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
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • 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
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles

Definitions

• the present invention relates to the field of detection of biological or chemical targets from liquid sample collection.
• the object of the present invention is to meet the need for collection and / or concentration of biological or chemical targets and thus be useful in the context of the detection of undesirable elements such as pathogenic microorganisms or chemical pollutants.
• samples such as river water, cooling tower fluids, sanitary or drinking water networks, etc.
• the biological or chemical targets may be present in very small quantities in the samples and their detection is not then possible only by the removal of large volumes (greater than 50 ml).
• Biological or chemical target means microorganisms, ie enveloped or non-enveloped viruses, Gram-positive and negative vegetative bacteria, spore-forming bacteria, protozoa, microscopic fungi and fungi. yeasts, microplankton, pollen, animal cells and plant cells; we also mean chemical molecules (fertilizers, drugs, chemical molecules polluting such as by-products of the chemical industry, phytosanitary products ...) or biochemicals (antigen, protein, hormones or disrupting compound of the endocrine system ...) .
• US Patent Application 2006/0141450 describes a method for capturing cells, organelles or viruses present in samples of physiological origin using the properties of unspecific paramagnetic adsorption of magnetic beads.
• the foregoing methods are directed to the processing of samples having a volume of about 1 ml, they are not suitable for processing samples larger than 50 ml.
• the Pathatrix device of Matrix Microscience proposes the capture of targets such as pathogenic microorganisms in 250 ml samples by a loop circulation of the sample in a tubular circuit in which magnetic functionalized capture beads are immobilized on a plane magnet. The beads being immobilized, this device leads to a low capture efficiency which does not allow in particular to detect targets present at a low concentration.
• the subject of the invention relates to an automatic device for treating a liquid sample of a volume V greater than or equal to 10 ml, preferably 50 ml and even more preferentially to 500 ml, comprising a reaction chamber, a volume v between 10 .mu.l and 5 ml, in which functionalized magnetic beads are placed allowing the capture of biological or chemical targets in an amount such that the ratio of the area of capture of the beads to the volume of the reaction chamber is between 0.2 and 200 m 2 / l, preferably between 0.2 and 20 m 2 / l and more preferably between 0.2 and 10 m 2 / l, said reaction chamber being equipped with: a system of non-invasive agitation allowing homogeneous dispersion of said beads; - a magnetic field that can be activated (ON position) or inactivated (OFF position); a fluid displacement
• FIG. 1 is a schematic representation of the device according to the invention comprising a reaction chamber (1) in which functionalized magnetic balls (2), a stirring system for example, an ultrasonic bath (3), a field are placed.
• magnetic (4) can be activated (ON position) or inactivated (OFF position) and a fluid displacement system (5) for introduction (51) and evacuation (52) of all or part of the contents of the chamber reaction.
• the reaction chamber has a volume of preferably between 50 and
• the reaction chamber is coated with a hydrophobic surface so as to avoid the adhesion of the capture beads on its inner walls; this hydrophobic surface is chosen from teflon, ETFE (ethylene trifluoroethylene), PFA (perfluoroalkoxy copolymer resin) and PTFE (polytetrafluoroethylene).
• teflon ethylene trifluoroethylene
• PFA perfluoroalkoxy copolymer resin
• PTFE polytetrafluoroethylene
• sample for example, from cooling circuit), environmental water (stream 3), or drinking water intended for human or animal consumption, and by extension any sample in which the target (s) biological or chemical detectors are in solution or in suspension.
• This sample may itself have been obtained from a sample or other sample likely to contain the desired biological or chemical targets, for example a food product, a body fluid, a sample of air, obtained by physical treatment and / or chemical, and / or biological according to any method adaptable by those skilled in the art.
• the device according to the invention has a capture capacity at very low target concentrations, it is therefore particularly suitable for the treatment of liquid samples that may contain only a very small quantity of biological or chemical targets; in particular, in the case of biological targets, the device according to the invention is thus capable of detecting biological targets present in an amount of less than or equal to 100 units per liter of sample, preferably in an amount less than or equal to 10 units per liter of sample, more preferably in an amount equal to one unit per liter of sample.
