EP1463744A4 - Dispositifs et procedes pour isoler et detecter des substances specifiques dans des matrices complexes - Google Patents

Dispositifs et procedes pour isoler et detecter des substances specifiques dans des matrices complexes

Info

Publication number
EP1463744A4
EP1463744A4 EP02805656A EP02805656A EP1463744A4 EP 1463744 A4 EP1463744 A4 EP 1463744A4 EP 02805656 A EP02805656 A EP 02805656A EP 02805656 A EP02805656 A EP 02805656A EP 1463744 A4 EP1463744 A4 EP 1463744A4
Authority
EP
European Patent Office
Prior art keywords
membrane
analyte
sample
reagent
eluant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02805656A
Other languages
German (de)
English (en)
Other versions
EP1463744A2 (fr
Inventor
Virginia C Gordon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SAFETEST Inc
Original Assignee
SAFETEST Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SAFETEST Inc filed Critical SAFETEST Inc
Publication of EP1463744A2 publication Critical patent/EP1463744A2/fr
Publication of EP1463744A4 publication Critical patent/EP1463744A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food

Definitions

  • the present invention pertains generally to methods and apparatus for analytical chemistry, and more particularly to test kits and methods for qualitatively or quantitatively determining one or more analytes present within a sample or matrix.
  • test substance may contain many diverse physical and/or chemical species, some or all of which may interfere with the intended analysis.
  • sample preparation steps in order to isolate and/or concentrate the particular analyte(s) of interest, prior to actually proceeding with analytical determination of the desired analyte(s).
  • test substance is a solid material (e.g., food) it is often necessary to chop or grind the solid material into particles, and to extract the desired analyte(s) from such particles by adding one or more liquid digestants, solvents or other fluids to form a slurry or suspension, and thereafter performing a "clean up" of the slurry or suspension by filtration or centrifugation to separate the analyte containing liquid from the extraneous solid matter.
  • microbiological culture techniques are used to test for the presence of undesirable microbial contaminants in foods and other substances. These microbiological culture techniques often take several days to complete and are subject to human error. While PCR and other genetic techniques have been developed to quickly test for the presence of specific microbial DNA or RNA, the use of those techniques can be problematic when the suspected microbial contamination is contained within a food or other complex matrix.
  • the present invention provides methods and systems (e.g., test kits) for qualitative and/or quantitative determination of one or more analytes present within a matrix that contains matter other than the analyte (e.g., solids, particulate matter, matter or substances that will interfere with the analysis, etc.).
  • These methods and apparatus are useable to detect or quantify specific analytes present in complex matrices such as foods, cosmetics or biologicals, organ/tissue homogenates, industrial waste, sewage, industrial fluids, microbiological or pharmaceutical incubator slurries, etc. to determine the quality, degradation, age, abuse, contamination, nutritional value, purity and other characteristics of the matrices.
  • a method and system for determining the presence of a single analyte.
  • This system comprises; a) a sample receiving vessel, b) a membrane and c) a reagent-containing well.
  • the test sample is initially prepared (e.g., chopped or ground if a solid) and is deposited in the sample-receiving vessel along with any desired diluent, digestion solution (e.g., enzymes), chelators, or chemical modifiers (e.g., antioxidants).
  • digestion solution e.g., enzymes
  • chelators e.g., chelators
  • chemical modifiers e.g., antioxidants
  • the type of membrane utilized in each embodiment will be selected based on the type and quantity of matter which is desired to be excluded from the prepared sample matter prior to analysis.
  • this initial membrane will be formed of microporous film having pores which are sized to present large particles of solid matter, proteins and other unwanted matter from passing therethrough, but which will allow a filtrate containing the desired analyte to drain into the reagent-containing well.
  • the analyte contained within the filtrate will react with the reagent in a manner which will permit the presence or quantity of analyte to be determined.
  • the analyte-reagent reaction will be a color forming reaction such that a visual determination may be made as to whether, or to what degree the desired analyte is present.
  • a method or system of the above- described character may be adapted for determination of two or more analytes by the addition of one or more additional membranes in series with the first membrane.
  • Each of these additional membranes is operative to capture and hold at least one analyte, while allowing a filtrate containing one or more other analyte(s) to pass therethrough.
  • Each of these additional membranes may subsequently be exposed to a wash or flush solution such that one or more eluants containing each of the additional analytes may be obtained.
  • Each such eluant may subsequently be combined with a reagent to provide an eluant-reagent admixture from which at least one analyte may be determined.
  • the present invention is adaptable for the qualitative or quantitative determination of two or more analytes from a single sample.
  • the analyte in situations where one or more analytes is/are present in a matrix at low concentrations (e.g., concentrations that are below the detection limit of the intended analytical test) the analyte may be captured on a membrane and may be subsequently eluted from that membrane with a volume of eluent that is substantially smaller than the volume of the original sample, thereby providing an analyte/eluant admixture wherein the concentration of the analyte is sufficiently high to permit its detection by the intended analytical method.
  • the starting concentration of the analyte in the original sample may then be determined by calculation based on the known volume of the original sample and the known volume of the eluant that was used to elute the analyte from the membrane.
