovation Solution
A method involving a reagent mixture with a solvent, a dye that changes states, an electron transfer agent, and a (D)-2-hydroxyglutarate dehydrogenase enzyme, allowing for the specific detection of (D)-2-hydroxyglutarate or (D)-2-hydroxyadipic acid by measuring the production of the reduced dye state, enabling rapid and efficient analysis in a 96-well or 384-well format.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current detection methods are used, then measurement precision is achieved, but productivity is reduced due to labor-intensive procedures and time-consuming sample preparation
Solution Approach 1:
The patent replaces complex mechanical/sample preparation systems with a biochemical detection system. The enzymatic assay using 2-hydroxyglutarate dehydrogenase and coupled with a colorimetric or fluorometric detection system eliminates the need for time-consuming sample preparation and complex instrumentation, enabling high-throughput analysis while maintaining measurement precision.
Solution Approach 2:
The patent changes the detection parameters by using enzymatic reactions that produce measurable signals (color change, fluorescence) directly from the sample. This allows for rapid detection without extensive sample preparation, transforming the detection process into a high-throughput format while preserving accuracy.
2Measurement precision
If current detection methods are used, then measurement precision is achieved, but loss of time increases due to labor-intensive procedures
Solution Approach 1:
The patent incorporates preliminary action by pre-mixing the enzymatic reagents and coupling systems before sample addition. The assay is designed so that all necessary components are prepared in advance, allowing rapid sample processing and elimination of time-consuming preparation steps during the actual detection process.
Solution Approach 2:
The patent replaces time-consuming mechanical sample preparation with a streamlined biochemical assay that can be performed directly on minimally processed samples, significantly reducing the time required while maintaining detection precision.
3Reliability
If current detection methods are used, then reliability is maintained, but device complexity increases due to specialized equipment requirements
Solution Approach 1:
The patent replaces complex specialized equipment with simple microplate-based enzymatic assays that can be performed using standard laboratory equipment. The detection system uses colorimetric or fluorometric readouts that can be measured with common plate readers, eliminating the need for complex specialized instrumentation while maintaining reliable detection.
Solution Approach 2:
The patent creates a universal detection platform that can be used for detecting 2-hydroxyglutarate and related compounds using the same enzymatic system and detection methodology. This multi-functional approach reduces device complexity by eliminating the need for multiple specialized instruments, as a single platform can handle various detection needs.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach provides a simple, robust, and fast enzymatic assay for specific determination of (D)-2-hydroxyglutarate, suitable for diagnosing associated diseases and monitoring biotechnological processes, with results available in 2-3 hours and capable of high-throughput analysis.
Implementation Method 1
a (D)-2-hydroxyglutarate dehydrogenase enzyme
Implementation Method 2
a dye having an oxidized state and a reduced state, wherein the reduced state can be distinguished from the oxidized state and wherein the dye is initially present in the oxidized state
Implementation Method 3
an electron transfer agent
Data Source
AI summary
The present invention relates to a method for detecting (D)-2-hydroxyglutarate or (D)-2-hydroxyadipic acid in a sample, the method comprising the steps of: a) contacting a sample with a reagent mixture, wherein said reagent mixture comprises: (i) a solvent, (ii) a dye having an oxidized state and a reduced state, wherein the reduced state can be distinguished from the oxidized state and wherein the dye is initially present in the oxidized state, (iii) an electron transfer agent, (iv) a (D)-2-hydroxyglutarate dehydrogenase enzyme, and (v) a cofactor; and b) detecting (D)-2-hydroxyglutarate or (D)-2-hydroxyadipic acid by measuring the production of the reduced state of the dye. The invention further pertains to a method for diagnosing and/or monitoring a (D)-2-hydroxy-glutarate-associated disease in a subject. Encompassed by the invention is also a method for diagnosing a mutation in an isocitrate dehydrogenase (IDH) gene or in a (D)-2-hydroxyglutarate (D2HG) dehydrogenase enzyme gene in a subject. In addition, the invention provides for a kit comprising (i) a solvent, (ii) a dye having an oxidized state and a reduced state, wherein the reduced state can be distinguished from the oxidized state and wherein the dye is initially present in the oxidized state, (iii) an electron transfer agent, (iv) a (D)-2-hydroxyglutarate dehydrogenase enzyme, and (v) a cofactor.