GB2316081A

GB2316081A - Dispensing of particles

Info

Publication number: GB2316081A
Application number: GB9616914A
Authority: GB
Inventors: Philip Shaw
Original assignee: BIO DOT Ltd; PRESSIUM Ltd
Current assignee: BIO DOT Ltd; PRESSIUM Ltd
Priority date: 1996-08-12
Filing date: 1996-08-12
Publication date: 1998-02-18
Anticipated expiration: 2016-08-12
Also published as: WO1998006821A1; GB9616914D0; GB2316081B

Abstract

A method and apparatus are provided for dispensing single particles. A dilute liquid suspension of particles is passed through a tube (20) whose diameter is such that the particles pass along the tube generally one at a time with a separation between the individual particles. Means (26) detects when a predetermined volume of liquid contains a single one of the particles after which the predetermined volume is dispensed onto a substrate. The particles may be e.g. single cells or they may be polymer beads used in combinational chemistry.

Description

DISPENSING OF PARTICLES 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.

In biochemical research, 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.

For example, in genetic engineering and Genome Research, it is important to be able to separate distinct strands of DNA. Previously known methods have involved inserting a mixture of strands in bacteria or yeast which are then grown to replicate the strands. In order to recover the different strands as separate entities, individual colonies of bacteria or yeast must then be picked out.

This process was, and still is, carried out by hand which both demands great skill and is labour intensive.

Attempting to automate the previous manual process does not bring about a sufficient reduction in the overall cost. It has been attempted to ink jet print bacteria the aforementioned process, but in no existing method of ink jet printing can it be guaranteed that each bacterial colony is derived from only a single organism and hence contains the same strand of DNA. There is therefore a need for a method of growing DNA in bacteria, yeast or other micro-organisms in which the individual colonies grow are easier to pick out and separate.

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.

However, 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. However, this specification contains much useful background material and its disclosure is incorporated herein by reference.

An analogous situation arises in the field of combinatorial chemistry, wherein libraries of compounds are produced on individual polymer beads, which need to be screened e.g. for biological activity. Combinatorial be screened e.g. for biological activity. Combinatorial chemistry has been reviewed by Michael Smith, New Scientist, 30 July 1996, pages 22 to 25, the disclosure of which is incorporated herein by reference. An example is given of growing a library of peptides on polymer beads about 10 gm across, and a difficulty which is identified in this article is identifying or tagging the beads so that they can be handled. Apart from the use of polymer beads in biochemical research, combinatorial chemistry is stated to be applicable in the field of inorganic chemistry e.g. for the identification of superconductors.

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 25 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 on average flow one-by-one separated by 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. With this arrangement the advantages of having only a single deposited particle at each site are obtained without the disadvantage of many empty deposition sites and without the presence of the single particle being decided by a statistical hit or miss.

In one aspect 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 become separated from one another along the length of the tube through which they mostly 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 whose diameter is such that as the liquid passes through the tube the particles become separated from one another along the tube through which they generally 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 nm to 500 gm. 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 plasmeds 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 polymer beads used in combinatorial chemistry, which may carry different biological compounds such as peptides or oligonucleotides. Such polymer 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 disperse inorganic material e.g. particles of pigment, particles of ceramic, microcapsule or the like. The most usual dispersion medium will be water or other aqueous liquid, but the use of organic solvent as dispersion media is not excluded.

The suspension of dilute liquid may be contained within a reservoir e.g. a closed pressurised reservoir, and where it contains organic or biological materials. 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 300 Rm, e.g. about 250 Clam. Such a diameter is preferably at least three times the nominal diameter of the individual particles. To avoid accidental passage of pairs or clumps of particles one behind the other past the detector, when an optical detector is used 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. In a further possibility there 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. As previously mentioned, the range of particles sizes is relatively narrow, so that the detector or detectors is or are generally arranged to accept particles of size + 20% 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, and detecting variations of light intensity caused by the particles passing through the variations in the light field. Where 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. Where the apparatus is used to disperse particles which are good light absorbers, then light absorbance or an obscuration method 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.

Although detection of 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. For example, individual particles may be tagged by a fluorescent dye or other marker so that different types of particle can be recognised e.g. by different fluorescences. 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. 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 for 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: (a) chemical reaction in a known specified or known unspecified sequence; or (b) construction of self-assembling objects.

Dispensing of a plurality of different single particles to different locations may be for the purpose of sorting and/or counting this particles. Dispensing of a plurality of single particles to the same or different locations may be for the purposes of: (a) counting the particles; or (b) dispensing an amount of material measured by size rather than weight or volume.

Various embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: 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; and Figure 5 is a simplified block diagram showing the sequence of operation of the apparatus of Figure 1.

In 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 about 250 zm. 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 WO93/16368 or is preferably a pair of such optical detectors arranged to look at the liquid passing through the line 22 in orthogonal directions.

From the detector 26 there depends a discharge portion 28 of the line 20 which is directed towards the substrate 14. 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 nondischarge position shown in solid lines to a dischargepermitting 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.

In the form of apparatus shown in Figure 1 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. In the alternative arrangement shown in Figure 2, 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.