  • a membrane is used to remove a positive or negative interferant from the sample to permit an analyte to be analyzed or detected by chemical or biochemical methods without interference.
  • an analyte is removed comprises a method and system wherein free fatty acids (FFA) are present in a sample (e.g., a food or oil) along with one or more inorganic acids.
  • FFA free fatty acids
  • the analytical method intended to be used to detect or to quantitate the presence of FFA will also detect the presence of inorganic acids. Therefore it is desired to remove the inorganic acid(s) from the sample prior to analysis for the FFA.
  • the sample is passed through at least one negatively charged membrane that captures inorganic acids but allows a filtrate containg any FFA's present in the sample to pass therethrough.
  • the FFA containing filtrate is then subjected to the analytical test for FFA's and an accurate quantitative or qualitative determination of FFA's is then obtained.
  • an eluant that releases the inorganic acid from the negatively charged membrane is used to elute the inorganic acid from the membrane on which it was captured, thereby providing an inorganic acid/eluant admixture from which the inorganic acid may be quantitatively or qualitatively analyzed.
  • the sample may be passed through a plurality of membranes, each of which has a binding affinity for a different type of inorganic acid, before the filtrate is analyzed for FFA.
  • a first membrane may be impregnated or coated with a substance which carries a sufficient negative charge to bind weak inorganic acids (e.g., acetic acid) and a second membrane may be impregnated or coated with a substance which carries a sufficient negative charge to bind stronger inorganic acids (e.g., citric acid).
  • the weak and strong inorganic acids that become bound to these membranes may then be separately eluted and analyzed, if desired.
  • the sample may be passed through an anionic membrane which will bind and hold metals present in the sample and the desired enzymatic analysis may then be performed on the metal free filtrate without interference from the previously present metals.
  • a membrane e.g., a membrane that is impregnated or coated with specific antibodies
  • the particular amino acid sequence may be selected on the basis of its known presence in the nucleic acid (e.g., DNA or RNA) of a particular organism or microbe (e.g., bacteria, virus, parasite, spore, prion, etc.), a genetically modified substance or a protein, that may be present in the sample.
  • the bound nucleic acid(s), genetically modified substance(s) or protein(s) are then eluted or released from the membrane and subjected to an analytical or detection technique, such as amplification and PCR, whereby a quantitative or qualitative determination of that nucleic acid or protein is made.
  • an analytical or detection technique such as amplification and PCR
  • This aspect of the invention is useable to determine the presence or concentration of certain pathogenic or deleterious microbes, toxic or deleterious proteins, or the presence of a prohibited or regulated substance (e.g., genetically modified plant substances or grain) in a food, beverage, water, medicine, cosmetic or other sample.
  • a sample is passed through a pre-weighed membrane which has a selective affinity to bind a certain substance.
  • the membrane with the substance bound thereto is then reweighed to determine the weight of the substance that was present in the sample.
  • a food or beverage sample may be passed through a membrane that has a specific binding affinity for proteins. Thereafter the membrane (with the protein bound thereto) may be weighed and the weight of the protein removed from the sample may be calculated. On this basis, one may also calculate the % protein present in the sample. Alternatively, the protein may be eluded from the membrane and analyzed as described herein.
  • Figure 1 is a schematic diagram of a single membrane device useable with some of the methods and systems of the present invention.
  • Figure 2 is a schematic diagram of a plural membrane useable with some of the methods and systems of the present invention.
  • Figure 3 is a table listing specific filtration and capture membranes that may be used in the present invention.
  • Figure 4 is a table listing specific detection reagents that may be used for detection or analysis of analytes in the present invention.
  • Figure 5 is a table listing specific test methods and systems and specifying the analytes, typical matricies in which the analyte is contained, specific membranes (cross-referenced to Figure 3) and the specific detection reagents (cross-referenced to Figure 4) useable in each test method and system.
  • the present invention includes a number of specific methods and systems (e.g., combinations of membranes, eluants and reagents; test kits) that may be used to obtain quantitative or qualitative determinations of specific analytes in foods, oils and other matrices.
  • the methods and systems may be used in conjunction with the devices described in copending United States Patent Application Serial No. 09/183,157 and previously issued United Stated Patent Nos. 5,958,714 and 6,489,123, the entireties of which are expressly incorporated herein by reference. Certain embodiments of these devices are commercially available as the SaftestTM Membrane Unit and the SaftestTM Filtration Unit from Saftest, Inc., 3550
  • Figures 1 and 2 show, in schematic fashion, examples of devices used in conjunction with the methods and systems of this invention.
  • Figure 1 shows a single membrane device 10.
  • This single membrane device 10 comprises a sample well 12, a membrane support 15, and a filtrate collection well 16.
  • the sample 18 comprises matrix that includes a liquid phase wherein the analyte as well as extraneous matter (e.g., solid particles or large molecular weight compounds)
  • a filtration membrane 13 having pores that are small enough to prevent passage therethrough of the extraneous matter but large enough to permit passage therethrough of the analyte-containg liquid phase is positioned on the membrane support.