• the liquid samples are preferably taken or prepared under clean conditions with sterile material.
• the liquid samples are prepared according to techniques known to those skilled in the art (Stachowiak JC et al., Anal Chem., 2007; 79 (15): 5763-70 ).
• the fluid displacement system for introducing and evacuating all or part of the contents of the reaction chamber makes it possible to introduce a fraction of liquid sample into the reaction chamber. in order to capture the biological or chemical targets and then evacuate this fraction of the liquid sample after the step capture. This system also makes it possible to introduce the functionalized magnetic beads at the beginning and to evacuate them at the end of the treatment of the liquid sample.
• this fluid displacement system preferably consists of a pressure regulating system (Fluigent) or a pump (53) such as a piston pump, a diaphragm pump or a peristaltic pump.
• a pressure regulating system Frluigent
• a pump such as a piston pump, a diaphragm pump or a peristaltic pump.
• the stirring system allowing homogeneous dispersion of the beads is non-invasive, that is to say that it does not require the introduction of a stirring accessory into the reaction chamber thus avoiding the risk of contaminating the reaction medium. liquid sample.
• It may be a stirring system of magnetic beads functionalized by a moving magnetic field implemented by one or more rotating magnets; by setting the balls in motion by causing the contents of the reaction chamber to be returned and returned by means of the fluid displacement system; or by ultrasonic agitation.
• the stirring system is preferably an ultrasonic stirring (elastic waves whose frequency is between 15 kHz and a few hundred MHz) implemented by ultrasound transmission by water in an ultrasonic bath or directly by a ultrasonic transducer (Borthwick et al (2005) Journal of Microbiological Methods 60, 207-216, Belgrader et al (1999) Analitical Chemistry 71 (19), 4232-4236).
• the ultrasonic bath has a power of between 5 and 100 W / l, preferably 40 W / l and an effective power of between 50 and 400 W.
• An ultrasonic bath that can be used for the device according to the invention is the ultrasonic washer S -line from Fisher Scientific.
• the device according to the invention may optionally comprise an ultrasonic stirring system and another non-invasive stirring system of the functionalized magnetic beads.
• the magnetic field is produced by a permanent magnet or an electromagnet.
• a magnet of dimensions L ⁇ l ⁇ p 20 ⁇ 13 ⁇ 10 mm and having as characteristics a neodymium iron boron (NdFeB) magnetization grade N38 and a nickel / copper / nickel coating.
• NdFeB neodymium iron boron
• the characteristics of the electromagnets are for example the following: 12Vdc traction electromagnets 10 W and 1.8 mm stroke.
• 12Vdc traction electromagnets 10 W and 1.8 mm stroke When a permanent magnet is used, it must be able to be positioned both near the reaction chamber to attract the functionalized magnetic beads (ON position) and at a distance from the reaction chamber sufficient for it no longer exerts attraction force on the balls (OFF position); the passage between these two positions can be obtained by different means of displacement of the permanent magnet.
• an electromagnet When an electromagnet is used, it is positioned against or near the reaction chamber and is either in operating mode (ON) in order to attract the functionalized magnetic beads, or inactive (OFF), it then exerts attractive force on the functionalized magnetic beads.
• the effectiveness of the magnetic attraction of the magnetic beads functionalized by the magnet or electromagnet can be improved by adding to the reaction chamber a metal foam as described in US Pat. No. 6,159,378, preferably nickel metal foams.
• the magnetic beads are microspheres having a diameter ranging from nanometers to a few micrometers; preferably, the functionalized magnetic beads used in the device according to the invention have a diameter of between 0.5 and 10 ⁇ m, and more preferably of 1 ⁇ m in diameter. Care should be taken to select beads of a size greater than or equal to that of the biological or chemical target (s).
• the magnetic nature of the beads allows to control them in a container using a magnet. Beads do not have a magnetic property in the absence of an external magnetic field, which prevents the formation of aggregates and allows their reuse. Any type of magnetic beads known to those skilled in the art can be used in the device according to the invention; as such, mention may be made of the functionalized magnetic beads marketed by the Dynal or Chemicell companies.
