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WO2009153074A1 - Bioodétecteur et procédé de détermination d'un poison, et utilisations du biodétecteur - Google Patents

Bioodétecteur et procédé de détermination d'un poison, et utilisations du biodétecteur Download PDF

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Publication number
WO2009153074A1
WO2009153074A1 PCT/EP2009/053861 EP2009053861W WO2009153074A1 WO 2009153074 A1 WO2009153074 A1 WO 2009153074A1 EP 2009053861 W EP2009053861 W EP 2009053861W WO 2009153074 A1 WO2009153074 A1 WO 2009153074A1
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WO
WIPO (PCT)
Prior art keywords
biosensor
sample
enzymes
enzyme
inhibitor
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.)
Ceased
Application number
PCT/EP2009/053861
Other languages
German (de)
English (en)
Inventor
Heike Barlag
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.)
Siemens AG
Siemens Corp
Original Assignee
Siemens AG
Siemens Corp
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 Siemens AG, Siemens Corp filed Critical Siemens AG
Publication of WO2009153074A1 publication Critical patent/WO2009153074A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C12Q1/46—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase involving cholinesterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/42—Poisoning, e.g. from bites or stings

Definitions

  • the present invention relates to a biosensor and a method for determining a poison in a sample. Also included are certain uses of the invention of the biosensor.
  • Toxic or toxic substances present an acute danger to humans and animals in drinking water, in food and in the air.
  • the test methods use living organisms such as fish, clams and microorganisms.
  • the test methods are based on the observation that the behavior of the whole organism changes with the addition of pollutants to the water (Brack et al., 1999).
  • Bacteria and algae also respond to pollutants by inhibiting their bioluminescence or photosynthetic activity.
  • Biological tests with living organisms are very complex and difficult to standardize due to physiological variations in the organism. In addition, the results of such tests are not clearly applicable to humans.
  • living organisms can be used to detect low concentrations of pollutants e.g. are often not used in the control of drinking water limit, because they do not respond sufficiently sensitive.
  • Chemical analytical techniques such as chromatographic techniques for the detection of toxic substances, are an alternative to tests with living organisms (Engst and Noske, 2006). However, they have proven to be very time-consuming and costly due to the large number of detectable substances. In addition, only known substances can be detected by these methods. In addition, the proof of Substances whose character and characteristics are only suspected, problematic.
  • Enzyme inhibitors are a class or group of toxic substances for humans. Their action on isolated enzymes makes them very well detectable in low concentrations.
  • the enzyme cyanidase is used, which decomposes cyanide into formic acid and ammonia. The degradation leads to a change in the pH, which can then be measured as an electrical potential difference (Keusgen et al., 2001).
  • An enzyme-based detection system also referred to as an enzyme inhibition assay, is independent of the substance, i. regardless of the chemical nature of the toxin. Thus, even unknown and unexpected toxins can be detected in a single detection method quickly, easily and with high sensitivity. By developing continuous flow systems, the detection of toxic substances by enzyme inhibition can be automated and highly efficient (Sole et al., 2003). A combination of different enzymes additionally increases the sensitivity of enzyme inhibition tests.
  • the present invention has the object, a new biosensor for determining the toxicity of a Propose a sample that can be used in a fully automatic system, low maintenance and easy to regenerate frequently, so that the sensor rarely needs to be replaced.
  • the biosensor should be addressable, with addressability meant the specific binding of individual enzymes to different sensors of the biosensor. By selecting different enzymes that respond to the same toxins, the sensor is calibrated to reflect the effect on humans.
  • a biosensor for determining a poison in a sample which comprises at least two enzymes which are arranged on the biosensor and which are brought into contact with a sample to be examined for the presence of at least one enzyme inhibitor, a measuring unit for measuring an enzyme inhibition caused by an interaction between the enzymes on the biosensor and at least one inhibitor in the sample, and an evaluation unit for evaluating the enzyme inhibition, the evaluation unit indicating a relationship between the measured enzyme inhibition and the toxicity of the sample to an organism and the enzymes are differently inhibited by the inhibitor in the sample at the same concentration.
  • the present invention relates to a method for determining a poison in a sample, comprising the steps of contacting the inventive biosensor with a sample to be tested, measuring enzyme inhibition caused by an interaction between the enzymes on the biosensor according to the invention and at least one Inhibitor in the sample, and evaluating the enzyme inhibition, wherein the evaluating indicates a relationship between the measured enzyme inhibition and the toxicity of the sample to an organism.
  • the invention relates to the use of the biosensor according to the invention for examining a sample for poison / e.
  • Figure 1 shows a sensor array with enzymes E from the different families of enzymes A, B, C to M.
  • Figure 2 shows the inhibition of esterase 1 as a function of the concentration of various inhibitors.
  • Figure 3 shows the inhibition of esterase 1 as a function of toxicity.
  • LD 50 value rat oral
  • Figure 4 shows the inhibition of esterase 2 as a function of toxicity with different sensitivities compared to esterase 1.
  • Figure 5 shows a biosensor response calculated from the measurements of esterases 1 and 2 with an approximately linear relationship between the amount of inhibitor at 50% inhibition and toxicity represented by LD50 (rat oral).
  • a first subject of the present invention is a biosensor for determining a poison in a sample comprising:
  • a measuring unit for measuring an enzyme inhibition caused by an interaction between the enzymes on the biosynthetic sensor and at least one inhibitor in the sample is caused
  • An evaluation unit for evaluating the enzyme inhibition wherein the evaluation unit indicates a relationship between the measured enzyme inhibition and the toxicity of the sample for an organism and the enzymes are inhibited to different degrees by the inhibitor in the sample at the same concentration.
  • enzymes with different sensitivity to the different substances of a group of inhibitors can be used.
  • the effect of inhibitors on individual enzymes may differ significantly from the toxicity of the substance to humans. Therefore, the signals of the different sensitive enzymes are weighted together.
  • Essential to the invention is that the biosensor indicates a relationship between all the enzymes used and the toxicity of the sample to the organism. Calibration measurements essentially do not establish the relationship between the concentration of inhibitors and the inhibitory effect, but between the toxicity of the inhibitors in the sample and the inhibitory effect. If the toxicity of a specific substance is not sufficiently known to humans, an animal model is used. Here, calibration to the LD 50 dose of the substance to the rat represents a possibility.
  • the sample has two or more inhibitors of different concentrations.
