Enzymatic microtiter plate-based nitrate detection in environmental and medical analysis.
Our microtiter plate assay is based on the enzymatic reduction of nitrate by dissimilatory nitrate reductase from Pseudomonas stutzeri [EC 1.7.99.4]. Exogenous redox mediators like methyl viologen, methylene blue, and cibachron blue were applied to reduce nitrate reductase. Concentrations of 0.02-0.9 mM nitrate can be detected with +/-6% standard deviation, by using a photometric Griess reaction for the formed nitrite. Nitrate reductase is stable in the pH range 6.5-9.0 and works in the temperature range 4-76 degrees C. The assay shows no interferences with salt content up to 1 M chloride or 11 mM chlorate, and serum albumin content up to 50 mg/ml. The time demand of our two-step procedure is 20 min/100 samples. Nitrate reductase could be conserved on site of the wells of microtiter plates for at least 6 months at room temperature. The nitrate assay was applied in environmental and consumer goods analysis, and for medical diagnostics in human plasma samples.
Borcherding
H
H
IMB, Institut für Molekulare Biotechnologie, Beutenbergstrasse 11, Jena, 07745, Germany.
Leikefeld
S
S
Frey
C
C
Diekmann
S
S
Steinrücke
P
P
D001322:Autoanalysis / Q000379:methods*; D001628:Beverages / Q000032:analysis; D001774:Blood Chemical Analysis / Q000379:methods*; D002704:Chlorates / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005504:Food Analysis / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008751:Methylene Blue / Q000494:pharmacology; D008832:Microchemistry; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000032:analysis; D009566:Nitrates / Q000032:analysis* / Q000097:blood; D009573:Nitrites / Q000032:analysis* / Q000097:blood; D010084:Oxidation-Reduction; D010269:Paraquat / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014867:Water / Q000032:analysis
D002704:Chlorates; D002712:Chlorides; D004791:Enzyme Inhibitors; D009566:Nitrates; D009573:Nitrites; D012709:Serum Albumin; D014867:Water; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D010269:Paraquat; D008751:Methylene Blue