EP2313755A1 - Appareillage et procédé de détection automatique de particules biologiques - Google Patents

Appareillage et procédé de détection automatique de particules biologiques

Info

Publication number
EP2313755A1
EP2313755A1 EP09780943A EP09780943A EP2313755A1 EP 2313755 A1 EP2313755 A1 EP 2313755A1 EP 09780943 A EP09780943 A EP 09780943A EP 09780943 A EP09780943 A EP 09780943A EP 2313755 A1 EP2313755 A1 EP 2313755A1
Authority
EP
European Patent Office
Prior art keywords
particles
collecting
unit
separating
particle
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Withdrawn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
EP09780943A
Other languages
German (de)
English (en)
Inventor
Ulrich Reidt
Alois Friedberger
Christoph Heller
Thomas Ziemann
Harald Waltenberger
Günter Müller
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.)
Airbus Defence and Space GmbH
Original Assignee
EADS Deutschland GmbH
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 EADS Deutschland GmbH filed Critical EADS Deutschland GmbH
Publication of EP2313755A1 publication Critical patent/EP2313755A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/286—Magnetic plugs and dipsticks disposed at the inner circumference of a recipient, e.g. magnetic drain bolt
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
    • 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
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2202—Devices for withdrawing samples in the gaseous state involving separation of sample components during sampling
    • 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
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2273—Atmospheric sampling
    • 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
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • the invention relates to a device and a method for the automatic detection of biological particles.
  • US Pat. No. 6,268,143 B1 and US Pat. No. 5,972,721 describe methods and devices for the automatic extraction and detection of biological particles, in particular microorganisms such as bacteria.
  • the principle of paramagnetic separation or immunomagnetic examination is used.
  • particles of separating particles which are coated in such a way that they bind to themselves special particles to be examined, of paramagnetic material are used.
  • This separation particles body - called beads - can be held in a magnetic field and thus extract. As a result, the special particles adhering to it are also extracted.
  • Airsampier which are equipped with nutrient media. These must be incubated after sampling for several hours or days before proof is available.
  • Systems for checking and accumulating microorganisms from the air into a liquid are also known, but these systems lack the possibility for fully automatic extraction and detection.
  • the object of the invention is to provide an apparatus and a method for the detection of particles in a particle-fluid mixture, which are fully automatic operable or feasible, universally applicable and can be implemented in a compact and simply constructed, preferably mobile system.
  • microorganisms e.g., bacteria, protozoa, fungi, viruses
  • biological particles e.g., spores
  • the enrichment, extraction and detection can be carried out both from gases, in particular from the air, as well as from liquids.
  • detection of biological materials enrichment, extraction and detection of non-biological or synthetic materials is possible, in particular explosives, liquid explosives and drugs.
  • paramagnetic beads in particular the use of paramagnetic beads (so-called beads) in conjunction with a collecting device, in particular an airsampler.
  • the beads are coated with antibodies, which in turn can bind molecules or particles of biological or non-biological origin.
  • the extreme concentration and immobilization of the beads thus loaded is achieved.
  • a fully automatic extraction and detection of the bound molecules or particles following the concentration is proposed.
  • the high concentration allows a highly sensitive detection of the analytes.
  • Figure 1 is a schematic representation for explaining the detection and binding of particles (antigens) by antibodies immobilized on a bead surface via biotin and streptavin.
  • FIG. 2 shows a schematic illustration for explaining a recognition and binding of bacteria by phage proteins which have been immobilized on a bead surface via biotin and streptavin;
  • 3a show various sectional views of an airsampler known per se, which can be used in the system presented here, in a corresponding application;
  • Figure 5 is a schematic representation of an enrichment of the paramagnetic see beads by a magnetic piston.
  • FIG. 6 shows a schematic representation of the enrichment of the paramagnetic beads via an expandable membrane
  • FIG. 7 shows a side view of an overall system as a device for detecting particles
  • Fig. 8 is a plan view of the entire system
  • Fig. 9 shows a DNA sequence on the model of a zipper
  • FIG. 10 shows a schematic representation of the accumulation of particles from liquids via a flow cell
  • FIG. 11 is a schematic representation of two interconnected microliter pipettes.
  • sampling sampling
  • enrichment extraction
  • extraction extraction and analysis of gases and liquids.
  • Primary target for detection are all particles, especially bacteria, viruses, spores, protozoa and biological toxins.
