WO1996014570A1 - Dispositif d'echantillonnage autonome generateur de signaux et ses modes d'utilisation - Google Patents
Dispositif d'echantillonnage autonome generateur de signaux et ses modes d'utilisation Download PDFInfo
- Publication number
- WO1996014570A1 WO1996014570A1 PCT/US1995/013147 US9513147W WO9614570A1 WO 1996014570 A1 WO1996014570 A1 WO 1996014570A1 US 9513147 W US9513147 W US 9513147W WO 9614570 A1 WO9614570 A1 WO 9614570A1
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- WO
- WIPO (PCT)
- Prior art keywords
- sampling
- lower housing
- sample
- signal generating
- nutrient
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000005070 sampling Methods 0.000 title claims description 65
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- 108060001084 Luciferase Proteins 0.000 claims abstract description 28
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- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 claims abstract description 26
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 claims abstract description 26
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 claims abstract description 26
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
- A61B10/0096—Casings for storing test samples
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
- A61B10/02—Instruments for taking cell samples or for biopsy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N2001/028—Sampling from a surface, swabbing, vaporising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
Definitions
- This invention relates in general to a device and methods for generating a signal in response to the testing of a sample which may contain biological material or toxic material wherein the device generating the signal is self- contained.
- sampling devices in various fields is known. For example, the sampling for contamination of food processing facilities; the sampling of contamination of the environment by heavy metals such as lead or cadmium; the collection of specimens from a patient to test for microorganism infection.
- the devices and methods previously used required various inconvenient steps. For example, batch preparation of a chemical which would provide a desired signal upon contact with a signal-generating sample, followed by pipetting of the batch preparation into multiple tubes. This preparation might take place in a food processing facility where contamination of the food being processed by foreign substances, such as a signal- generating chemical is a major concern. Often, for reasons of personnel turnover or otherwise, the personnel doing the sampling may lack significant experience preparing chemical formulations, thereby introducing the risk of error in preparation of the signal-generating composition. Other devices have required shipment back to laboratories for further processing and/or analysis of samples obtained. For example, food processing facilities often use bacterial plating and counting techniques which could take 24-48 hours to provide results. Environmental sampling for biological contamination often takes up to five days.
- Some devices have attempted to overcome these problems by designing a self-contained testing unit.
- signal generating chemicals such as luciferase/luciferin
- their design is believed not to offer the same degree of stability or ease of use of the luciferase/luciferin as in the present invention.
- contamination of the testing device prior to use must be avoided.
- the present invention provides for the protection of a sampling means prior to use, whereas other devices leave the sampling means relatively accessible to pre-test contamination.
- the device of the present invention fulfills this need.
- This invention in one aspect features a device which allows for the rapid and convenient testing of surfaces, solutions or atmospheres for biological contamination by assaying for the presence of adenosine triphosphate ("ATP") .
- ATP adenosine triphosphate
- the device will allow for the testing of surfaces in, for example, food processing factories, supermarkets and fast food restaurants to determine if such surfaces contain biological material.
- a device which tests for the presence of specific species of bacterial or other procaryotic or eucaryotic contamination is provided.
- a device which indicates the presence of toxic material by either the generation or cessation or diminution of bioluminescence generated by bioluminescent bacteria is provided.
- the device in one embodiment comprises a sampling means; a sampling means washing means; a signal generating means, wherein said signal generating means is stably packaged; a means for separating said signal generating means from a sample washed from said sampling means by said sample means washing means; and a sample and signal generating means mixing means.
- biological material is meant procaryotic, such as bacterial, or eucaryotic, such as molds, yeast, plant, animal and other eucaryotic organisms, cellular material wherein this material has associated with it ATP.
- sampling means is meant a device which allows one to obtain all of or a portion of a sample which may be present on a surface, in a solution or in an atmosphere to be tested.
- the sampling means may be a swab comprising a shaft and an absorbent tip.
- the shaft of the sampling means may further comprise a venting means .
- the swab tip may be comprised of natural or synthetic materials so long as deposition of a sample thereto may occur.
- the material may be but is not limited to nylon, dacron, rayon, porex, absorbent polypropylene, absorbent polyethylene, nitrocellulose, cotton, wool, cellulose or sponges.
- the swab shaft is hollow.
- the swab is provided for use in a pre- moistened form.
- This pre-moistening assists in the adsorption of biological or toxic material onto the surface of the sampling means.
- the moistening fluid may be a simple buffer or water.
- the fluid may also act to release ATP from within cellular matter which may comprise the biological material adsorbed from a sampled surface.