  • the sample 18 then passes from the sample well 12 and through the filtration membrane 13, whereby the extraneous matter is retained above the membrane and a filtrate 16 containing the analyte passes through the filtration membrane 13 and into the filtrate collection well 16.
  • a desired analytical or detection technique may then be used to quantitatively or qualitatively determine the analyte in the filtrate 20. In some instances, such analysis will require one or more reagents to be mixed with the analyte-containing filtrate 20.
  • the neat filtrate 20 may be used for the analysis (e.g., examined microscopically, placed in an analytical instrument such as a spectrophotometer or chromatograph or applied to an indicator (e.g., pH paper, paper or dip sticks which indicate the presence of the analyte, etc.)
  • the sample18 may be substantially free of extraneous matter that must be removed by a filtration membrane 13 (e.g., a clean oil or liquid solution) but, instead, the sample 18 may contain two analytes that must be separated or some interferant that will interfere with analysis for the analyte and must therefore be separated from the analyte prior to analysis.
  • a capture membrane 14 will be mounted on the membrane support rather than a filtration membrane 13.
  • This capture membrane 14 may be selected so as to capture (e.g., chemically bond to or otherwise hold) a first analyte while allowing a second analyte to pass therethrough in the filtrate 20.
  • the first analyte may subsequently be eluted (e.g., released) from the capture membrane and determined separately and the first analyte contained in the filtrate 20 may also be determined.
  • the capture membrane may also be used to capture an interferant while allowing a filtrate containg the analyte to pass therethrough or vice versa.
  • FIG. 2 shows, in schematic fashion, a two membrane device .
  • the top membrane is either a filtration membrane 13 (for samples 18 which contain extraneous matter that must be filtered out) or a capture membrane 14 (for samples that contain multiple analytes or interferants).
  • the bottom membrane is a capture membrane 14.
  • the sample 18 passes through the top membrane which removes extraneous matter or captures a first analyte or interferant.
  • the filtrate that has passed through the top membrane then passes through the bottom membrane which captures an analyte or interferant and the filtrate 20 that has passed through both membranes then collects in the filtrate well 20.
  • An analyte contained in the final filtrate 20 may be determined as described above.
  • analyte(s) may be eluted from the membrane(s) and determined separately.
  • FIG 2 shows that the bottom capture membrane 14 is transferred to a second membrane support 15a.
  • An eluant 22 is then passed through the capture membrane 14 so as to elute (e.g., release) the analyte from that membrane 14.
  • An eluant/analyte admixture 24 is then collected in a collection well 26.
  • the second analyte may then be quantitatively or qualitatively determined from the eluant/analyte admixture.
  • the membrane may contain an indicator that changes to indicate the presence of the analyte thereon or the membrane may be weighed to determine the weight of the analyte contained thereon.
  • membranes 13, 14 may be used to capture and optionally analyze virtually any number of analytes or inerferants.
  • a method and system for citric acid and free fatty acid determinations in any sample for example, an oil.
  • the sample e.g., oil
  • the sample is passed through a positively charged anionic membrane for capture of the citric acid from the oil and detection of free fatty acids in the filtrate.
  • the citric acid that has become bound to the positively charged anionic membrane is then eluted or released from the membrane using a high salt solution (e.g., 0.5 M NaCI in water) as the eluant.
  • the eluant/citric acid admixture is then combined with sulfanilic acid hydrochloride with a nitrite activator (e.g., 0.2% sulfanilic acid and 5% sodium nitrite).
  • this citric acid/free fatty acid system can be used for determinations in various other matrices including food.
  • the food may be soluablized such that the lipids are dissolved in a liquid phase.
  • a first membrane may be used to remove solid extraneous matter.
  • the liquid, lipid-containing filtrate is then passed through the capture membrane such that the citric acid becomes bound to the capture membrane.
  • the free fatty acids are then measured in the filtrate that passes through the capture membrane.
  • the citric acid is then released from the capture membrane by elution with a salt solution as described above.
  • the eluant/citric acid admixture may then be contained in a second vessel and the presence and/or amount of citric acid may be analyzed as described above.
  • a method and system for determining acetic acid and free fatty acid can be used for determinations in other food matrices and encapsulated lipids in foods where the food is solubilized.
  • a first filtration membrane is used to remove particles and other solid matter.
  • the acetic acid containg bound to the capture membrane, the free fatty acids measured in the effluent and then the acetic acid released from the capture membrane with high salt solution into a second vessel and quantitated.
  • Test kit for alkenal acid determinations in oil A first filtration membrane and oil in food using a particulate removing filtration membrane and then a methyl indole or methylphenyl indole detection system with a very strong acid such as methane sulfonic acid.
  • kits s to be used in conjunction with a second test for malonaldehyde utilizing a methyl indole reagent with weak acid such as small amount of HCI 6.
  • kits to be used with a second test for lipid peroxides in the eluant using an iron catalyzed electron transfer to xylenol orange 7.
  • a test kit for protein determination on filtered and unfiltered oils in conjunction with the citric acid determination by using one membrane to bind citric acid and one to bind protein and eluting each membrane separately and detecting the analyte.