  • a use for the analysis of biological and non-biological substances is likewise possible with the device described here.
  • the only requirement is the presence of specific binding molecules (e.g., antibodies) with sufficient affinity. Since an antibody can be formed against almost any substance, the device described herein can be used to detect a variety of substances.
  • separator particles used to separate or separate the particles of interest here are paramagnetic beads which are loaded with antibodies and to which the particles or microorganisms bind with high affinity.
  • the beads themselves have i. d. R. size in the range of less than 0.5 microns to 10 microns and consist mainly of a paramagnetic core and a shell of silicate, latex or polystyrene.
  • various methods can be used: a) passive adsorption (eg via hydrophobic interactions), b) direct chemical coupling (crosslinking) via peptide bond or the like, c) coupling via immobilized antibody proteins, eg protein A or protein G and / or d) coupling via biotin-streptavidin binding.
  • the technique mentioned under d) is shown in FIG. It uses the high affinity (binding force) of two biological molecules to each other: biotin 18 and streptavidin 20.
  • the antibody 10 is labeled with the molecule biotin 18 at the side facing away from its specific binding site (the so-called Fc domain 22).
  • the protein streptavidin 20 is coupled to the surface 14 of the paramagnetic beads 16.
  • antibodies 10 are bound virtually irreversibly to the spherical surface 14,
  • the antibodies 10 After the immobilization of the antibodies 10 on the paramagnetic beads 16, they can be used for the targeted "capture” of the microorganisms or particles, in which case the antibodies bind to their so-called antigens 12 (the particles 13 to be detected); Spezieile surface structures of microorganisms.
  • certain phage proteins 24 can be used for the targeted recognition and binding of some bacterial species - a bacterium 26 is shown as an example.
  • Phages are viruses that attack only bacteria and can dock with their special envelope proteins on the bacterial surface. Using biotechnological methods, it is possible to produce large amounts of these phage proteins 24 and also to mark with biotin 18.
  • these phage proteins 24 can also be coupled to streptavadin-coated paramagnetic beads 16, with the phage proteins 24 bound in turn interacting with the bacterial docking sites (surface proteins of the bacteria 26).
  • step 1) and step 2) take place simultaneously.
  • the air flow 39 through the sampler is e.g. 12.5 L / min; at a sampling time of e.g. 10 minutes, this corresponds to a total volume of 125 liters of air. With a volume of the collection liquid of typically 5 mL, this results in a concentration by a factor of 25,000.
  • FIGS. 3 and 3 a show two cross-sectional drawings illustrating the air sampler 30 with inlet 32, outlet 34, sump 36, and tangent nozzles 38.
  • Fig. 3a shows a cross section along the line MN of Fig. 3.
  • the central axis 1 1 and a tangent 15 are further illustrated to illustrate the arrangement and orientation of the tangential nozzles 38.
  • the collection liquid 40 in the collection container 36 is mixed in the system described here with paramagnetic beads 16 described above.
  • the particles 13 (analytes) transferred from the air stream 39 (FIG. 4) into the collection liquid 40 can bind to the specifically activated paramagnetic beads 16 during the sampling.
  • the beads 16 are attracted by means of a magnetic field and enriched in a small volume within the metering volume 43 of a metering unit 41 (FIG. 4).
  • This volume is typically about 50 ⁇ l, which achieves a further concentration by a factor of 100. With the overall system can thus be a total of z. B. achieve a 2.5 million-fold enrichment of airborne germs (or other particles).
  • the third step ie the enrichment of the paramagnetic beads 16 from the collecting container 36 of the air sampler 30, can take place via three different technical methods, which are explained in more detail below.
  • a magnet 44 is attached to the outer wall 46.
  • the collecting liquid 40 with the paramagnetic beads 16 is drawn from the air sampler 30 into the dosing unit 41 via an outlet channel 45.
  • the beads 16 automatically deposit on the inner wall 48 of the dosing unit 41 in the region of the magnetic field.
  • the collection liquid 40 is pushed out of the syringe 42 again.
  • the beads 16 are held and retained by the magnet 44.
  • the Beads 16 can be washed by receiving and dispensing fresh buffer several times.
  • the smallest possible buffer volume is left in the syringe 42 in the last washing step and the magnet 44 is removed from the outer wall 46 of the dosing unit 41.
  • the collecting liquid 40 is likewise drawn from the air sampler 30 via an outlet channel 45 into the dosing unit 41, preferably a syringe 42.
  • the syringe 42 has a hollow syringe plunger 50, in which a second, magnetic piston - magnetic piston 52 - or punch - with the magnet 44 can be moved up and down (telescope principle).
  • the magnetic piston 52 is fully retracted into the syringe plunger 50.
  • the paramagnetic beads 16 can thus accumulate on the Kobenboden 54.