- Fluids as are known in the art, for example Extralight ® supplied by Analytical Luminescence Laboratory of San Diego, California may be used. Other fluids suitable for this purpose are set forth in United States Patent 5,004,684, herein incorporated by reference.
- the sampling means may function through capillary action, for example a capillary tube or tubes.
- the sampling means may comprise a pipetting means.
- the sampling means may comprise a chamber which captures a sample of an atmosphere, such as the atmosphere present in an enclosed work space.
- sampling means washing means is meant a means which allows the removal of all or a part of a sample present on the "sampling means".
- an upper housing means comprising a reservoir containing a fluid or dry substance in which the fluid or dry substance may be selectively released as desired, ordinarily to release a sample that has been obtained.
- the fluid may comprise the following: water, surfactants, salts, a neutralizing compound such as sodium thiosulfate.
- the fluid may be a bacterial or other procaryotic or eucaryotic nutrient-indicator medium as described in more detail below.
- a bacterial-regeneration fluid may be used to rehydrate lyophilized bacteria, when they are used as the signal-generating means.
- the dry substance may be but is not limited to a luciferase/luciferin composition, a lyophilized bacterial composition, a nutrient-indicator composition or other substance as appropriate, as set forth in more detail below.
- the upper housing means may contain a container such as, but not limited to, an ampule or a packet .
- the ampule or packet may contain a fluid or dry substance as described above.
- the upper housing may contain two containers, both or either comprising, for example, but not limited to an ampule or packet, containing the same or different fluids or dry substances.
- a fluid may be directly contained in the upper housing and a container containing a fluid or dry substance be contained therein.
- signal generating means is meant a composition which upon . interaction with the sample may generate a signal which may be detected by human visual inspection or by detection in a device.
- the signal comprises a change in the light emission or absorption characteristics of the sample.
- the alteration of the luminescence (including amplitude, polarization, and other properties) of a signal is used.
- a luciferase/luciferin composition is used to generate a signal.
- the luciferase/luciferin composition is preferably lyophilized and stably packaged.
- the luciferase/luciferin composition is highly sensitive and can detect the presence of as little as 10 "16 moles of ATP.
- signal generating means such as bioluminescent bacteria and nutrient-indicator media may also be used.
- Other signal detection systems may be employed. Examples of such signal detection systems include, but are not limited to, spectrophotomers, colorimeters, luminometers, fluorometers, and devices that measure the decay of radioisotopes.
- stably packaged is meant that the signal generating means may be stored prior to use for prolonged periods of time, for example, over one year if stored at 2-8 degrees Centigrade, and still provide a signal upon activation.
- the signal generating means is stably packaged within a sealed glass ampule.
- the ampule may be a borosilicate glass, for example Pyrex®. It may be an "onionskin” type of glass ampule.
- a moisture proof foil or plastic packet may be used. More generally, any packaging which provides a good barrier to the loss or introduction of moisture may be used.
- separation means is meant a means for separating said signal generating means from a sample washed from said sampling means by said sample means washing means until the mixing of sample and signal generating means is desired.
- separation means is also meant that a breachable barrier exists between the signal generating means and the sample.
- the separation means may be a porous plastic or hydrophobic material- filter, however, the porosity is not such that the sample would filter through without the application of a force, other than gravity, on the sample.
- the separation means may be a one way valve, a puncturable membrane, a temperature or chemical sensitive dissolvable membrane.
- a sample and signal generating means mixing means is meant a means of generating the force required to move the sample across the separation means set forth above.
- a deformable lower section of a lower housing of the device that is a region below the separation means and the sample, and which contains the signal generating means may be compressed and will allow for the passage of air or another gas from the lower section of the lower housing through the sample.
- the pressure on the top of the sample will be greater than that below it. This pressure differential will drive the sample through the separation means.
- the sampling means washing means is activated. In one embodiment, this is done by bending a bulb containing the sample washing fluid, thereby breaking a seal and allowing the fluid to wash down the hollow interior of the swab.
- the fluid washes the sample off of the swab.
- the fluid may also act to permeabilize any cells present in the sample, thereby releasing ATP contained therein.
- the sample is stably contained in the upper section of the lower housing of the device by the walls of the lower housing and the means for separating said signal generating means ⁇ from a sample washed from said sampling means by said sample means washing means until the generation of a signal is desired.
- a signal may be generated.
- luciferase/luciferin is the signal generating means used, a signal will be generated which is proportional to the amount of ATP, if any, present in the sample.
