Enzymatic method for detecting polyaromatic hydrocarbons
Polyaromatic hydrocarbons (PAH) from industrial waste and oil spills are a recurrent threat to food safety due to their stability and toxicity. Existing methods to establish the concentrations of PAH in environmental and food samples rely on expensive and laborious methodology. The teachings herein provide methods and kits for detecting the presence or absence of PAH. In certain embodiments, the quantity of PAH in a sample can be determined.
1. A method for detecting the presence of polyaromatic hydrocarbon (PAH) in a sample, comprising:
combining a sample with at least one PAH dioxygenase and at least one NAD(P)H cofactor to farm a first reaction solution;
incubating the first reaction solution under conditions suitable for forming dihydroxydihydro PAH and NAD(P)+;
adding at least one electron acceptor, at least one electron carrier, and at least one dihydroxy-dihydro PAH dehydrogenase to form a second reaction solution;
incubating the second reaction solution under conditions suitable for generating a detectable signal; and
measuring the detectable signal, thereby detecting the presence of PAH in the sample.
2. The method of claim 1 , wherein the at least one PAH dioxygenase comprises a recombinant cell lysate comprising naphthalene-1,2-dioxygenase from Pseudomonas putida.
3. The method of claim 2 , wherein the cell lysate further comprises at least one NAD(P)H deactivator.
4. The method of claim 1 , wherein the at least one dihydroxy-dihydro PAH dehydrogenase comprises 1,2-dihydroxy-1,2-dihydronapthalene dehydrogenase from Pseudomonas putida.
5. The method of claim 1 , wherein the at least one electron acceptor comprises resazurin and the at least one electron carrier comprises at least one diaphorase.
6. The method of claim 5 , wherein the at least one diaphorase comprises Clostridium kluyveri diaphorase.
7. The method of claim 1 , wherein the at least one electron acceptor comprises at least one tetrazolium salt and the at least one electron carrier comprises at least one derivative of phenazinium methyl sulfate.
8. The method of claim 6 , wherein the at least one derivative of phenazinium methyl sulfate comprises at least one of 1-methoxy-5-methylphenazinium methyl sulfate or phenazine methosulfate.
9. The method of claim 7 , wherein the at least one tetrazolium salt comprises thiazoyl blue tetrazolium bromide.