WO1998006821A1 - Distribution de particules - Google Patents

Distribution de particules Download PDF

Info

Publication number
WO1998006821A1
WO1998006821A1 PCT/GB1997/002070 GB9702070W WO9806821A1 WO 1998006821 A1 WO1998006821 A1 WO 1998006821A1 GB 9702070 W GB9702070 W GB 9702070W WO 9806821 A1 WO9806821 A1 WO 9806821A1
Authority
WO
WIPO (PCT)
Prior art keywords
particles
substrate
particle
dispensing
detection
Prior art date
Application number
PCT/GB1997/002070
Other languages
English (en)
Inventor
Philip Shaw
Original Assignee
Pressium Limited
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 Pressium Limited filed Critical Pressium Limited
Publication of WO1998006821A1 publication Critical patent/WO1998006821A1/fr

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Classifications

    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C12M33/07—Dosage or metering devices therefore
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B07—SEPARATING SOLIDS FROM SOLIDS; SORTING
    • B07C—POSTAL SORTING; SORTING INDIVIDUAL ARTICLES, OR BULK MATERIAL FIT TO BE SORTED PIECE-MEAL, e.g. BY PICKING
    • B07C5/00—Sorting according to a characteristic or feature of the articles or material being sorted, e.g. by control effected by devices which detect or measure such characteristic or feature; Sorting by manually actuated devices, e.g. switches
    • B07C5/36—Sorting apparatus characterised by the means used for distribution
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/00459—Beads
    • B01J2219/00468—Beads by manipulation of individual beads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties

Definitions

  • the present invention relates to a method and apparatus for dispensing single particles, and to a dilute liquid suspension of particles for use with the aforesaid method or apparatus.
  • fluids are dispensed into miniature vessels or onto reaction surfaces in order to assess a vast number of possible chemical or biochemical reactions.
  • Such reactions include growing bacteria, drug screening, assays, use of enzymes and the like, and all require fluid to be dispensed in a controllable weight or volume.
  • Patent Specification No WO 92/18608 discloses forming an array of bacteria, yeast or bacteriophage for the purpose of identifying particular constituents thereof .
  • An array of different particles is formed by directing a stream of droplets each containing on average one or a few biological particles at spaced locations on the array.
  • no means is provided for discriminating the number of particles per droplet, and the patentees prefer not to work in the range where there is a high likelihood of there being only one biological particle per droplet because there is then a large preponderance of empty droplets.
  • this specification contains much useful background material and its disclosure is incorporated herein by reference.
  • the applicants Biodot Limited manufacture automated dispensing equipment including a computer controlled X-Y tables and dispensers which may be of the "airbrush” type, may be a "drop on demand” airless dispenser for droplets of volume 2 nl or above, may use a syringe pump, and may be used to print patterns or droplets in a matrix array.
  • the present invention controllably deposits single particles by forming a liquid stream along which the particles flow in liquid, testing when a predetermined volume of liquid in said stream contains a single particle, and depositing the volume of liquid containing the single particle.
  • the invention provides a method for dispensing one or more different single particles onto a substrate.
  • a dilute liquid suspension of the particles is provided, and the liquid is passed through a tube whose diameter is such that the particles may become separated from one another along the length of the tube through which they often pass one a time.
  • a detector is provided for detecting when a predetermined volume of the liquid contains a single one of the particles, and means is provided for dispensing said predetermined volume onto a substrate.
  • the invention also concerns apparatus for dispensing a single particle, which comprises a reservoir for a dilute liquid suspension of the particles, and means for withdrawing the liquid from the reservoir and passing it through a tube such that as the liquid passes through the tube the particles tend to become separated from one another along the tube through which they often pass as individuals with a separation of liquid between the individual particles.
  • a detector is provided for detecting when a predetermined volume of the liquid contains a single one of the particles, and means is provided for dispensing said predetermined volume onto a substrate.
  • the invention is also concerned with a dilute liquid suspension of particles for use in the aforesaid method and apparatus and with the use of arrays of biological or other particles as aforesaid in microorganism cloning, screening a library of compounds made by combinatorial chemistry, or the like.
  • the particles in the liquid suspension or dispersion are typically of size from 100 n to 500 ⁇ m. They may comprise biological cells of e.g. bacteria, yeast or blood which may contain particular lengths of DNA e.g. in the form of plasmids which it is desired to grow. In such cell dispersions, which may contain a library of variants of the genetic material, each variant being associated with the single cell, it is important to be able to deposit just one cell in a particular location and then grow that cell into a colony in which that variant of the genetic material has become amplified.
  • the particles may also be viruses e.g. bacteriophages .
  • the particles may be beads used in combinatorial chemistry, which may carry different chemical compounds such as peptides or oligonucleotides .
  • Such beads e.g. of polystyrene are now commercially available e.g. from Bangs Laboratories in the USA or from Rapp Laboratories in Germany, and are manufactured to known sizes in which the particle size distribution about the nominal value is very sharp, typically ⁇ 5%. It is also possible to dispense other material e.g. particles or beads of pigment, ceramic, or microcapsules , or plant seeds or the like.
  • the most usual dispersion medium will be water or other aqueous liquid, but the use of other solvents, for example DMSO, THF or alcohol as the dispersion medium is not excluded.
  • the dilute suspension may be contained within a reservoir e.g. a closed pressurised reservoir.
  • the suspension is preferably uncontaminated with foreign matter and subsequent operations are preferably carried out while maintaining freedom from contamination.
  • the suspension is withdrawn from the reservoir by means of a capillary tube having a diameter of typically 100 to 2000 ⁇ m. Such a diameter is preferably at least three times the nominal diameter of the individual particles.
  • detection is preferably by means of first and second detectors looking at the tube in different directions e.g. in generally orthogonal directions .
  • a control system responsive to the outputs of the detectors is arranged to recognise a single particle as being present only when both of the detectors shows the presence of a single particle of the correct size.
  • the tube may be a plurality of sensor arrays dispersed along the tube to ensure that multiple particles are not shadowed one behind the other or to detect non-spherical particles.
  • the range of particle sizes is relatively narrow, so that the detector or detectors is or are generally arranged to accept particles of size no more than ⁇ 24% of the nominal value.
  • Detection is preferably optical and may work by passing the particles in the liquid medium past a light source which generates a light field having a plurality of variations in intensity spaced along the tube or a plurality of light beams spaced along and/or across the tube, and detecting variations of light intensity caused by the particles passing through the variations in the light field.
  • the particles being detected are bacteria or other particles that are poor absorbers of light
  • the light detected is preferably that scattered by the particles.
  • the apparatus is used to disperse particles which are good light absorbers, then light absorbance or an obscuration method, possibly using a continuous light field and a video detection system or possibly using a plurality of light beams and detectors, may be employed.
  • a method and apparatus for measuring particles in this way is disclosed in WO 93/16368, the disclosure of which is incorporated herein by reference.
  • an individual particle on the basis of its size may be carried out optically, the invention is not limited to this mode of individual particle detection and e.g. ultrasound may be used.
