Fluorometric methods are roughly 500 times more sensitive than colorimetric methods. CD BioGlyco provides various fluorometric assays to quantify sialic acid for our clients worldwide.
Fluorometric methods for the analysis of sialic acid and its derivatives typically involve the reaction of sialic acid with an oxidant or acid, and the resulting compounds further react to form a fluorophore. It is then excited, and the emission is measured to determine the content of sialic acid in a sample.
Sialic acid is hydrolyzed and successfully labeled, while minimal interfering compounds reacted with the 3,5-diaminobenzoic acid. Each 100 μL of 3,5-diaminobenzoic acid solution can effectively label 5 μmol of sialic acid. This method is highly specific for sialic acid and therefore provides accurate results in the quantification of sialic acids in human samples.
Sialic acids are released from glycans using mild acid hydrolysis and then reacted with thiobarbituric acid to form a fluorogen rather than a chromogen. This method is 500-fold more sensitive than the traditional thiobarbiturate method, allowing for analysis of sialic acid in the picomole range.
Pyridoxamine dihydrochloride is heated in methanol at 70 °C for 45 min in the presence of zinc acetate and pyridine to react with the α-keto acid generated by the ring-opening of sialic acid to form a fluorophore. This method is specific for free sialic acid, but bound sialic acid can also be measured by performing a previous acid release step.
Fig.1 Reaction of formylpyruvic acid with pyridoxamine in the presence of zinc ions. (Cheeseman, 2021)
The bound sialic acid is oxidized with periodic acid to form formaldehyde, which reacts with acetylacetone solution (acetylacetone, ammonium acetate, glacial acetic acid, and distilled water) at 60 °C for 10 min to obtain a fluorescent compound that emits light at 510 nm. This method could be used to quantify O-acetylated sialic acid.
The oxidization of sialic acid with periodic acid generates formaldehyde. Due to concerns about its toxicity, excess periodate is neutralized by adding sodium thiosulfate instead of sodium arsenite. Formaldehyde is then reacted with acetanilide and ammonium acetate for 10 min at room temperature to form a fluorophore. This method is extremely sensitive and can measure quantities of sialic acid as low as 1 μg and as high as 90 μg.
Plasma or serum sialic acid has been established as a potential tumor marker, and a variety of methods are available to determine serum or plasma sialic acid. CD BioGlyco has developed rapid fluorimetric methods for sialic acid quantification, including 3,5-diaminobenzoic acid, thiobarbiturate method, pyridoxamine, acetylacetone/periodic acid, and acetoacetanilide/ammonia.
CD BioGlyco is committed to providing high-quality sialic acid analysis services using fluorometric assays for our clients. If you are interested in our services, please feel free to contact us for more details.
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