- the signal may be detected by a sampling device specifically adapted to be used with the sampling devices of this invention.
- the presence of ATP indicates that the area sampled contains biological material and may need to be cleaned, re-cleaned or sanitized.
- Other specific applications of the invention are as follows: The testing of water samples to determine if they contain biological material. Testing of water samples to determine if they contain toxic material such as compounds which have bacteriocidal activity. Testing of samples for specific bacterial species (e.g., Salmonella, E. coli , Listeria) or other procaryotic or eucaryotic organisms using a nutrient-indicator medium as the signal generating means.
- toxic material material which has been associated with a detrimental effect on living organisms.
- detrimental effect is meant a condition of decreased reproduction rate, less than optimal health, a disease or death.
- Compounds which activate "stress” inducible promoters of bacterial species are also included within this definition.
- Examples of toxic material include but are not limited to heavy metals, such as tin, lead, cadmium, mercury and chromium; organic chemicals, such as pesticides and industrial waste products.
- Bacteriocidal activity is meant a decrease or cessation in the rate of reproduction or the death of some or all of the bacteria exposed to a particular compound.
- nutrient-indicator medium is meant a medium which provides for the growth of specific target organisms but not others. The presence of the specific bacterial, mold or yeast species is made known by a signal generated on the basis of the cleavage of the nutrient-indicator releasing the indicator portion of the molecule by a specific enzyme, particular to a specific, bacterial, mold or yeast species.
- a sample to be tested for toxic material is collected, by a sampling means, for example a swab in the case of a surface or a pipette for a water sample.
- the sample is placed in the upper section of the lower housing of the device .
- the sample is washed off the sampling means by the sample means washing means, the washing means may contain additional reagents, for example bacterial reconstitution fluid. In the case of a liquid sample, washing may not be required but the addition of reagents may be desirable.
- a stable packaging means for example, a glass ampule containing lyophilized bioluminescent bacteria may be provided in the lower section of the lower housing of the device.
- the bacteria could be present in an ampule in the upper housing means and which would be reconstituted there, for addition to the sample below.
- a signal may be generated if toxic material are present.
- the signal may be the increase or decrease of luminescence depending upon the bacterial system used as described in more detail below.
- a nutrient- indicator medium as defined herein is provided in a stable packaging means, such as a glass ampule. "A sample to be tested is added to the device, by swab or pipette or other sampling means. The sample is washed off the sampling means.
- Figure 1 is a cross-sectional illustration of one embodiment of the present invention.
- Figure 2 is a schematic illustration of the components of one of the embodiments of the invention.
- Figures 3A-3E illustrate alternative embodiments for providing means for packaging a fluid or dry substance in the device of the present invention.
- Figures 4A-4D illustrate alternative embodiments for providing a sampling means to sample surfaces or fluids or gases.
- Figures 5A-5E illustrate alternative embodiments for providing a separation means.
- Figures 6A-6H illustrate the alternative embodiments for providing means for packaging a fluid or dry substance in the lower section of the device.
- Figure 1 illustrates an embodiment of the present invention which includes a top housing 10; an upper barrier means 11 between the top housing 10 and the upper section 13A of a lower housing 14C; a means 12 for breaking the upper barrier means 11; a sampling means 15A comprised of a shaft 15B and a surface sampling means 15C; a sampling means engaging means 13B which may optionally fit inside of the upper section 14A of the lower housing 14C; a separation means 17 separating the upper section 14A of lower housing 14C from a lower section 14B of the lower housing 14C; a signal means packaging means 18.
- the top housing 10 and upper barrier 11 define a chamber 19 which optionally may contain a sealed or partially sealed container as shown in Figure 3.
- Figure 2 illustrates a top housing 20, an upper barrier means 21 between the top housing 20 and the upper section 22 of a lower housing 27.
- the top housing 20 and upper barrier means 21 define a chamber 28 which optionally may contain a sealed or partially sealed container as shown in Figure 3.
- a separation means 24 separating the upper section 22 of lower housing 27 from a lower section 25 of the lower housing 27.
- Figure 3 illustrates alternative embodiments for providing means for packaging a fluid or dry substance in the chamber 28.
- Figure 3A illustrates a container 29 within chamber 28.
- Figure 3B illustrates a fluid 30 directly contained by the walls of the top housing 20 and upper barrier means 21.
- Figure 3C illustrates two containers 29A and 29B containing the same or different fluids.
- Figure 3D illustrates two containers 29C and 29A, 29C containing a dry substance and 29A a fluid.