  • the collecting liquid 40 can be exchanged for another liquid (so-called washing). Finally, a minimum volume of liquid is drawn up (eg 50 ⁇ l_). For elution, the magnetic piston 52 is pulled upward, as a result of which the magnetic field in the syringe 42 virtually disappears and the beads 16 detach from the piston head 54.
  • This concept offers the advantage that when the magnet 44 is raised, the syringe 42 functions as a normal metering unit 41.
  • a digestion module in particular an ultrasound device 56, is further attached in the lower region of the dosing unit 41. This is particularly preferred in connection with the magnetic piston 52, since then no magnet has to be mounted in the lower region and space is created for the disruption module.
  • the magnet 44 (preferably a bar magnet 58) is dipped directly into the collecting liquid 40.
  • the magnet 44 is movably mounted and protected by a stretchable membrane 60.
  • the magnet 44 is immersed in a new vessel 62 with a small volume of liquid.
  • the magnet 44 is removed and the beads 16 may detach from the membrane 60.
  • ultrasound which is transferred to the bar magnet, a detachment of the beads can be facilitated. Also by the use of an external ultrasound device, a facilitated detachment of the beads is possible.
  • the air sampler 30 shown in FIGS. 3, 3 a has two components, a collecting vessel - in the form of the collecting container 36 - and an attachment with nozzles - nozzle attachment 64.
  • a lifting and pivoting unit 66 By using a lifting and pivoting unit 66, the collecting container can ter 36 automatically separated from the nozzle attachment 64 and pivoted. As a result, easy access to the collection liquid 40 is possible.
  • Fig. 6 shows the under III. C. in combination with the lifting and pivoting unit 66.
  • the lifting and pivoting unit 66 can also be used in combination with those described in III. A. or III. B. described methods can be used.
  • a special outlet channel 45 from the reservoir 36 is therefore no longer required.
  • bead-bonded particles 13 are now available for further analysis. They are further digested, for example, for molecular biological detection, in particular PCR or hybridization.
  • FIG. 11 shows an alternative embodiment of the dosing unit 41.
  • a microliter pipette 140 with extended pipette tip 142 and two suction units 144, 145 for different volume ranges both the washing of the beads 16 and a precise pipetting in small volumes is possible.
  • the beads 16 are retained during flushing on the inside of the pipette tip by an externally pivoted-magnet 44.
  • the pipetting system 146 described here can be easily integrated into the overall system. Also, an operation with the Hub pivot unit 66 is possible.
  • the enrichment concepts described above can be combined with other elements to form an overall system.
  • the overall system shown in more detail in FIGS. 7 and 8 forms a device 70 for the automatic detection of, in particular, biological particles and has as components a collecting device 72, a transfer unit 74, the metering unit 41, the magnet 44, a group 76 of FIG Reservoir, a drive unit 78, a Auf gleich acquired 80, possibly with tempering 82, a detection 84 and a control unit 86 on.
  • the collecting device 72 used is preferably the air sampler 30, in particular an air sampler 30 from the company SKC (see FIG. 3 and patents US 5,902,385 and US 5,904,752) or the Bertin company.
  • the air sampler 30 transfers particles 13, in particular microorganisms (bacteria, viruses) and toxins from the gas phase into the collecting liquid 40.
  • the transfer unit 74 preferably has the lifting pivot unit 66. Since the preferred air sampler 30 has a modular structure, and in particular consists of at least two components, the nozzle attachment 64 can be separated and the collection container 36 can be transferred to the enrichment position. Here, the paramagnetic beads 16 can be taken up and enriched with one of the methods described in Section 3.
  • the metering unit 41 is preferably designed as a syringe 42. With the dosing unit 41, the collection liquid 40 is raised. As dosing unit 41 can also in III. C. or III. D described constructions serve.
  • the magnet 44 serves as a separator to concentrate the paramagnetic beads 16 in or on the dosing unit 41. Thus, the beads 16 can be separated from the liquid surrounding them.
  • the group 76 has multiple reservoirs (vessels) 91-98 with different liquids needed to process the particles 13.
  • a rest position 99 is provided.
  • the following liquid reservoirs are provided: ⁇ solution with paramagnetic beads (first reservoir 91) "equilibration solution (second reservoir 92) ⁇ first digestion solution (third reservoir 93)" second digestion solution (fourth reservoir 94) ⁇ collecting liquid, eg water (fifth reservoir 95) ⁇ cleaning solution (sixth reservoir 96)
  • the reservoirs 91 - 98 are preferably aligned together with the rest position 99 and the collector 72 - in a line. As a result, the metering unit 41 can be moved linearly between the reservoirs 91 - 98, possibly the rest position 99 and the collecting device 72, by means of a linear drive 100 of simple construction.
  • the drive unit 78 has the drives explained below under F) to I):
  • the disruption device 80 preferably has the abovementioned ultrasound device 56, in particular in the form of an ultrasound bath 110, for the mechanical disruption of the particles, in particular microorganisms.
  • the ultrasonic bath 110 is filled with liquid and the dosing unit 41 can dip into this liquid.