  • test kit for protein determination on refined oil concentrating the protein on a protein binding membrane by passing 1 to 200ml of oil through the membrane and eluting the protein off into another tube using a salt solution in 1ml.
  • a test kit for protein determination in meals by digesting the meal with phosphoric or another strong acid and using a particulate removing membrane to remove debris and then testing the filtrate for protein.
  • a test kit for protein determination on tallows or greases by using a membrane to bind protein and eluting the membrane and detecting the analyte.
  • a test method and kit to determine oxidation of beverages and determination efficacy of certain additives and/or stabilizers on oxidation using the alkenal test The beverage, carbonated or not, is separated through protein binding membrane and the filtrate tested with methylindolis solution with sulfonic acid.
  • a test for rapid determination of the quality of cooking oils and fats by testing for lipid peroxides using a peroxidase and iron catalyzed reagent and complexed to xylenol orange and alkenals using the methylindole reagent with a very strong acid added This test can be used on beer or beverages and predict quality and shelf life of beverages.
  • a test method and kit to detect specific microbes or viruses in foods or tissues by emulsifying the food and releasing the nucleic acids using surfactants or osmotic changes to lyse the membranes and cells and using a particulate binding membrane followed by a nucleic acid binding membrane. The DNA is released and then amplification of a sequence specific to the target organism to detect its presence performed.
  • a test method and kit to detect afiatoxins in foods or tissues by emulsifying the food and releasing the afiatoxins using surfactants or osmotic changes to lyse the membranes and cells and after filtering out particulates using a second membrane coated with an antibody specific to multiple or particular afiatoxins.
  • the afiatoxins are released and then detected using peroxidase conjugated antibodies
  • a test method and kit to detect specific live microbes or viruses in foods or tissues by emulsifying the food and releasing the nucleic acids using surfactants or osmotic changes to lyse the membranes and cells and using a particulate binding membrane followed by a ribonucleic acid binding membrane. The RNA is released and then amplification of a sequence specific to the target organism to detect its presence performed.
  • the following examples demonstrate methods of detecting various analytes contained in samples, in accordance with the invention disclosed hereinabove.
  • the analytes may be removed from a sample using a device or system incorporating one or more membranes for filtering the sample, such as devices and systems disclosed in commonly owned PCT International Patent Publication No. WO 99/20396 and U.S. Patent No. 6,489,132, and the publicly available SafTestTM Filtration Unit available from Saftest, Inc. (Phoenix, AZ).
  • PCT International Patent Publication No. WO 99/20396 and U.S. Patent No. 6,489,132 are expressly incorporated herein by reference.
  • This example demonstrates free fatty acids contained in an oil sample.
  • the oil sample also contains citric acid. It is desirable to separate the citric acid from the sample prior to assay of the FFA content as the presence of inorganic acids such as citric
  • a 1 ml_ sample of soybean oil is applied to a membrane of a filtering device.
  • the membrane is a strongly basic anionic membrane, such as the Q membrane adsorber membrane with quaternary ammonium groups (Q-MA membrane) publicly available from Sartohus (Sartohus North America, Inc., Edgewood, NY).
  • Q-MA membrane quaternary ammonium groups
  • the membrane containing the citric acid is removed from the container and is washed with 1 ml_ of 0.5 M NaCI in water.
  • the eluant is collected in a second container.
  • One ml_ of the eluant containing citric acid is mixed with 0.3 mL of a reagent containing 0.2% sulfanilic acid and 5% sodium nitrite.
  • the reaction occurs for about 30 minutes an elevated temperature (approximately 42-45 °C).
  • the presence of citric acid in the sample results in a yellow color which can be measured by examining the reaction mixture with a spectrometer at
  • a test kit suitable for performing this citric acid assay is commercially available under the name CitriSafeTM from Saftest, Inc. (Phoenix, AZ). The CitriSafeTM test kit is generally described in Appendix A to this patent application.
  • the amount of free fatty acids originally present in the soybean oil is determined by measuring the acidity of the oil after the removal of the citric acid using the methodology described in incorporated parent application Serial No. 09/183,157 and commercially available as a test kit under the name FASafeTM from Saftest, Inc. (Phoenix, AZ).
  • a 5 gram sample of mackerel is solubilized to create a slurry.
  • the slurry is heated to approximately 40-45 °C and filtered to remove particulates from the slurry.
  • Two (2) mL of the filtered slurry is applied to a membrane structure of a filtering device.
  • the membrane structure includes two stacked membranes one disposed on top of the other.
  • the upper membrane is a weakly basic membrane, such as the D membrane adsorber with diethylamine groups (the MA-D membrane), and the lower membrane is a strongly basic membrane, such as the membrane used in Example 1.
  • These membranes are publicly available from Sartohus (Sartorius North America, Inc., Edgewood, NY).
  • acetic acid, and other weak inorganic acids are retained by the upper membrane, and citric acid, and other strong inorganic acids are retained in the lower membrane.
  • the remaining slurry containing free fatty acids is collected in a container.