• the magnetic beads are functionalized, that is to say that their outer surface is covered with molecules having the property of binding more or less specifically with one or more biological or chemical targets; these molecules are called capture material.
• the surface of the balls covered by the capture material constitutes the capture surface.
• this capture area is between 1.10 3 and 10 10 -2 m 2 .
• the electrostatic interactions the targets are captured according to the charges present on their surface.
• the capture surfaces contain end groups anion exchangers or cations of greater or lesser strength. It may be carboxyl, sulfone, phosphate, di-ethylaminoethyl (DEAE), poly-lysine, polyethyleneimine (PEI) and more generally charged polymers (Deponte et al., Anal Bioanal Chem 2004: 379: 419-426); hydrophobic interactions: the targets are captured according to the difference in hydrophobicity of surfaces.
• the hydrophobic regions of targets such as proteins, peptides or nucleic acids in an aqueous medium preferentially bind to a hydrophobic capture material such as a hydrocarbon chain from 1 (methyl) to 18 (octadecyl) carbon atoms; covalent bonds: functional end groups grafted onto the beads make it possible to bind the target ligands covalently; it is in particular amino group, hydroxyl, thiol, polyglutaraldehyde ....; the affinity bonds: the beads are covered with ligands allowing highly specific and selective binding such as interactions between an antigen and an antibody, hybridization between two nucleotide fragments, between biotin and streptavidin ...
• a hydrophobic capture material such as a hydrocarbon chain from 1 (methyl) to 18 (octadecyl) carbon atoms
• covalent bonds functional end groups grafted onto the beads make it possible to bind the target ligands covalently; it is in particular amino group,
• the magnetic beads are functionalized with two or more different capture materials for capturing two or more targets.
• Magnetic beads coated with several different capture materials are used; or several types of magnetic beads, each type of beads being covered with a different capture material.
• the functionalized magnetic beads are introduced into the reaction chamber in an amount such that the ratio of the capture surface to the volume of the the sample is between 0.2 and 200 m 2 / l, preferably between 0.2 and 20 m 2 / l, more preferably between 0.5 and 10 m 2 / l and most preferably between 1 and 5 m 2 / l, for example 2.8 m 2 / l.
• the device according to the invention has the advantage of allowing the treatment of liquid samples of large volume, greater than or equal to 10 ml, preferably 50 ml and even more preferably 500 ml, while keeping a small reaction chamber (Maximum of a few milliliters) thus allowing to retain the advantages of a capture of biological or chemical targets in small volume, that is to say: a smaller footprint and low cost of the device due to its small size; - greater ease and speed of recovery of the magnetic beads;
• This device is also advantageous in that it offers a good capture surface ratio with respect to the volume of the reaction chamber and a wide range of capture materials chosen according to the biological or chemical targets sought; finally, the functionalized magnetic beads can be recovered and reused.
• the automated processing of high volume liquid samples by the device according to the invention advantageously replaces the usual techniques for treating such samples, such as filtration or ultrafiltration.
• This device thus makes it possible to take a sample fraction likely to contain biological or chemical targets, to capture said targets and to concentrate in order to process them to confirm or even quantify their presence.
• this device can be positioned at the sampling location so that it performs regular sampling and analysis.
• the device according to the invention may be supplemented by a liquid sample collection system consisting of a fluid sampling system (61) such as a pump and a storage tank (62) of the sample. sample upstream of the reaction chamber ( Figure 2).
• the liquid sample collection system is connected either directly or via one or more intermediate reservoirs to the fluid displacement system for introducing a fraction of the liquid sample into the reaction chamber (51).
• the present invention also relates to a method for capturing biological or chemical targets likely to be present in a liquid sample of volume V of greater than or equal to 10 ml, preferably 50 ml and more preferably 500 ml, at the same time.
• the automation of the method according to the invention is programmed and controlled by a suitable computer system.