  • the ultrasound bath 110 may be used at low power for resuspending the paramagnetic beads 16.
  • the disruption device 86 further has a first temperature control unit 82, which can be operated jointly or separately with the ultrasonic bath 110.
  • the temperature control unit 82 serves to support biochemical processes for the digestion of the particles 13, in particular microorganisms (for example enzymatic digestion). Thermal digestion processes close to the boiling point are also possible with the temperature control unit.
  • a temperature control of the entire system is provided.
  • the reagent reservoirs 91-97, the waste vessel 98 and the collection container 36 are tempered. This is z. B. by a example in Fig. 8, indicated as a heating coil second temperature control unit 19 1 possible.
  • the detection unit 84 is provided at the end of the process chain. Depending on the type of sample preparation, all known analysis methods can be integrated into the overall system.
  • ⁇ PCR Polymerase Chain Reaction
  • ⁇ ELISA enzyme-linked immunosorbent assay
  • hybridization method The most important methods for the detection and analysis of biological molecules are listed below: ⁇ PCR (Polymerase Chain Reaction), ⁇ ELISA (enzyme-linked immunosorbent assay), ⁇ hybridization method.
  • the control unit 86 serves to control and monitor the entire system.
  • a control unit 86 for example, a computer or data processing device is provided, in which the individual control steps for the fully automatic implementation of the detection method in the form of control commands are stored as software.
  • a data transfer for example via the Internet (online) possible.
  • the data transfer is used to synchronize the results via a database or to alarm. It is also possible to control the entire system online, so that the system can be operated over greater distances.
  • the chemical digestion can be carried out via chaotropic salts, in particular guanidinium hydrochloride or guanidinium thiocyanate. These are automatically recorded in the illustrated system in the dosing unit 41 (preferably syringe 42) in order to unlock the adhering to the beads 16 particles 13.
  • the ultrasound device 56, 110 and / or a heating module - tempering unit 82 and / or 1 19 - could be integrated into the overall system.
  • an extraction by shear forces glass beads or tissue homogenizer
  • the loaded beads 16 are transferred to a special homogenizer, and forces between the glass beads or the homogenizer wall can be effectively disrupted.
  • a very successful method for digesting biological particles 13 is the biochemical extraction.
  • enzymes in particular proteases and RNases, can be used. It is very common to use proteinase K for cell digestion and lysozyme for bacterial digestion.
  • the PCR (Polymerase Chain Reaction) method is a process that replicates the smallest amounts of a DNA segment in a chain reaction can (amplification).
  • the PCR method is very often used today, if on the basis of certain DNA sequences evidence should be performed, such as: ⁇ in forensics or the paternity test, ⁇ in microbiology for the detection of microorganisms (bacteria and viruses), «in the medical Diagnostics if it is necessary to detect viral DNA or RNA in the blood, or
  • PCR reverse transcription - PCR
  • RT-PCT reverse transcription - PCR
  • ELISA enzyme-linked immunosorbent assay
  • ELISA enzyme-linked immunosorbent assay
  • ELISA tests are widely used today in medical diagnostics. But they are also used in many other areas when specific proteins or biological toxins are detected. In the case of bacterial or viral detection, the specific surface proteins are recognized by the antibody.
  • a DNA double helix can be thought of as a "zipper” ( Figure 9)
  • the "teeth” of this zipper are the bases adenine (A), cytosine (C), guanine (G), and thymine (T).
  • the information containing the DNA is encrypted in the order of these four letters along the "zipper".
  • Opposite "teeth” always form only either AT or GC pairs.
  • the ACGCT sequence for example, has the base sequence TGCGA as a complementary counterpart, and the "zipper” is opened by heating so that single strands are present.
  • Short, single-stranded pieces of DNA, called probes can now find their matching counterpart on the long single strand. Upon cooling, these probes bind at the appropriate site, one then speaks of hybridization. This can be visualized by means of markings (eg by a fluorescent dye). In this way it can be found whether or not specific sequences, for example specific genes, are present in the DNA under study.
  • Well-known hybridization methods are in situ hybridization, in particular fluorescence in situ hybridization (FISH) and hybridization on microarrays.
  • FISH fluorescence in situ hybridization
  • the overall system flowchart shown includes several subsections (A-E, see below) needed for immunodetection (ELISA).
  • ELISA immunodetection
  • Airsampler on / off Airsampler 30 on / off
  • Ultrasonic bath on / off ie on / off
  • Syringe T go to Pos. waste ⁇ ; Syringe I; Piston iti.
  • Syringe t go to Pos. waste ⁇ ; Syringe i; Piston i.
  • Syringe t go to Pos. H 2 O * -; Syringe i; Piston t. Syringe f; go to Pos. waste ⁇ ; Syringe i; Piston i.
