Spectrophotometric method for olefine acid detection
A technology of enoic acid and concentration, which is applied in the preparation of test samples and the measurement of color/spectral properties, etc., can solve the problem of difficult to achieve rapid and efficient screening of aspartate ammonia lyase and acrylate amino acid, and achieve intuitive results. Effect
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Embodiment 1
[0043] Embodiment 1: the selection of oxidizing agent and the determination of detection wavelength
[0044] KMnO with a concentration of 6mM was prepared in ultrapure water respectively 4 Aqueous solution, K 2 Cr 2 o 7 aqueous solution, acrylic acid aqueous solution, and β-alanine aqueous solution; take 30 μL of the above solutions in a 96-well plate, and add 170 μL ultrapure water to a final concentration of 0.9 mM. Use a microplate reader at 37°C to detect the scan pattern of the full band of 200-750nm. Combine the scanning images of each solution, and select the oxidant (KMnO 4 、K 2 Cr 2 o 7 ) and the hydrogenation reaction substrate acrylic acid and the hydrogenation reaction product β-alanine have no overlapping absorption region as the detection wavelength candidate region. Such as figure 1 As shown, KMnO 4 There is a strong absorption at 520-540nm, and the corresponding absorption values of acrylic acid and β-alanine are extremely low, which is an ideal det...
Embodiment 2
[0049] Example 2: Effect of ammoniation reaction product β-alanine and ammoniation reaction substrate ammonia on color reaction
[0050] 1. Effect of β-alanine on oxidation reaction
[0051] In order to investigate the effect of the ammoniation reaction product β-alanine on KMnO 4 Influenced, the reaction system shown in Table 2 was designed. Take 30 μL of 6mM KMnO 4 Mix the aqueous solution with different volumes of 6mM β-alanine aqueous solution in a 96-well plate, add ultrapure water to make up the system to 200 μL, KMnO 4 The final concentration of β-alanine was 0.9mM, the final concentration of β-alanine was 0.6mM and 0.3mM, respectively, and 0.9mM KMnO without β-alanine 4 The solution was used as a reagent blank, reacted in a 96-well plate at 37°C for 4 minutes, and scanned at 200-750nm with a microplate reader at 37°C. Such as Figure 5 As shown, KMnO 4 Compared with different concentrations of β-alanine mixture and reagent blank, there is no significant change in...
Embodiment 3
[0057]Embodiment 3: determination of reaction conditions
[0058] 1. Determination of reaction time
[0059] Build KMnO separately 4 Color reaction system with different concentrations of acrylic acid, add ultrapure water to make up the system to 200μL, KMnO 4 The final concentration of acrylic acid was 0.9mM, and the final concentrations of acrylic acid were 0.9mM, 0.6mM and 0.3mM, respectively. At a final concentration of 0.9mM KMnO 4 The aqueous solution is the reagent blank, react in a 96-well plate at 37°C for 4min, and detect the reagent blank absorbance A at 37°C with a microplate reader 0 and reaction solution A 540 Change of absorbance value at 540nm within 10min, read data every 30s, and calculate ΔA 540 =A 0 -A 540 . Use a microplate reader to measure three groups of parallel experiments, and calculate ΔA accordingly 540 mean and standard deviation. Such as Figure 7 As shown, the obtained curve tends to be stable after 2min, so the reaction time is sele...
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