• the treatment of the biological or chemical targets captured on the functionalized magnetic beads may be carried out in the reaction chamber of the device according to the invention or in a tank connected to the fluid displacement system allowing the contents of the reaction chamber (52 ).
• this treatment consists of:
• the biological or biochemical analysis may for example consist in the cultivation on selective media of the biological targets eluted in the case of microorganisms and their characterization; or else the detection by antibodies of the biological targets ...
• the eluted biological targets are lysed in order to release their nucleic acids.
• the methods of lysis are widely known to those skilled in the art, it may be a chemical lysis using a lysis buffer containing detergents such as sodium dodecyl sulphate (SDS), lithium dodecyl sulphate (LiDS), sarcosyl or chaotropic agents such as guanidium hydrochloride (GHCl), guanidium thiocyanate (GTC), sodium iodide (NaI), perchlorate ..., enzymatic lysis with using proteinase K or lysozyme ... or mechanical lysis for example by coupling the effect of beads and ultrasound or by thermal gradient.
• GHCl guanidium hydrochloride
• GTC guanidium thiocyanate
• NaI sodium iodide
• perchlorate ... enzymatic lysis with using proteinase K or lysozyme ... or mechanical lysis for example by coupling the effect of beads and ultrasound or by thermal gradient.
• the treatment consists of: elution of the chemical targets using an elution elution buffer that the person skilled in the art according to said target then the analysis of the targets recovered by conventional chemical techniques such as nuclear magnetic resonance, chromatography, UV or IR spectroscopy, electrochemistry ...
• the detection is carried out by the techniques available to humans of the job he chooses according to the biochemical target, for example, a coupling of the target with a radioactively labeled antigen or by fluorescence etc.
• the invention also relates to a method for detecting biological or chemical targets comprising the steps a) to f) of the above capture method characterized in that it further comprises the steps of: g) elution said targets; and h) detecting said eluted targets in step g).
• step h) may consist in the lysis of said targets and in the detection of nucleic acids released during lysis.
• Figure 1 is a schematic representation of the device according to the invention.
• Figure 2 is a schematic representation of the device according to the invention further comprising a liquid sample collection system.
• Figure 3 illustrates the mounting of the device according to the invention used in the following example; said device comprises two identical assemblies in parallel each consisting of a reaction chamber (1), the two reaction chambers are positioned in an ultrasound bath (3) equipped with a magnet (4), they are connected, on the one hand , to a waste container (7) via a pump (53) and, on the other hand, to a reservoir (9) containing the sample, the sample supply of the reaction chambers is via solenoid valves (8).
• Figures 4 to 7 are histograms representing and comparing the amount of genomic units identified in samples according to the method of the invention (N) or initially present in the samples (NO).
• Example catching bacteria in a river water sample
• Fluid circulation is ensured by an Ismatec IPC-N peristaltic pump connected to PFA flexible tubes with an external diameter of 1.6 mm and an internal diameter of 1 mm.
• These flexible tubes act as a reaction chamber in which the magnetic beads are stored.
• the balls are controlled by a magnet.
• the position of the magnet is controlled by the arm of an electromagnet; in its ON position, the magnet is in contact with the hose, in the OFF position, it is moved away from it.
• the region of the flexible reaction chamber and where the beads are positioned is immersed in an ultrasonic bath whose function is to disperse the aggregates formed by the beads after their capture in the magnetic field.
• the flexible tube Downstream, the flexible tube is connected to a 6-way valve (Upchurch V-1471-DC) to select the inlet liquid.
• a 6-way valve Upchurch V-1471-DC
• This volume v is positioned in the hose at the balls; the magnet is then put in the OFF position; the ultrasounds are lit and allow the beads to disperse in the fraction of volume v. The ultrasounds are then extinguished; the sample is moved in the flexible tube in order to resuspend the beads possibly deposited on the wall by ultrasonic action.
• the beads are left in contact with the sample for 45 seconds allowing target capture.
• the magnet is then set to ON, the balls are then captured by the magnetic field and agglomerated against the inner wall of the hose.