  • Syringe t go to Pos. waste ⁇ ; Syringe i; Piston i. Syringe t; go to Pos. Equilibration Solution ⁇ -; Syringe i; Piston t. Syringe t; go to Pos. waste ⁇ ; Syringe i; Piston i. Syringe t; go to Pos. Equilibration Solution ⁇ -; Syringe i; Piston T. syringe f; go to Pos. waste ⁇ ; Syringe I; Piston i.
  • control device For loading the air sampler 30, the following commands are executed controlled by the control device:
  • Syringe t go to Pos. mag. Beads ⁇ -; Syringe i; Piston T4 / i ⁇ stroke. Swivel unit 4 ⁇ T syringe t; go to Pos. Stroke swivel unit ⁇ -; Syringe I; Piston i. Syringe t; go to Pos. rest position ⁇ -; Syringe I.
  • Syringe T go to Pos. waste ⁇ ; Syringe i; Piston i.
  • Routine VII.A.3 Rinse with H 2 O, 3 x 5 ml. Syringe t; go to Pos. rest position ⁇ -; Syringe I.
  • Syringe t go to Pos. Cleaning ⁇ ; Syringe 4-; Piston t. Syringe t go to Pos. Waste ⁇ ; Syringe i; Piston i. Syringe f go to Pos. Cleaning ->; Syringe i; Piston T. syringe t go to Pos. Waste ->; Syringe i; Piston I. Syringe t go to Pos. Cleaning ->; Syringe i; Piston t. Syringe f go to Pos. Waste ->; Syringe i; Piston i.
  • Routine VII.A.3) rinsing with H 2 O, 3 ⁇ 5 m
  • Routine VII.A.3 rinsing with H 2 O, 3 ⁇ 5 ml
  • Syringe T go to Pos. rest position ⁇ -; Syringe I.
  • Syringe t go to Pos. Preservation ⁇ ; Syringe i; Piston t. Syringe t go to Pos. Waste ⁇ ; Syringe i; Piston i. Syringe t go to Pos. Preservation ⁇ ; Syringe 4-; Piston t. Syringe t go to Pos. Waste ⁇ ; Syringe i; Piston i. Syringe t go to Pos. Preservation ⁇ ; Syringe i; Piston t. Syringe t go to Pos. Waste - »; Syringe 1; Piston i. Syringe t go to Pos. Rest position ⁇ -; Syringe i.
  • Another possibility is to apply the beads 16 to a membrane, preferably to a micromechanical filter (not shown), the surface of which can then be used as a detection platform.
  • Detection on the surface of a micromechanical filter has already been described in detail in German Patent Application 10 2006 026 559.5 and in 10 2007 021 387.7.
  • a refined process is also the subject of a German patent application entitled "Optical Particle Filter and Detection Method” filed in parallel with this application on the same day, in which the company EADS Deutschland GmbH is also a notifier Referenced patent applications.
  • the air sampler 30 is replaced by a filtration unit 120, which allows a high liquid flow rate.
  • a flow cell 122 bounded by two diaphragms 124,126.
  • the pore size of the membranes 124, 126 should be selected so that the particles 13, in particular microorganisms, can pass, but the paramagnetic particles 16 are retained. Between the membranes 124, 126 are the paramagnetic beads 16 to which the particles 13 effectively bind.
  • stirrer 128 (rotor), which is driven by means of the flow 134, is located in the flow cell 122.
  • An automatic removal of the beads 16 is effected by a closure in the flow cell 122, in particular by a septum 130, which can be pierced with an injection needle 132.
  • a use of non-paramagnetic beads is also conceivable in the overall system. Enrichment of the beads after "air sampling” could take place by means of a magnetic field over a porous membrane, preferably a micromechanical filter, which would retain the beads but allow liquids to pass through for immunodetection (ELISA) are necessary to be pumped through these micromechanical filters.
  • ELISA immunodetection
  • nucleic acid-coupled beads Another possibility for sampling microorganisms is the use of nucleic acid-coupled beads in the overall system.
  • the microorganisms from the air are enriched and digested (for extraction methods see above).
  • the beads coated with nucleic acids are added to the lysate and the genetic material of the microorganisms can hybridize with the nucleic acid on the beads.
  • Non-specific binding of the extracted DNA to glass milk (silica matrix) is also possible.
  • an appropriate amount of glass milk is added to the lysate after cell lysis and the liberated DNA can bind nonspecifically to the silica particles.
  • the airsampler from SKC was integrated.
  • the flexible and modular design of the overall system also allows the integration of other types of air samplers (see publication Hogan et al., 2005).
  • the following air sampler types would also be suitable for integration into the overall system:
  • a fully automatic cleaning and disinfection program can be established in the overall system. All conceivable cleaning and disinfecting solutions can be used in the robust system. The following solutions may preferably be used:
  • Airsampler 30 all components of the Airsampler 30 are automatically cleaned by adding the solutions. Subsequently, a rinsing step is possible. This is done by a fluidic system that provides the reagents, on or brings and then resumes.
  • the system is disinfected by UV irradiation.
  • several UV tubes are mounted above the system, which sterilize the entire system in a relatively short period of time.
  • the entire system opens up a variety of applications.
  • the following application possibilities are conceivable: ⁇ Medical applications in diagnostics, in particular rapid detection of infectious disease pathogens from body fluids, in particular blood, saliva, tear fluid and urine; »Military applications, in particular integration of the entire system into military vehicles, ships, submarines and aircraft; »Application as a mobile system in all military and civilian areas; ⁇ Application in the area of "homeland security", in particular to ward off terrorist attacks with biological weapons;

