Fluorescent microplate analysis of amino acids and other primary amines in soils

October 4, 2012 – 3:18 pm

Our paper on a popular soil amino acid technique just came out in Soil Biology and Biochemistry. We got interested in improving this method and adapting it for microplates a couple years ago and after quite a bit of lab work this is the result. The abstract:

In studies of soil nitrogen (N) cycling, there is growing demand for accurate high-throughput analyses of amino acids and other small organic N compounds. We adapted an existing fluorometric amino acid method based on o-phthaldialdehyde and β-mercaptoethanol (OPAME) for use in 96-well microplates, and tested it using standards and field samples. While we started with an existing protocol, we made one critical change: instead of using a 1-min incubation period, we used a 1-h incubation period to deal with differences in reaction timing among microplate wells and to reduce interference from ammonium. Our microplate method is similar in sensitivity to existing protocols and able to determine leucine standard concentrations as low as ∼0.5 μM. Finally, we demonstrate that the OPAME reagent fluoresces in the presence of primary amines other than amino acids, such as amino sugars and tyramine. Because of this broad sensitivity to primary amines, descriptions of the measured pool should be revised from total free amino acids (TFAA) to total free primary amines (TFPA).

Reaction kinetics of primary amines and ammonium with OPAME. a. Fluorescence levels of leucine, a mixed amino acid standard, and ammonium over 3 h at 1.5 min intervals. b. Ratio of ammonium:leucine fluorescence for 20 μM standards over 3 h (mean ± 95% CI, n = 5). c. Fluorescence levels of leucine, glucosamine, tyramine, and N-acetylglucosamine over 3 h at 1.5 min intervals.

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