FMN-Dependent Lactate Dehydrogenase for Oxygen-Independent Lactate Detection
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Solution Overview
Problem
Current lactate detection methods, particularly biosensors, suffer from oxygen interference due to the use of oxygen-dependent enzymes like lactate oxidases, limiting their applicability and accuracy, especially in environments where oxygen levels fluctuate.
Innovation Solution
Employing a specific class of flavin mononucleotide (FMN)-dependent lactate dehydrogenases with distinct amino acid sequences that can oxidize lactate and transfer electrons to redox mediators, rather than oxygen, thereby reducing oxygen interference and enabling accurate lactate detection in the presence of oxygen.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If lactate oxidase is used for lactate detection, then the biosensor can operate with simple enzyme system, but oxygen interference occurs and measurement accuracy deteriorates
Solution Approach 1:
The patent introduces an electron mediator (such as ferrocene derivatives, viologens, or quinones) as an intermediary substance that facilitates electron transfer from the reduced enzyme to the electrode. This mediator acts as a bridge, allowing the biosensor to function without direct oxygen involvement, thereby eliminating oxygen interference while maintaining simple enzyme system architecture
Solution Approach 2:
The patent changes the operational parameters of the biosensor by operating at low potentials (below 0.2V vs Ag/AgCl) where oxygen reduction does not occur. This parameter change allows the use of lactate oxidase while avoiding oxygen interference, as the electrode potential is controlled to prevent oxygen from competing as an electron acceptor
2Device complexity
If first-generation biosensor with oxygen as electron acceptor is used, then the system is simple and does not require mediators, but oxygen dependence limits applicability in varying oxygen environments
Solution Approach 1:
By introducing electron mediators, the system transitions to a configuration where electron transfer does not depend on oxygen. The mediator accepts electrons from the reduced enzyme and transfers them to the electrode independently of oxygen presence, enabling the biosensor to function in both aerobic and anaerobic environments
Solution Approach 2:
The patent creates a universal biosensor system that can operate across different oxygen conditions (aerobic, anaerobic, and varying oxygen tension) by using mediators that enable electron transfer independent of oxygen. This multi-functional capability allows the same biosensor design to be applied in diverse physiological and industrial environments
3Measurement precision
If second-generation biosensor with redox mediators is used, then oxygen interference is avoided, but the device complexity increases due to mediator requirements
Solution Approach 1:
The patent employs thin film modifiers on the electrode surface that incorporate redox mediators in a structured, stable matrix. This thin film approach organizes the mediators in a controlled manner, reducing their leaching and improving stability, while the film structure itself provides a simple, integrated platform that reduces overall system complexity
Solution Approach 2:
The patent uses composite materials combining conductive polymers, nanoparticles, or other functional materials with redox mediators to create integrated electrode modifications. These composite structures provide both the electron transfer function and structural stability, reducing the need for separate components and simplifying the overall biosensor system
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
The method provides oxygen-independent lactate detection and quantification, enhancing the reliability and versatility of biosensors for continuous lactate measurement in various samples, including food, beverages, and biological fluids.
Implementation Method 1
L-lactate is oxidized to pyruvate in the first (reductive) half-reaction while FMN is reduced
Implementation Method 2
In the second (oxidative) half-reaction, oxygen is used as an electron acceptor to re-oxidize FMN and is itself reduced to hydrogen peroxide (H2O2)
Implementation Method 3
amperometric biosensors have been proposed based on O2 consumption and H2O2 formation which is detected on electrodes
Data Source
AI summary
The present invention relates to methods and means for detecting and/or quantifying lactate. Specifically, the present invention is directed to a method for detecting and/or quantifying lactate in a sample comprising the steps of: a) providing an electrode comprising a lactate dehydrogenase (LDH) comprising a flavin mononucleotide (FMN) and an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 2, comprising the following sequence T-(Xx)n1-F-(Xx)n2-X1-X2-(Xx)n3-X3-X4 (SEQ ID NO: 1), wherein T is T122 of SEQ ID NO: 2, or corresponding to T122, F is F152 of SEQ ID NO: 2, or corresponding to F152, Xx is any amino acid, n1 is an integer of 25 to 35, n2 is an integer of 40 to 45, n3 is an integer of 5 to 20, X1 is N or F, X2 is L or F, X3 is G, T, or S, and X4 is I or V; b) contacting the sample with the electrode; and c) detecting the oxidation of lactate by the LDH, wherein the oxidation of lactate is performed in the presence of a redox mediator.