  • the evaluation unit comprises a multivariate data analysis.
  • a correlation between the signal caused by the evaluation unit of the biosensor and the toxicity of the sample can be easily and simply established.
  • the biosensor comprises at least one enzyme substrate, preferably acetylcholine and / or para-aminophenyl butyrate.
  • This embodiment thus relates to the determination of the toxicity of a sample due to acetylcholinesterase inhibitors.
  • acetylcholinesterase inhibitors may also be present in the mixture in the sample.
  • Various non-human esterases are immobilized on the biosensor. They are particularly stable compared to human acetylcholinesterase.
  • the substrate Paraaminophenylbutyrat can be used, so that with a, in this embodiment, electrochemically operating biosensor according to the invention the enzyme product can be measured particularly easily.
  • the enzymes may also be further modified to better immobilize them to the sensor. All enzymes are contacted with the sample, and the inhibitory effect on the different esterases is detected separately. By comparing the biosensor response by the evaluation unit with previous calibration measurements, the toxicity of the sample to humans is determined or calculated.
  • the biosensor comprises at least one control enzyme.
  • Particularly preferred is a phosphatase.
  • the enzymes used are selected from the group consisting of natural enzymes, synthetic enzymes, mutant enzymes, and modified ones
  • mutated or modified enzymes are particularly suitable, which may differ significantly in their reaction from the underlying naturally occurring variants. This allows statements to be made about the relevance of certain mutations for the function of an enzyme.
  • the enzymes belong to the same group of enzymes. They are preferably ester acids.
  • a system By simultaneously using a group of enzymes that are all inhibited by the same kind of toxins, but to varying degrees, a system is created. which indicates the sensitivity of the human organism to this group of substances.
  • the term "same type of poisons" in the context of the present invention does not mean a similar chemical structure, but an identical mode of action of the poisons, in this embodiment, the inhibition of acetylcholinesterase, thereby making it possible to determine, without elaborate chemical analysis, whether the sample can be dangerous to humans due to the addition of a certain type of toxin, such a system has the particular advantage of providing a reliable result in a very short time, usually less than 15 minutes
  • the sensor can also be used, for example, in mobile field devices.
  • Embodiments in which the enzyme is an esterase and the enzyme inhibitor is an acetylcholinesterase inhibitor are preferred.
  • the acetylcholinesterase inhibitors are of particular interest because they are used as pesticides and as warfare agents.
  • the esterase is selected such that it is particularly stable, in particular temperature-stable.
  • the enzymes are arranged on the biosensor in an array, which allows an even faster and more varied measurement.
  • the biosensor is calibrated prior to its use.
  • Another object of the invention relates to a method for determining a poison in a sample, comprising the steps:
  • two or more enzyme inhibitors of different concentration are measured.
  • Another object of the present invention is the use of a biosensor according to the invention for testing a sample for poison / e.
  • the biosensor according to the invention is suitable for monitoring various liquids, in particular for determining poisons in water, preferably drinking water,
  • Sewage water, surface water, well water, ballast water of ships and / or cooling water Sewage water, surface water, well water, ballast water of ships and / or cooling water.
  • biosensor can be used to monitor extracts dissolved from solids, especially pesticides, biological and / or chemical warfare agents, and / or heavy metals.
  • the sample may also be a gas, preferably air, e.g. in air conditioners or respiratory masks.
  • E stands for enzyme
  • A, B, C to M are the different family of enzymes, eg esterases, phosphatases, etc.
  • the numbers relate to the different variants of the individual enzymes in the enzyme families.
  • the variations of the enzymes serve to simulate the human response to the corresponding family of enzyme inhibitors.
  • Fig. 2 shows the inhibition of an esterase (esterase 1) as a function of the concentration of various inhibitors.
  • Fig. 3 as a characteristic of the measured inhibitory effect for an inhibitor, the amount of inhibitor in the sample at 50% inhibition is used.
  • the LD 50 value for the rat is used orally.
  • the acute reference dose ArfD
  • WHO World Health Organization
  • ADI for "acceptable daily intake”
  • NOEL for "no observed effect level”
  • the amount of inhibitor applied at 50% inhibition against toxicity there is no simple correlation. In the case of an unknown inhibitor, therefore, it is not possible initially to conclude from the measured inhibition on the toxicity. The aim is therefore to find variations of enzymes that react differently to the inhibitors of the same family.
  • Fig. 4 shows the measurement of the inhibition of a second esterase with reduced sensitivity to paraoxon and increased susceptibility to omethoate, quinalphos and methidation.
  • Fig. 5 shows the response of the sensor calculated from the measurements of the two esterases 1 and 2. From the two measurements, the response of the biosensor is calculated by averaging over the reciprocal values. This results in an overall response with an approximately linear relationship between the inhibitor level at 50% inhibition and the toxicity represented by LD50 for rat orally. By using more than two esterases, the linearization can be improved even more.
  • the linearization of the sensor system response establishes a clear correlation between the signal and the toxicity of the sample, regardless of the inhibitor. This means that the toxicity can be calculated without the knowledge of the inhibitor from the signal of the biosensor according to the invention. This is also possible for the mixtures of inhibitors, since the inhibition effect is additive.
  • Limit values for drinking water can be derived from the LD50 value or, more specifically, the acute reference dose for humans.
  • the system can be used as alarm device for drinking water using these limits. If the limit value is exceeded, an alarm is issued and appropriate measures are taken to protect the population.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un biodétecteur servant à déterminer un poison dans un échantillon comprenant au moins deux enzymes qui sont disposées dans le biodétecteur et qui sont amenées en contact avec un échantillon à analyser quant à la présence d'au moins un inhibiteur d'enzyme; une unité de mesure destinée à mesurer une inhibition d'enzyme qui est occasionnée par une interaction entre les enzymes sur le biodétecteur et au moins un inhibiteur dans l'échantillon; et une unité d'évaluation servant à évaluer l'inhibition de l'enzyme. L'invention est caractérisée en ce que l'unité d'évaluation fournit une relation entre les inhibitions d'enzymes mesurées et la toxicité de l'échantillon pour un organisme. L'invention concerne en outre un procédé de détermination d'un poison dans un échantillon, ainsi que des utilisations, conformes à l'invention, du biodétecteur. Fig. 2.
PCT/EP2009/053861 2008-06-19 2009-04-01 Bioodétecteur et procédé de détermination d'un poison, et utilisations du biodétecteur Ceased WO2009153074A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200810029174 DE102008029174A1 (de) 2008-06-19 2008-06-19 Biosensor und Verfahren zum Bestimmen eines Gifts sowie Verwendungen des Biosensors
DE102008029174.9 2008-06-19