  • Individual particles may be tagged by a fluorescent dye, visible colour or other marker so that different types of particle can be recognised e.g. by different fluorescences or visible colour. This would enable different particle types or substances to be deposited in a known sequence on a substrate e.g. in a single location or would enable a given particle type to be deposited at a given location. It is possible to detect a single particle by measuring the intensity of fluorescences emitted by particles and analysing a video image to identify single particles.
  • particles may also be measured by the intensity of fluorescence emitted by the particles or by computer measurement of the image captured by a video camera.
  • the individual particles may also be made detectable by virtue of an electrical charge applied thereto e.g. by ionisation in an aqueous medium, or they may contain a particle of a magnetic material such as magnetite by which they may be recognised.
  • the combination of means tor recognising the presence of a single particle within a volume of fluid and means for recognising the type of that particle adds to the possible versatility of the apparatus. This plurality of different single particles may be dispensed to the same location for the purposes of:
  • Figures 1 and 2 are diagrammatic perspective views of first and second embodiments of the invention
  • Figure 3 is an enlarged diagrammatic view of the tip of a third form of the deposition apparatus
  • Figures 4A and 4B are diagrammatic views of the deposition tip according to alternative forms of the invention.
  • Figure 5 is a simplified block diagram showing the sequence of operation of the apparatus of Figure 1 ;
  • Figure 6 is a diagrammatic view of a continuous field light source and the resulting image in a variant of the apparatus of figure 1;
  • Figure 7 is a diagrammatic view of a multiple beam light source and the resulting image in a variant of the apparatus of figure 1.
  • a single particle dispensing apparatus 10 is supported above an X-Y-table 12 which carries a substrate 14 which may, for example, be a culture plate for yeast or bacteria.
  • the substrate 14 and the dispensing apparatus 10 are relatively movable in indexed steps as indicated by the arrows 16.
  • the dispensing apparatus includes a pressurised tank 18 which contains a sparse dispersion of biological or organic particles, and the dispersion is fed from the tank through an outlet line 20 of internal diameter of e.g. 1 mm.
  • Flow of fluid through the line 20 may be interrupted by an in-line valve 24 which may be a high speed solenoid operated valve, for example an INKA valve available from the Lee Company of Connecticut, USA.
  • the line 20 then passes through an optical detector 26, which may, for example, be a single optical detection system as disclosed in W093/16368 or is preferably a pair of such optical detectors arranged to look at the liquid passing through the line 22 in orthogonal directions.
  • a gutter 30 may be positioned to catch droplets discharged from the portion 28, and to return them to the tank 18 by the action of a pump 32 e.g. a peristaltic pump.
  • the gutter 30 is displaceable from the non- discharge position shown in solid lines to a discharge- permitting position shown in dotted lines by the action of a solenoid 34 controlled by a control unit 36.
  • Operation of the pump 22 and the valve 24 are controlled by lines 38, 40 from the control unit 36, and the output signals from the optical detector 26 are fed to the controller 36 through line 42.
  • the controller 36 also controls the solenoid 34 through line 44.
  • a pump which can deliver the fluid in constant volumes, e.g. a measuring pump available from Fluid Metering Inc. of Oyster Bay, New York, USA or a syringe pump available from Cavro Inc. of Sunnyvale, California.
  • the dispensing apparatus 10 carries a gutter 30 via which portions of the fluid stream which are not to be dispensed can be recycled to a holding tank 18.
  • the dispensing apparatus 50 does not contain a gutter but instead is maintained (except when fluid is to be dispensed) in register with a discharge port 52 located away from the X-Y table 12, the discharge line 52 discharges to waste, and the dispensing apparatus 50 may be moved between its stand-by position and its dispensing position as indicated by arrow 54.
  • Figure 3 shows an alternative arrangement for handling droplets discharged from the end of discharge portion 28.
  • An electrical field applied across plates 60 normally causes the droplets to be deflected into a gutter 62, except when they are to pass undeviated onto the substrate 14.
  • the liquid passing along the holding or discharge portion 28 of the outlet line 20 may notionally be divided into individual portions or length 1,, 1 2 , 1 3 , each corresponding to one period of flow of liquid along the line 20.
  • discharge is permitted when a portion 1 2 containing a single particle is bounded by previous or subsequent portions 1,, 1 3 in which no particle is present.
  • the portion 1 3 of liquid containing a single particle is bounded by two or more previous portions 1,, 1 2 and two or more subsequent portions 1 4 , 1 5 in which no particle is present.
  • the portion 1 2 of Figure 4A or 1 3 in Figure 4B is discharged onto the substrate, but empty portions e.g. 1 2 , 14 in Fig. 4B, or parts thereof, may also be discharged onto the substrate.
  • the portions 1, to 1 3 or 1, to 1 5 preferably correspond to individual droplets which form at the end of tube 28, these typically being of volume about 100 nl per drop for 1 ⁇ m particles or the constant volume being delivered by the pump. These volumes are chosen to give optimal speed and accuracy for any given particle size. Thus the total volume of the portion 28 may typically be 300-500 nl. If desired, the individual portions 1 1 — 1 3 or 1 ,—1 5 of fluid may be physically separated from one another as they pass along the portion 28 e.g. by bubbles of air which may be formed before, during or after detection of the presence of a particle in a given length of fluid passing along the output tube 28.
  • FIGS. 6 and 7 variants of the detector 26 are shown in Figures 6 and 7.
  • a continuous field light source 70 is provided to one side of the tube portion 28, and passage of a particle produces a shadow in the resulting image 72 which is captured for example by a video camera.
  • the continuous field light source provides planar illumination like that from a fluroescent light tube.
  • Figure 7 there is provided a multiple beam light source to one side of the tube portion 28 which casts image 82 to the other side of the light source which is detected by a detector or a by multiplicity of detectors for the respective beams.
  • a suitable multiple beam light source is available from Image Optics Components Ltd, Basildon, Essex, part QBS101 Quartz Beam Splitter.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et un appareil de distribution de particules uniques. On introduit une suspension liquide et diluée de particules dans un tube (20) dont le diamètre est tel que les particules passent en général dans le tube l'une après l'autre avec un intervalle entre les particules individuelles. Un dispositif (26) détecte le moment où un volume prédéterminé de liquide contient une seule des particules, après quoi le volume prédéterminé est distribué sur un substrat. Les particules peuvent par exemple être des cellules isolées ou des perles de polymère utilisées en chimie combinatoire.
PCT/GB1997/002070 1996-08-12 1997-08-01 Distribution de particules WO1998006821A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9616914A GB2316081B (en) 1996-08-12 1996-08-12 Dispensing of particles
GB9616914.9 1996-08-12