  • the membrane containing the acetic acid is removed from the container and is washed with 2 mL of 1 M NaCI in water.
  • the eluant is collected in a second container. 100 ⁇ l of the eluant containing acetic acid is mixed with 1.0 mL of a reagent containing 0.1 % xylenol orange in neutralized isopropanol.
  • the reaction occurs for about 10 minutes at an elevated temperature (approximately 42-45 °C).
  • the presence of acetic acid in the sample is determined by examining the reaction mixture with a spectrometer at 570 nm.
  • the amount of acetic acid present in the sample is determined by comparing the results to one or more standards.
  • the citric acid is removed from the lower membrane using the procedure disclosed in Example 1.
  • the amount of free fatty acids originally present in the fish slurry is determined by measuring the acidity of the oil after the removal of the inorganic acids using the FASafeTM publicly available from Saftest, Inc. (Phoenix, AZ).
  • the FASafeTM test kit is useable in conjunction with devices described in copending United States Patent Application Serial No. 09/183,157 and previously issued United Stated Patent Nos. 5,958,714 and 6,489,123, the entireties of which are expressly incorporated herein by reference. Certain embodiments of these devices are commercially available as the SaftestTM Membrane Unit and the SaftestTM Filtration Unit or SaftestTM Work Station, from Saftest, Inc.
  • This example demonstrates the determination of fat content or the percent of fat in foods.
  • the homogenate is prefiltered to remove particulates using a cellulose acetate membrane having a pore size of 0.45 microns.
  • the filtered homogenate is passed through a membrane that binds proteins, such as the polyethersulfone (PES) membrane sold by Sartohus, Inc, and then the filtered homogenate is passed through a membrane that binds surfactants, such as the MA-Q or MA-S membranes from Sartorius.
  • the MA-S membrane has sulfonyl groups on the membrane surface for binding surfactants.
  • a portion of the filtrate (20 ⁇ L) that is free of proteins and surfactants is mixed with 1.0 mL of lipase (Sigma, St. Louis, MO) in phosphate buffer to enzymatically cleave the fatty acids from glycerol.
  • the amount of glycerol present in the filtrate is measured enzymatically using a series of enzyme reactions using glycerol kinase and ATP to produce glycerol 1 -phosphate and glycerol-1 phosphatase to produce dihydroxyacetone, which is detected with a peroxidase catalyzed reaction with aminoantipyrine to produce a measurable quinoneimine dye. This reaction is complete in 10 minutes at 42 °C.
  • a test kit for this percent fat assay is commercially available as Percent Fat Kit MSA from Saftest, Inc. (Phoenix, AZ) and is described in Appendix B to this patent application.
  • the Percent Fat Kit MSA is useable in conjunction with devices described in copending United States Patent Application Serial No. 09/183,157 and previously issued United Stated Patent Nos. 5,958,714 and 6,489,123, the entireties of which are expressly incorporated herein by reference. Certain embodiments of these devices are commercially available as the SaftestTM Membrane Unit and the SaftestTM Filtration Unit or SaftestTM Work Station, from Saftest, Inc.
  • the present invention is used to determine total protein content in a refined oil such as soy bean oil.
  • a 5 mL sample of refined and genetically modified soy bean oil is heated to approximately 40 °C and is mixed with 5 mL of 100% isopropanol.
  • the warm mixture is applied to a membrane that binds proteins, such as the membrane used in Example 1.
  • the protein in the oil/alcohol mixture binds to the membrane, and the fatty acids contained in the mixture pass into a container.
  • the protein-containing membrane is moved to another container and is washed to release the protein into the container with 1 mL of buffered, low salt solution (0.05 M NaCI in phosphate buffer at a pH between 7 and 9).
  • buffered, low salt solution 0.05 M NaCI in phosphate buffer at a pH between 7 and 9.
  • One (1) mL of the concentrated filtrate is mixed with
  • the amount of protein is quantified by comparing the blue color of the mixture to one or more standards, and/or by using a spectrometer at 570nm.
  • test kit for this protein content assay is commercially available as ProteSafeTM from Saftest, Inc. (Phoenix, AZ) and is described in
  • the ProteSafeTM test kit is useable in conjunction with devices described in copending United States Patent Application Serial No. 09/183,157 and previously issued United Stated Patent Nos. 5,958,714 and 6,489,123, the entireties of which are expressly incorporated herein by reference. Certain embodiments of these devices are commercially available as the SaftestTM Membrane Unit and the SaftestTM Filtration Unit or SaftestTM
  • This example demonstrates methods to identify the presence of one or more microbes, including pathogenic and non-pathogenic bacteria and viruses, in food products.
  • the microbes are detected by binding nucleic acids to one or more membranes, and amplifying the nucleic acids using nucleic acid primers having a desired nucleotide sequence for the microbes.
  • ground beef Ten grams of ground beef is prepared for determination of the presence of ecoli H157.
  • the ground beef is homogenized with a buffered solution, such as phosphate buffer, containing 1-2% sodium dodecyl sulfate (SDS) in a ratio of approximately 1 to 4, of beef to diluent, to disrupt the cellular component and to release nucleic acids contained within the beef.