Publications (1)

Publication Number Publication Date
WO2009153074A1 true WO2009153074A1 (fr) 2009-12-23

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WO (1) WO2009153074A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031563A1 (fr) * 2001-10-11 2003-04-17 Agentase Llc Capteurs destines a la detection d'un analyte
WO2004025244A2 (fr) * 2002-09-16 2004-03-25 Sense Proteomic Limited Reseaux de proteines et leurs utilisations

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE340266T1 (de) * 1999-04-26 2006-10-15 Us Army Med Res Mat Command Immobilisierte enzyme als biosensoren fur chemische toxine
EP1283902A4 (fr) * 2000-05-05 2007-05-02 Us Army Medical Res And Materi Analyse pour la detection, la mesure et le controle des activites et des concentrations de proteines et ses procedes d'utilisation
WO2004085672A2 (fr) * 2003-03-21 2004-10-07 Tiax Llc Moniteur d'alarme pour agents chimiques
WO2008123763A1 (fr) * 2007-04-10 2008-10-16 Khiazh Sdn Bhd Dosage pour des composés inhibant la cholinestérase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031563A1 (fr) * 2001-10-11 2003-04-17 Agentase Llc Capteurs destines a la detection d'un analyte
WO2004025244A2 (fr) * 2002-09-16 2004-03-25 Sense Proteomic Limited Reseaux de proteines et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHU HENG ET AL: "Analysis of yeast protein kinases using protein chips", NATURE GENETICS, vol. 26, no. 3, November 2000 (2000-11-01), pages 283 - 289, XP002538557, ISSN: 1061-4036 *

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