DNA Aptamer Biosensors for Enzyme-Free L-Lactate Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting lactate concentration, such as those using lactate oxidase and lactate dehydrogenase, are prone to inaccuracies due to the production of H2O2 and require oxygen or NAD+, making them unreliable and difficult to calibrate, and there is a lack of aptamers for lactate detection.
Innovation Solution
Development of DNA aptamers, specifically Lac201 and Lac202, with sequences SEQ ID NO: 1 and SEQ ID NO: 2, that bind to L-lactate with high affinity and specificity, enabling biosensors to detect lactate without the need for additional substrates, and simultaneous detection of lactate and glucose using fluorophore-labeled sensors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If enzyme-based methods (lactate oxidase or lactate dehydrogenase) are used for lactate detection, then lactate concentration can be measured, but inaccuracies occur due to H2O2 production and dependence on oxygen or NAD+ availability
Solution Approach 1:
The patent replaces the enzymatic detection system (lactate oxidase or lactate dehydrogenase) with a DNA aptamer-based detection system. The aptamer binds directly to lactate through molecular recognition, eliminating the need for enzymatic reactions that produce H2O2 or require oxygen and NAD+. This substitution of the detection mechanism resolves the technical contradiction by providing a more reliable and accurate measurement that is not limited by enzyme activity or co-substrate availability.
2Adaptability or versatility
If enzyme-based biosensors are used for concurrent detection of lactate and glucose, then both analytes can be monitored, but calibration becomes difficult due to enzyme denaturation and variable activity
Solution Approach 1:
The patent replaces enzyme-based biosensors with DNA aptamer-based sensors for concurrent detection of lactate and glucose. The aptamers provide stable molecular recognition without the issues of enzyme denaturation or variable activity. This substitution makes calibration significantly easier while maintaining the ability to detect multiple analytes simultaneously, thus resolving the technical contradiction between versatility and ease of manufacture.
3Quantity of substance
If traditional aptamer selection methods are used, then aptamers can be obtained for high-affinity targets, but isolation of aptamers for low epitope molecules like lactate has been perceived as difficult
Solution Approach 1:
The patent employs SELEX (Systematic Evolution of Ligands by EXponential enrichment) with optimized parameters including specific buffer conditions (pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% Tween 20), controlled temperatures (4°C for washing, room temperature for binding), and iterative selection rounds. These parameter optimizations enable successful isolation of high-affinity aptamers for lactate, a low epitope molecule, by creating ideal conditions for aptamer-target complex formation and selection.
Solution Approach 2:
The patent uses a biotinylated lactate analog as an intermediary target during the SELEX process to facilitate aptamer selection. The biotin label enables easy capture and enrichment of binding sequences without interfering with the fundamental aptamer-lactate interaction. This intermediary approach simplifies the detection and isolation of high-affinity binders while maintaining the specificity for lactate recognition.
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 aptamers provide accurate and selective detection of lactate with a low limit of detection (LOD) and high selectivity, allowing for continuous and multiplexed monitoring of lactate and glucose concentrations, suitable for biomedical diagnosis, food and beverage industry, and environmental monitoring.
Implementation Method 1
Aptamers are single-stranded DNA molecules that have been shown to bind to a diverse range of target molecules with high affinity and specificity
Data Source
AI summary
Aptamers specific for L-lactate are provided. Also provided are biosensors comprising such aptamers and methods for detecting L-lactate using such aptamers and/or biosensors. Also provided is a method for multiplexed detection of L-lactate and D-glucose.