Publications (1)

Publication Number Publication Date
WO1998006821A1 true WO1998006821A1 (fr) 1998-02-19

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ID=10798370

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/002070 WO1998006821A1 (fr) 1996-08-12 1997-08-01 Distribution de particules

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GB (1) GB2316081B (fr)
WO (1) WO1998006821A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPQ629100A0 (en) * 2000-03-16 2000-04-15 Btf Pty Ltd Process for preparing controlled samples of particles such as microorganisms and cells
GB2551116B (en) * 2016-05-31 2019-05-15 Solentim Ltd Dispensing method and apparatus
GB2602105A (en) * 2020-12-18 2022-06-22 Solentim Ltd Cell dispenser

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3826364A (en) * 1972-05-22 1974-07-30 Univ Leland Stanford Junior Particle sorting method and apparatus
US4230558A (en) * 1978-10-02 1980-10-28 Coulter Electronics, Inc. Single drop separator
US4279345A (en) * 1979-08-03 1981-07-21 Allred John C High speed particle sorter using a field emission electrode
US4538733A (en) * 1983-10-14 1985-09-03 Becton, Dickinson And Company Particle sorter with neutralized collection wells and method of using same
US4661913A (en) * 1984-09-11 1987-04-28 Becton, Dickinson And Company Apparatus and method for the detection and classification of articles using flow cytometry techniques
US4793705A (en) * 1987-10-07 1988-12-27 The United States Of America As Represented By The United States Department Of Energy Single molecule tracking
DE3925093A1 (de) * 1989-07-28 1991-01-31 Roland Dipl Phys Hartig Vorrichtung zur trennung von an magnetischen partikeln (beads) gebundenem biologischem material in einem magnetfeld
US5206143A (en) * 1985-11-01 1993-04-27 Smithkline Beecham Corporation Method and reagents for performing subset analysis using quantitative differences in fluorescence intensity
EP0626455A1 (fr) * 1993-05-28 1994-11-30 Enea Ente Per Le Nuove Tecnologie, L'energia E L'ambiente Méthode d'analyse, de séparation et de microencapsulation d'éléments cellulaires
WO1997038318A1 (fr) * 1996-04-04 1997-10-16 Novartis Ag Dispositif servant a compter de petites particules et appareil de tri comprenant ce dispositif

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4347935A (en) * 1979-05-16 1982-09-07 The United States Of America As Represented By The United States Department Of Energy Method and apparatus for electrostatically sorting biological cells
GB9108066D0 (en) * 1991-04-16 1991-06-05 Amersham Int Plc Method and apparatus for forming an array of biological particles
GB9202887D0 (en) * 1992-02-12 1992-03-25 Cambridge Consultants A particle sizing system based on incoherent structured illumination

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3826364A (en) * 1972-05-22 1974-07-30 Univ Leland Stanford Junior Particle sorting method and apparatus
US3826364B1 (fr) * 1972-05-22 1984-09-25
US4230558A (en) * 1978-10-02 1980-10-28 Coulter Electronics, Inc. Single drop separator
US4279345A (en) * 1979-08-03 1981-07-21 Allred John C High speed particle sorter using a field emission electrode
US4538733A (en) * 1983-10-14 1985-09-03 Becton, Dickinson And Company Particle sorter with neutralized collection wells and method of using same
US4661913A (en) * 1984-09-11 1987-04-28 Becton, Dickinson And Company Apparatus and method for the detection and classification of articles using flow cytometry techniques
US5206143A (en) * 1985-11-01 1993-04-27 Smithkline Beecham Corporation Method and reagents for performing subset analysis using quantitative differences in fluorescence intensity
US4793705A (en) * 1987-10-07 1988-12-27 The United States Of America As Represented By The United States Department Of Energy Single molecule tracking
DE3925093A1 (de) * 1989-07-28 1991-01-31 Roland Dipl Phys Hartig Vorrichtung zur trennung von an magnetischen partikeln (beads) gebundenem biologischem material in einem magnetfeld
EP0626455A1 (fr) * 1993-05-28 1994-11-30 Enea Ente Per Le Nuove Tecnologie, L'energia E L'ambiente Méthode d'analyse, de séparation et de microencapsulation d'éléments cellulaires
WO1997038318A1 (fr) * 1996-04-04 1997-10-16 Novartis Ag Dispositif servant a compter de petites particules et appareil de tri comprenant ce dispositif

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
B. STIEGLITZ ET AL: "MECHANIZED SYSTEMPS FOR MEDIA DISPENSING, INOCULATION, AND REPLICATION OF MICROORGANISMS", BIOTECHNOLOGY AND BIOENGINEERING, vol. 28, no. 9, 1986, USA, pages 1310 - 1317, XP002052235 *

Also Published As

Publication number Publication date
GB2316081A (en) 1998-02-18
GB9616914D0 (en) 1996-09-25
GB2316081B (en) 2001-05-09

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