• the sample is pushed by the pump towards a trash at a speed of 300 ⁇ l / min and a new fraction of volume v of the sample is positioned in the reaction chamber where the beads are. This capture cycle is repeated 50 to 100 times.
• the system comprising several valves, it is possible to select a new input connected to a reservoir containing the elution buffer (or lysis if necessary). By action of the pump, this buffer is positioned at the storage area of the balls.
• ultrasound is applied to disperse the beads in the buffer.
• the targets are then eluted from the surface of the beads and optionally lysed.
• the magnet is then turned ON; the beads aggregate and are retained while the sample of elution buffer and, optionally, lysis comprising the targets is recovered through the pump.
• Detailed protocol The principle of this capture was implemented on two liquid samples, one of 20 ml of 10 mM pH 8 Tris-HCl buffer and the other of 10 ml of a river water (Rhône) taken on same day, these two samples were doped with targets: cells of Escherichia coli and Bacillus subtilis at about 10 6 genomic units (corresponds to about 10 6 bacteria).
• the magnetic beads used are super paramagnetic beads coated with a layer of polyethyleneimine (SiMAG-PEI Chemicell) which allows electrostatic type interactions with targets (bacterial cells); indeed, the bacteria are globally negatively charged and the PEI is a strong anion exchanger.
• the bead area / volume ratio of the sample fraction is 2.8 m 2 / l.
• the beads are recovered in 100 ⁇ l of 10 mM Tris-HCl. Then the DNA extraction is carried out directly on the beads: the lysis of the cells is carried out at 37 ° C. in a lysis buffer (5M guanidine-HC, 20 mM Tris pH 8, 1% sarcosyl). After 10 minutes of incubation, the mixture is placed for two minutes in an ultrasonic bath at 37 ° C.
• a lysis buffer 5M guanidine-HC, 20 mM Tris pH 8, 1% sarcosyl.
• the beads are pooled with a magnet and the supernatant is recovered and placed in 4 times its volume of 3M guanidine HCl solution, 20 mM Tris-HCl, 80% ethanol (v / v) pH 4; the DNA is then captured on 2.5 .mu.l of magnetic beads functionalized with silanol (SiMAG-Silanol, Chemicell). After two rinsing with a solution of 2 mM NaCl, 10 mM Tris-HCl, 75% ethanol (v / v), the purified DNA is eluted in 10 .mu.l of 10 mM Tris-HCl, pH 8 after 2 minutes in an ultrasonic bath .
• the DNA extraction and purification protocol may also be performed automatically with the instrumental device used in the previous capture step.
• the amount of DNA extracted is determined by quantitative PCR and is expressed in genomic unit. For this, 5 .mu.l of extracted DNA suspension are mixed with 5 .mu.l of the following PCR reagents: 0.3 .mu.M primers, 2.5 .mu.M GoId Taq polymerase, BSA (1.4 mg / ml), buffer IX (delivered with AmpliTaq Gold® DNA Polymerase from Applied Biosystems), MgCl 2 3 M, dNTP 200 ⁇ M, betaine 0.65 mM.
• the primers used have the following sequence: Esche ⁇ chia coli (16S ribosomal RNA gene) CoIiTQF Forward 5'-CATGCCGCGTGTATGAAGAA coliTQR Reverse 5'-CGGGTAACGTCAATGAGCAAA
• the number of genomic units initially present in the sample is determined by PCR by mixing 5 .mu.l of the starting liquid sample previously sonicated for 10 minutes with 5 .mu.l of the PCR reagent mixture described above. . Results target detection in Tris-HCl buffer samples
• results obtained show that the differences observed between the amount of DNA captured by the device according to the invention (N) and the amount of DNA present in the corresponding starting sample (NO) are lower than at 0.41 klo, which means that almost all the cells initially present were captured. target detection in river water samples
• results obtained show that the differences observed between the quantity of DNA captured by the device according to the invention (N) and the quantity of DNA present in the corresponding starting sample. (NO) are less than 0.21 g / l, which again shows that almost all the cells initially present were captured.

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