- Figure 3E illustrates a fluid 30 directly contained by the walls of the top housing 20 and upper barrier means 21 and a container 29C containing a dry substance.
- Figure 4 illustrates alternative embodiments for providing a sampling means to sample surfaces or fluids or gases.
- Figure 4A illustrates a shaft 40A with a central channel 40B and channels 40C extending into a surface sampling means 41.
- Figure 4B illustrates a solid shaft 42 attached to a surface sampling means 43.
- Figure 4C illustrates a pipetting means 44 for sampling fluids.
- Figure 4D illustrates that a separate sampling means, such as a pipette 45 may be used.
- Figure 5 illustrates alternative embodiments for providing a separation means.
- Figure 5A illustrates a semi-porous filter element 50.
- Figure 5B illustrates a one-way valving means.
- Figure 5C illustrates a puncturable membrane 52 and a puncturing device 53.
- Figure 5D illustrates a porous separation means 54.
- Figure 5E illustrates a temperature or solvent dissolvable membrane 55.
- Figures 6A to 6H illustrate alternative embodiments for providing means for packaging a fluid or dry substance in a chamber 60 of the lower section 25 of lower housing 27.
- Figure 6A illustrates a container 61 containing a fluid or dry substance within chamber 60.
- Figure 6B illustrates a plastic or foil packet 62 containing a fluid or dry substance within chamber 60.
- Figure 6C illustrates a dry substance 63 directly contained within chamber 60.
- Figure 6D illustrates a fluid 64 directly contained within chamber 60.
- Figure 6E illustrates an ampule 65 within an ampule 66 or a packet 66 within a packet 66.
- the ampule or packet 65 may contain a fluid or dry substance.
- the ampule or packet 66 may contain a fluid or dry substance.
- the fluid or dry substance in ampule or packet 65 or 66 may be the same or different.
- Figure 6F illustrates an ampule 67 and an ampule 68 or a packet 67 and a packet 68.
- the ampule or packet 67 may contain a fluid or dry substance.
- the ampule or packet 68 may contain a fluid or dry substance.
- the fluid or dry substance in ampule or packet 67 or 68 may be the same or different.
- Figure 6G illustrates a fluid 69B directly contained in chamber 60 and a container 69A containing a fluid or dry substance.
- Figure 6H illustrates an ampule or packet 70 containing a first substance 70A and a second substance 70B.
- the present invention in one embodiment, describes a device and methods for the rapid and convenient testing of surfaces, solutions or gases for biological contamination by assaying for the presence of adenosine triphosphate ("ATP") .
- ATP adenosine triphosphate
- the "sampling means washing means” may comprise for example, a reservoir containing a fluid in which the fluid may be selectively released after a sample has been obtained.
- the fluid may comprise the following: water, surfactants, salts, a neutralizing compound, such as sodium thiosulfate.
- a suitable formulation is as follows: 50 mM Triz a base, 0.5 mM MgCl 2 , 0.5% w/v Tween 80, 0.01 g/L Sodium Thiosulfate, pH to about 7.7 to 8.0 with, for example, acetic acid, a preservative, such as Rohm and Haas' Proclin 300 may be added to about 10-40 ppm, preferably 15 ppm. Further, an additional preservative, such as sodium Azide, may be added to 0.02% w/v.
- a “signal generating means” such as a luciferase/luciferin composition
- a suitable luciferase/luciferin composition comprises the following: Luciferase at a concentration of from about 50-100 ⁇ g/ml (the higher end of this range is preferable) ; Luciferin at a concentration of from about 0.1-4.0 mg/ml (the middle to high end of this range is preferable) ; EDTA at a concentration of from about 0.1 - 2.0 mM, (the middle of this range is preferable) ; DTT at a concentration of from about 2.0 - 4.0 mM (the middle of this range is preferable) ; BSA at about 5.0 - 50.0 mg/ml, (the middle of this range is preferred) ; Trizma at about 25 - 75 mM, (the middle of this range is preferred) ; MgCl 2 at a concentration of about 25.0 - 100.0 M (the middle of this
- luciferase 76 ⁇ g/ml BSA 25 mg/ml
- Luciferin 2 mg/ml EDTA ImM
- DTT 3 M Trizma 50 M
- MgCl 2 50 m The luciferase/luciferin composition is preferably lyophilized and stably packaged.
