# US 2014/0154723 A1 · Rapid detection of petroleum in food > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154723 A1 - Document ID: US20140154723A1 - Status: Published application - Kind: A1 - Application number: 14/086,396 - Filed: 2013-11-21 - Published: 2014-06-05 - Earliest claimed: 2012-11-21 - Assignees: Bioo Scientific Corporation - Inventors: Joseph F. Krebs; John Duvall-Jisha; Sol Resnick - Web page: https://patentgrep.com/patent/US20140154723A1 ## CPC - C12Q 1/26: involving oxidoreductase - C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)} - G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Described herein is a rapid coupled enzymatic assay to detect polycyclic aromatic hydrocarbon (PAH) compounds in samples. The method uses a detection composition that includes: a buffered solution comprising NADH; naphthalene dioxygenase, naphthalene 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase, and 1,2-dihydroxynaphthalene dioxygenase. The presence of PAH is determined by noting a color change of the detection composition, either visually or spectrophotometrically. ## Claims ### Claim 1 A method of detecting polycyclic aromatic hydrocarbons comprising: adding a sample suspected of containing one or more polycyclic aromatic hydrocarbons to a detection composition, wherein the detection composition comprises: NADH; naphthalene dioxygenase; naphthalene 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase; and 1,2-dihydroxynaphthalene dioxygenase; determining if the color of the detection composition has changed due to the presence of polycyclic aromatic hydrocarbons. ### Claim 2 (depends on 1) The method of claim 1, wherein the sample is derived from food. ### Claim 3 (depends on 1) The method of claim 1, wherein the sample is obtained from an environment. ### Claim 4 (depends on 1) The method of claim 1, wherein the naphthalene dioxygenase is stabilized naphthalene dioxygenase. ### Claim 5 (depends on 4) The method of claim 4, wherein the stabilized naphthalene dioxygenase is produced by the method comprising: obtaining naphthalene dioxygenase from a bacterial source; and dialyzing the obtained naphthalene dioxygenase using dialysis tubing having a molecular weight cut off (MWCO) of less than or equal to about 7500. ### Claim 6 (depends on 5) The method of claim 5, wherein the dialysis is performed in the presence of dithiothreitol (DTT) and/or glycerol. ### Claim 7 (depends on 1) The method of claim 1, wherein the naphthalene 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase is desalted before use in the detection composition. ### Claim 8 (depends on 1) The method of claim 1, wherein the 1,2-dihydroxynaphthalene dioxygenase is desalted before use in the detection composition. ### Claim 9 (depends on 1) The method of claim 1, wherein a change in the color of the detection composition is determined by detecting a change in the specific absorbance of ultraviolet or visible light by the detection composition. ### Claim 10 (depends on 1) The method of claim 1, wherein a change in the color of the detection composition is determined by detecting a change in the specific absorbance at wavelengths of between about 360 nm to about 550 nm. ### Claim 11 A method of producing stabilized naphthalene dioxygenase comprising: obtaining a naphthalene dioxygenase fraction from lysed cells of a bacterial strain which can produce all four subunits of naphthalene dioxygenase; dialyzing the obtained naphthalene dioxygenase using dialysis tubing having a molecular weight cut off (MWCO) of less than or equal to about 7500. ### Claim 12 (depends on 11) The method of claim 11, wherein the bacterial strain is Pseudomonas putida. ### Claim 13 (depends on 11) The method of claim 11, wherein the naphthalene dioxygenase is produced recombinantly in active form from E. coli. ### Claim 14 (depends on 13) The method of claim 13, wherein the E. coli is strain JM109 (DE3) (PDTG141). ### Claim 15 (depends on 11) The method of claim 11, wherein obtaining the naphthalene dioxygenase fraction comprises: growing the bacterial strain; separating the grown cells from the growth medium; lysing the separated cells; and separating the naphthalene dioxygenase fraction from the insoluble fraction of the lysed separated cells. ### Claim 16 (depends on 15) The method of claim 15, wherein the naphthalene dioxygenase fraction obtained from the lysed separated cells is subjected to dialysis prior to any substantial storage or freezing of the naphthalene dioxygenase fraction. ### Claim 17 (depends on 11) The method of claim 11, wherein the dialysis is performed in the presence of dithiothreitol (DTT) and/or glycerol. ### Claim 18 (depends on 11) The method of claim 11, wherein the dialysis is performed in a buffered solution having a pH of about 8, at a temperature of less than 25° C. ### Claim 19 A detection composition for detecting the presence of polycyclic aromatic hydrocarbons in a sample, comprising: a buffered solution comprising NADH; naphthalene dioxygenase, naphthalene 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase, and 1,2-dihydroxynaphthalene dioxygenase. ### Claim at position 20 20-24. (canceled) ### Claim 25 A test kit to detect polycyclic aromatic hydrocarbons in a sample comprising: a buffered solution comprising NADH; naphthalene dioxygenase, naphthalene 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase, and 1,2-dihydroxynaphthalene dioxygenase.