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Abstract

L'invention porte sur un appareillage (70) et sur un procédé de détection automatique de particules (13), en particulier de particules biologiques telles que des microorganismes. L'appareillage comprend un dispositif destiné à lier les particules (13) à des corps de particules de séparation, pouvant se lier sélectivement aux particules, un dispositif pour extraire les corps de particules de séparation (16), avec les particules (13) qui y sont liées, à partir d'un liquide collecteur (40), et une unité de détection (84) destinée à détecter un nombre et/ou une concentration des particules (13) ainsi séparées. Le dispositif destiné à lier les particules (13) aux corps de particules de séparation est un dispositif collecteur (72) destiné à collecter les particules (13) à partir d'un mélange particules-fluide (39, 134) à étudier, dispositif qui peut être traversé par le mélange particules-fluide, et qui peut être rempli automatiquement d'un liquide collecteur (40) auquel on a ajouté les particules de séparation (16).
EP09780943A 2008-07-31 2009-07-22 Appareillage et procédé de détection automatique de particules biologiques Withdrawn EP2313755A1 (fr)

Applications Claiming Priority (2)

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DE102008035771A DE102008035771B4 (de) 2008-07-31 2008-07-31 Vorrichtung und Verfahren zur automatischen Detektion von biologischen Partikeln
PCT/EP2009/059446 WO2010012641A1 (fr) 2008-07-31 2009-07-22 Appareillage et procédé de détection automatique de particules biologiques

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EP2313755A1 true EP2313755A1 (fr) 2011-04-27

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EP (1) EP2313755A1 (fr)
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WO (1) WO2010012641A1 (fr)

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US20110263044A1 (en) 2011-10-27
WO2010012641A1 (fr) 2010-02-04
DE102008035771B4 (de) 2011-04-07

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