  • SDS sodium dodecyl sulfate
  • the slurry of homogenized ground beef is applied to a first membrane, such as a polytetraflouroethylene membrane (available from Sartorius), to remove particulates from the ground beef.
  • the filtered slurry is then applied to a second membrane that is configured to bind DNA or RNA.
  • Anionic membranes such as
  • MA-Q membranes from Sartorius or membranes having one or more types of nucleic acid binding antibodies, such as MA-A membranes (Sartorius), which has crosslinked antibodies attached to it by glutaraldehyde crosslinking, or the MA-I Iminodiacetic acid membranes (Sartorius), which are reacted with the protein amino groups of the antibodies.
  • the slurry is passed through two additional nucleic acid binding membranes to increase the amount of nucleic acid removed from the homogenate. The filtrate is then discarded.
  • RNA and DNA are then amplified using polymerase chain reaction (PCR) and one or more nucleic acid primers that have sequences for ecoli H157.
  • PCR methods are conventionally known to persons of ordinary skill in the art, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd Edition, 2001.
  • the PCR products are labeled by incorporating a fluorescent marker during the amplification steps, and the presence of ecoli H157 is determined by measuring the fluorescence contained in the PCR products.
  • CitriSafeTM STD assay measures the citric acid concentration in samples as parts per million (ppm).
  • the SarTesf 1 * Analyzer will report results as parts per million (ppm) citric acid in the sample.
  • the dilution factor must be taken into account. Multiply the instrument results by the dilution factor to obtain the value of the sample.
  • Ranges for the controls are found on the package insert provided in the Control Kit. Assay values for the controls should approximate these ranges. However, it is advised that each lab establish control ranges using their own equipment and personnel.
  • the Percent Fat assay is intended to determine the amount ol lipid or (at in a sample.
  • the Percent Fat assay measures the (at level in samples using e ⁇ zymalic hydrolysis ol triglycendes to glycerol. followed by an e ⁇ zymaticcolonmetric measurement ol the glycerol released.
  • the t ⁇ glyce ⁇ de concentration is qua ⁇ litated in grams (g) per 100 grams ol sample.
  • Kit Contents The Percent Fat kit comes packaged with all the reagents and membrane units needed to run the assay.
  • the kit contains Reagent A. one Calibrator Se ⁇ es. and one Control. Each kit comes with the appropriate number ol disposable membrane holders and membrane units. The kit should be stored at 4*C.
  • the Percent Fat reagent will stain clothing and equipment. When used as directed, the Percent Far reagents, controls, and calibrators should present no hazard to the user. As a normal laboratory precaution, avoid contact with eyes and skin. Do not oioel by mouth.
  • Sample preparation will van/ based on the particular atnx material being tested. Prepare and/or dilute the sample appropriately, according to the sample preparation section in the SatTett"' Quick Stan Card.
  • Test Method t Dispense one aliquot of Percent Fat Reagent A into each of the calibrator tubes, the reagent blank, the control tube, and each of the sample tubes.
  • control value falls within the range, insert the sample tubes in the designated order to analyze in the SafTest"' Analyzer. Wipe each test tube with a knt-free tissue p ⁇ or to insertion in the SafTest'" Analyzer.
  • the SafTest"' Analyzer wiD use the calibrators to calculate the tngtycerid ⁇ concentration in grams (g) of t ⁇ gryce ⁇ d ⁇ per 100 grams of oil.
  • sample value is greater than Ih ⁇ value of the highest calibrator, the instrument will Hag the result as HI.' The sample must be diluted al a higher dilution and retested. Values that are Nagged 'HI* are inaccurate and should not be reported.
  • the range for the control is found on the package insert provided with the Percent Fat Oil Control
  • the assay value for the control should approximate this range.
  • the control result should be multiplied by 400 to gel the result. However, it is advised that each lab establish a control range using its own equipment and personnel
  • the Safety Associates, Inc. Percent Fat Test Kit measures the fat level in samples using an enzymatic hydrolysis of triglycendes to glycerol, followed by an enzymatic/colorimetric measurement of the glycerol released. The triglyceride concentration is quantitated in grams (g) per 100 grams of sample.
  • control values fall within the ranges, insert the sample tubes and analyze in the SafTestTM Analyzer in the designated order (see SafTestTM Analyzer Instructions for detailed instructions). Wipe each test tube with a lint-free tissue prior to insertion in the SafTestTM Analyzer. Multiply your result by the dilution to get the % fat.
  • the SafTestTM Analyzer will use the calibrators to calculate the Percent Fat content as percent fat in the sample.
  • Ranges for the Control are found on the package insert provided in the Control Kit. Assay values for the controls should approximate these ranges. However, it is advised that each lab establish control ranges using their own equipment and personnel.
  • the Percent Fat assay is intended to determine the amount of lipid or rat in a sample
  • the Percent Fat assay measures Ihe fat level in samples using enzymatic hydrolysis of triglycendes to glycerol, followed by an enzymaUc colonmet ⁇ c measurement of the glycerol released
  • the tnglyceride concentration is quanlitated m grams (g) per 100 grams of sample.
