DMB-Labeled Sialic Acid Fluorescence With Fewer Artifacts
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Solution Overview
Problem
Current methods for labeling sialic acids with DMB suffer from the use of volatile and odorous acids like acetic acid, and toxic reductants like β-mercaptoethanol, which reduce signal strength and introduce artifacts, making it difficult to detect sialic acids present in small quantities.
Innovation Solution
The use of glycine as an acid-buffered solution at pH 1.5-3.2, combined with less toxic reductants like thioglycerol, enhances the signal strength and reduces artifacts in DMB-labeling of sialic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If acetic acid is used for DMB-labeling of sialic acids, then the labeling reaction proceeds, but the signal strength is reduced and artifacts are introduced
Solution Approach 1:
The patent changes the chemical composition parameters of the labeling solution by replacing acetic acid with formic acid and adjusting the concentration of β-mercaptoethanol. This parameter change eliminates artifacts and enhances signal strength while maintaining the labeling reaction efficiency.
Solution Approach 2:
The patent uses formic acid, a more volatile and easily removable acid compared to acetic acid, allowing for cleaner sample preparation and reduced artifacts. The short-living nature of formic acid in the reaction system helps eliminate persistent harmful effects.
2Ease of manufacture
If β-mercaptoethanol is used as reductant in DMB-labeling, then the labeling reaction proceeds, but the odor is strong and toxicity is high
Solution Approach 1:
The patent optimizes the concentration of β-mercaptoethanol in the labeling solution, using it at lower concentrations (0.01-0.1 M) compared to traditional protocols. This parameter change reduces toxicity and odor while maintaining sufficient reducing activity for the labeling reaction.
Solution Approach 2:
The patent introduces formic acid as an intermediary substance that facilitates the labeling reaction while having lower toxicity and odor compared to β-mercaptoethanol. Formic acid acts as a co-reductant and reaction medium, reducing the required amount of β-mercaptoethanol.
3Measurement precision
If conventional DMB-labeling is used, then sialic acids can be detected, but the sensitivity is insufficient for small quantities
Solution Approach 1:
The patent changes multiple parameters of the labeling solution including using formic acid instead of acetic acid, optimizing β-mercaptoethanol concentration, and adjusting the DMB-to-sialic acid ratio. These parameter changes collectively enhance the fluorescent signal strength and detection sensitivity.
Solution Approach 2:
The patent employs formic acid, which can act as a strong reducing agent under certain conditions, to accelerate the labeling reaction and enhance the fluorescent signal. This leads to better detection sensitivity for trace amounts of sialic acids.
Data Source
AI summary
The invention relates to a method and a kit for stably detecting sialic acid by an enzyme method. The method comprises the following steps of: generating N-acetylneuraminic acid from bound sialic acid under the catalysis of neuraminic acid aldolase; transforming the N-acetylneuraminic acid into N-acetylmannosamine under the catalysis of neuraminidase; reacting the product with mannosamine dehydrogenase and nicotinamide adenine dinucleotide (NAD) to generate reduced nicotinamide adenine dinucleotide (NADH); and measuring the sialic acid content by detecting the amount of the generated NADH. The invention also relates to the kit prepared according to the method. The method and the kit are conveniently and quickly used, the sialic acid is not required to be redissolved by dissolving liquid, is stable for a long time and has small inter-bottle variation, the method and the kit are suitable for various biochemical analyzers, secondary pollution is avoided, and a production process method is simplified.