- luciferase/luciferin composition suitable for use in one embodiment of this invention may be done as follows: a 25 ⁇ l aliquot of the "luciferase/luciferin cocktail", (the luciferase/luciferin cocktail compositions are obtainable, for example, from Sigma chemical corporation or JBL or Boehringer Mannheim; by "cocktail” is meant the luciferin and luciferase and other elements that allow the system to produce a signal upon exposure to ATP) , is deposited and frozen in a thin-walled glass onion-skin tube (“OST”) . The aliquot is lyophilized to a moisture content of about less than 4%. The OST is then removed from the lyophilizer to a dry environment where the ends of the OST are melted in a high temperature flame, hermetically sealing the material inside the glass ampule.
- luciferase/luciferin cocktail the luciferase/luciferin cocktail compositions are obtainable, for example, from Sigma chemical corporation or
- bioluminescent bacteria would be lyophilized as is known in the art. These bioluminescent bacteria would be placed into a stable packaging means in the device of the present invention. A sample would be added, as described elsewhere regarding the operation of the device. Upon reconstitution these bioluminescent bacteria will "bioluminesce” , thereby indicating their viability. Upon addition of a sample containing a toxic material of a particular threshold concentration the bacteria will cease exhibiting bioluminescence, indicating the presence of a toxic material.
- Bacterial reagents as are known in the art for example the Microtox ® reagent provided by Microbics Corporation of Carlsbad, California may be utilized. Ecotoxicology Monitoring, Section 3, Chapters 13-23, Mervyn Richardson Editor. (1993) is hereby incorporated by reference.
- the luciferase gene may be cloned behind a promoter that is activated by various substances, for example, heavy metals. Upon the addition of a sample containing activating toxic material, for example, heavy metals, luciferase will be produced providing a detectable signal. Construction of a bacterial plasmid may be done as is known in the art and as set forth in Molecular Cloning, a Laboratory Manual Second Edition, Sambrook et al . , incorporated herein by reference. Heavy metal inducible promoters and/or bacterial bioluminescent sensor strategies are disclosed in the following publications: Abstracts of the American Society for Microbiology, 93 general Meeting 16-20 May 1993, Atlanta Georgia; Abstract No.
- the testing of samples for specific procaryotic bacterial species e . g . , Salmonella, E. coli , Listeria
- the signal generating means comprises a nutrient-indicator media, such as Colilert® nutrient-indicator media, available from IDEXX Laboratories of Westbrook, Maine. United States Patent 4,925,789 detailing the composition and use of nutrient- indicator media is incorporated herein by reference.
- a liquid sample is obtained with for example a pipette. The sample is mixed with the nutrient-indicator media.
- the whole device is incubated at a required temperature, dependent on the microorganism which one wishes to detect, for a specified time. For example, 35 degrees Centigrade for E. coli . If specific species of target bacteria are present a signal will be generated. More specifically, E. coli contain the enzyme ⁇ -glucorindase, while coliform bacteria contain the enzyme -galactosidase. In the presence of the nutrient-indicator 4-methylumbelliferyl-/3- D-glucuronide (MUG) , E. coli will metabolize this compound, releasing an indicator portion, which when cleaved fluoresces when excited by an ultraviolet light source.
- a required temperature dependent on the microorganism which one wishes to detect, for a specified time. For example, 35 degrees Centigrade for E. coli . If specific species of target bacteria are present a signal will be generated. More specifically, E. coli contain the enzyme ⁇ -glucorindase, while coliform bacteria contain the enzyme -gal
- coliform in the presence of the nutrient-indicator o-nitrophenyl- ⁇ -D-galactoside (ONPG) will metabolize this compound, releasing an indicator portion, which when cleaved colors a sample yellow.
- ONPG o-nitrophenyl- ⁇ -D-galactoside
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Abstract
L'invention concerne un dispositif autonome générateur de signaux et ses modes d'utilisation. Le dispositif et ses modes d'utilisation peuvent détecter la présence de matières biologiques ou toxiques, et peut faire appel, entre autres compositions, à une composition de luciférine/luciférase, à des milieux indicateurs de nutriment ou à des bactéries bioluminescentes pour émettre un signal indiquant la présence de matière biologique ou toxique.
Applications Claiming Priority (2)
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US33685194A | 1994-11-07 | 1994-11-07 | |
US08/336,851 | 1994-11-07 |
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WO1996014570A1 true WO1996014570A1 (fr) | 1996-05-17 |
WO1996014570A9 WO1996014570A9 (fr) | 1996-09-19 |
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PCT/US1995/013147 WO1996014570A1 (fr) | 1994-11-07 | 1995-10-17 | Dispositif d'echantillonnage autonome generateur de signaux et ses modes d'utilisation |
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