  • Kit Contents The Percent Fat kit comes packaged with all Ihe reagents and membrane units needed to run the assay.
  • the kit contains Reagent A. one Calibrator Se ⁇ es, and one Control Each kit comes with the appropnate number of disposable membrane holders and membrane units The kit should be stored at 4*C
  • Sample preparation will vary based on the particular matrix material being tested. Prepare and/or dilute Ihe sample appropriately, according to the sample preparation section in the SafTestTM Quick Start Card
  • control tube Following successful calibration, insert Ihe control tube. The control should be run every time the calibrators are run. To ensure the instrument and reagents are performing properly, the control value should fall within Ihe range stated on Ihe package insert for the lot of controls used (see Package Insert - Control for Percent Fat Kits). If the control falls significantly outside the range, rerun the assay
  • control value falls within the range, insert the sample tubes in the designated order to analyze in the SafTest'" Analyzer. Wipe each test tube with a Imt-free tissue prior to insertion in the SafTesf"* Analyzer. ⁇ . At the end of the day, store calibrators, control, and reagent bottles with dispensers attached at room temperature (2-6'C).
  • the SafTest" Analyzer will use the calibrators to calculate Ihe tnglyceride concentration in grams (g) of triglyce ⁇ de per 100 grams of oil.
  • the range for the control is found on the package insert provided with Ihe Percent Fat Oil Control.
  • the assay value for the control should approximate this range.
  • the control result should be multiplied by 400 to get the result However, it is advised that each lab establish a control range using its own equipment and personnel.
  • New food matrices with no established protocols are considered special applications and should be checked for interference and spike recovery Refer to Ihe Sample Preparation table found in Ihe S_fTe-l m Binder.
  • Step l Label a (jlass test tube lor each sample and place the tubes inside Ihe acrylic base on the S ⁇ tTestTM Filtration Unit
  • Dispenser Volume is set to 1.0 ml.
  • the Safety Associates, Inc. ProteSafeTM STD assay measures the protein concentration in oil samples as milligram per deciliter (mg/dl) or ppm (10ppm is 1 mg/dL)
  • the SafTestTM Analyzer will report results as milligram per deciliter (mg/dl) of protein in the sample. This can be converted to ppm by multiplying by 10.
  • the dilution factor must be taken into account. Multiply the instrument results by the dilution factor to obtain the value of the sample. 3. If the sample value is greater than the value of the highest calibrator ⁇ the instrument will flag the results as "HI.” The sample must be diluted to a higher dilution and retested.
  • Ranges for the controls are found on the package insert provided in the Control Kit. Assay values for the controls should approximate these ranges. However, it is advised that each lab establish control ranges using their own equipment and personnel.
  • the Safety Associates, Inc. ProteSafe M assay is a rapid method that measures the protein content in an oil, tallow or grease by determining protein content captured on a chemically modified membrane which is then washed to release and concentrate the protein for analysis. Samples should be kept at 40"C when sampling, placing on the membrane and aliquoting the filtrate.
  • Reagents and calibrators and controls are stored cold until used. The first day of use allow reagents to stand at room temperature (18-25°C)for 30-40 minutes before beginning assay. Once brought to room temperature leave at room temperature until all reagents are used except for calibrators and controls which are kept in the refrigerator.
  • Dispenser volume is adjustable. To prepare a 12ml sample, set the dispenser at 2ml and use three aliquots. Combine with 6ml of the oil. Warm and vortex well.
  • Elution Buffer is added to the membrane in two SOO ⁇ l aliquots using a 1ml pipetter.
  • control value is within the expected range, insert the sample tubes and analyze in the SafTestTM Analyzer in the designated order (see Section 4 for detailed instructions). Wipe each test tube with a lint-free tissue prior to insertion in the SafTesfTM 1 Analyzer.
  • the SafTesfTM Analyzer will use the calibrators to determine the protein concentration of the control and samples.
  • Range for the Control is found on the package insert provided in the Control Kit. Assay values for the control should be within 10% of these values. However, it is advised that each lab establish control ranges using their own equipment and personnel.
  • the Percent Seasoning assay is intended to determine the amount of seasoning on a chip.
  • the Percent Seasoning assay measures the protein level n seasoning on chips using a microprot ⁇ in assay with cobjrmetnc endpoints.
  • the protem concentration is qua ⁇ titated in milligrams (mgj per ol and converted to % seasoning by comparison to a 6% equivalent seasoning control.
  • Kit Contents The Percent Seasoning kit comes packaged with all the reagents and membrane units needed to run the assay.
  • the kit conlains Reagent A.
  • Each kit comes with the appropriate number of disposable membrane holders and membrane units.
  • the kit should be stored al 4*C.unl ⁇ l used and left out after first use for up to one month at room temperature.
  • Preparation Reagent Salts mto 5Ute ⁇ ol distilled water.
  • Sample preparation will vary based on Ihe particular seasoning being tested. Prepare andVor dilute the chip sample appropriately, according to the sample preparation section in the SafTest"' Quick Start Card.
  • Test Method 1. For Ih ⁇ first assay of the day turn on the SafTesf" Analyzer for 5 minutes Place the 570/S90 filter in Ihe filter compartment Select the "SEA FL" program.
  • control tube Following successful calibration, insert the control tube.
  • the control should be run every time the samples are run. To ensure the instrument and reagents are performing properly, the control value should fall within the range staled on the package insert for the lot of controls used (see Package Insert - Control for Percent Seasoning Kits). II the control fans ignificantly outside the range, rerun the assay.
  • the SafTest"* Analyzer will use the calibrators to calculate Ihe protein concentration in mg/dL of controls and the solubilized chip.
  • the range for the control is found on the package insert provided with the Percent Seasoning Control.
  • the assay value lor the control should approximate this range.

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Abstract

Procédés et systèmes pour déterminer la présence d'un ou plusieurs analytes dans divers types de matrices tels qu'aliments, huiles, fluides corporels, etc. On utilise des membranes pour évacuer des matières étrangères telles que des particules solides et/ou pour lier des interférants ou analytes spécifiques. Des dosages spécifiques servent à déterminer l'acide citrique, les lipides totaux (ou le % de graisse), les acides gras libres, la teneur en protéines ou en acides spécifiques indiquant la présence d'ADN ou d'ARN de certains microbes (tels que les pathogènes) ou de substances génétiquement modifiées (telles que les graines obtenues par génie génétique).
EP02805656A 2001-12-20 2002-12-20 Dispositifs et procedes pour isoler et detecter des substances specifiques dans des matrices complexes Withdrawn EP1463744A4 (fr)

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JP2006153488A (ja) * 2004-11-25 2006-06-15 Canon Chemicals Inc 光学的測定法
EP2024742B1 (fr) * 2006-05-22 2020-03-04 3M Innovative Properties Company Système et procédé de préparation d'échantillons
FR3109585A1 (fr) 2020-04-28 2021-10-29 Withings Plaquette de test et système de test biologique automatisé
FR3126046A1 (fr) 2021-08-04 2023-02-10 Withings Support de test biologique amélioré

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2649411A1 (fr) * 1989-07-07 1991-01-11 Biosem Determination quantitative et identification de bacteries
EP0605003A2 (fr) * 1992-12-31 1994-07-06 Vicam, L.P. Méthode d'essai pour détecter la présence de bactéries
EP0617284A2 (fr) * 1993-03-18 1994-09-28 Mochida Pharmaceutical Co., Ltd. Appareil et procédé pour mesure simplifiée
US5438128A (en) * 1992-02-07 1995-08-01 Millipore Corporation Method for rapid purifiction of nucleic acids using layered ion-exchange membranes
JPH10290691A (ja) * 1997-02-19 1998-11-04 Dainippon Printing Co Ltd 遺伝子増幅方法および装置
WO1999020396A1 (fr) * 1997-10-22 1999-04-29 Safety Associates, Inc. Procedes et appareils pour determiner les analysats dans differentes matrices
WO2002099139A1 (fr) * 2001-06-04 2002-12-12 Cuno, Incorporated Membrane microporeuse modifiee pour capture d'acide nucleique non specifique, et specifique d'une sequence, et procedes d'utilisation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591580A (en) * 1994-03-31 1997-01-07 Johnson & Johnson Clinical Diagnostics, Inc. Method, test element and test kit for semi-quantitative detection of target nucleic acid
US5958714A (en) * 1996-10-02 1999-09-28 Safety Associates, Inc. Test kits for determining at least two specific analytes in foods and other complex matrices
EP1204859B1 (fr) * 1999-07-16 2006-11-22 The Board Of Regents, The University Of Texas System Methode et appareil de distribution d'echantillons a un reseau de capteurs chimiques

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2649411A1 (fr) * 1989-07-07 1991-01-11 Biosem Determination quantitative et identification de bacteries
US5438128A (en) * 1992-02-07 1995-08-01 Millipore Corporation Method for rapid purifiction of nucleic acids using layered ion-exchange membranes
EP0605003A2 (fr) * 1992-12-31 1994-07-06 Vicam, L.P. Méthode d'essai pour détecter la présence de bactéries
EP0617284A2 (fr) * 1993-03-18 1994-09-28 Mochida Pharmaceutical Co., Ltd. Appareil et procédé pour mesure simplifiée
JPH10290691A (ja) * 1997-02-19 1998-11-04 Dainippon Printing Co Ltd 遺伝子増幅方法および装置
WO1999020396A1 (fr) * 1997-10-22 1999-04-29 Safety Associates, Inc. Procedes et appareils pour determiner les analysats dans differentes matrices
WO2002099139A1 (fr) * 2001-06-04 2002-12-12 Cuno, Incorporated Membrane microporeuse modifiee pour capture d'acide nucleique non specifique, et specifique d'une sequence, et procedes d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 1999, no. 02 26 February 1999 (1999-02-26) *

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AU2002365053A1 (en) 2003-07-09
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JP2005533239A (ja) 2005-11-04
WO2003054516A3 (fr) 2004-02-12

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