As shown in Figures 4A and 4B, the liquid passing along the holding or discharge portion 28 of the outlet line 20 may notionally be divided into individual portions or length 11, 12, 13 as shown in Figure 28, each corresponding to one period of flow of liquid along the line 20. In a preferred arrangement, discharge is permitted when a portion 12 containing a single particle is bounded by previous or subsequent portions 1l, 13 in which no particle is present. In an alternative arrangement shown in Figure 4B, the portion 13 of liquid containing a single particle is bounded by two or more previous portions 11, 12 and two or more subsequent portions 14, 15 in which no particle is present. In each case, preferably on the portion 12 of Figure 4A or 13 in Figure 4B is discharged to waste, but empty portions, or parts thereof, may also be discharged onto the substrate.

Provided that only a single particle is dispensed, it is significant how much fluid is dispensed with it. The portions 11 to 13 or 11 to 15 preferably correspond to individual droplets which form at the end of tube 28, these typically being of volume about 100 nl per drop.

Thus the total volume of the portion 28 may typically be 300-500 nl. If desired, the individual portions I 3 or lj-15 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.

The operation of the machine is shown in simplified form in Figure 5 which is believed to be self explanatory.

Thus particles of a known constant single size can be suspended sparsely in a fluid which is passed to the holding tube 28 by means of the pump 22 and controlled by delivery valve 24. At the start of the tube is located the particle size detector 26 which operates by interrogating the particles with structured light or with a sensor array. Any particle measured as the correct size must therefore be a single particle, and any differently sized particle must be multiple particles.

If a single particle is detected and is identified as being alone in the delivery tube 28, it is held there in the fluid and can be dispensed and the fluid subsequently dried or filtered away if it is not required for maintaining the stability or integrity of the particle.

If multiple particles are present the fluid can be directed to waste or can be recirculated.

Various changes can be made to the embodiments described above without departing from the invention.

Claims

1. A method for dispensing a single particle which comprises: providing a dilute liquid suspension of particles; passing the liquid through a tube whose diameter is such that the particles pass along the tube generally one at a time with a separation between the individual particles; detecting when a predetermined volume of liquid contains a single one of the particles; and dispensing said predetermined volume onto a substrate.

2. The method of claim 1, wherein the particles are single cells.

3. The method of claim 2, wherein the particles are bacteria, yeast cells or blood cells.

4. The method of claim 1, wherein the particles are polymer beads.

5. The method of claim 4, wherein the polymer beads carry chemical, biochemical or biological compounds, reagents or organisms.

6. The method of any preceding claim, wherein detection of an individual particle is on the basis of its size.

7. The method of claim 6, wherein the detection accepts particles of size within + 20% of the nominal size of the particles.

8. The method of claim 6 or 7, wherein the detection is optical.

9. The method of claim 8, wherein the tube is larger than the individual particles, detection is by means of first and second detectors looking at the tube in generally orthogonal directions, and a single particle is recognised as present only when both detectors indicate a single particle of the correct size.

10. The method of claim 8 or 9, wherein detection is 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 direction of relative movement and detecting variations in light intensity caused by the particles passing through the variations in the light field.

11. The method of claim 10, wherein the light detected is light absorbed by or scattered from the particles.

12. The method of any of claims 1 to 5, wherein detection is by fluorescence or phosphorescence of the particles.

13. The method of any of claims 1 to 5, wherein detection is by ionic charge or magnetic or dielectric properties of the particles.

14. The method of any preceding claim, when used to deposit successive particles in a single place on the substrate.

15. The method of any of claims 1 to 13, comprising moving the substrate so that successive particles are deposited in different locations on the substrate.

16. The method of claim 15, wherein the successive particles are deposited along a track.

17. The method of claim 15, wherein successive particles are deposited in a two-dimensional array.

18. The method of any preceding claim, wherein volumes of liquid not containing a single one of the particles are discharged to waste.

19. The method of any of claims 1 to 17, wherein volumes of liquid not containing a single one of the particles are recycled to a reservoir.

20. A method for depositing one or more single particles, substantially as hereinbefore described with reference to the accompanying drawings.

21. Apparatus for dispensing a single particle, which comprises: a reservoir for a dilute liquid suspension of particles; means for feeding liquid from the reservoir through a tube whose diameter is such that in use the particles generally pass along the tube one at a time with a separation between the individual particles; a detector for detecting when a predetermined volume or liquid contains a single one of the particles; and dispensing means for dispensing said predetermined volume onto a substrate.

22. The apparatus of claim 21, wherein the detector is arranged to accept particles of size within + 20% of a nominal size for the particles.

23. The apparatus of claim 21 or 22, wherein the detection means is optical.

24. The apparatus of claim 23, wherein the tube is larger than the individual particles, first and second detectors look at the tube in generally orthogonal directions, and the detectors are connected to a means which signals the presents of a single particle only when both detectors have indicated a single particle of the correct size.

25. The apparatus of claim 23 or 24, wherein the detection means comprises a light source for generating a light field having a plurality of spaced variations in intensity, means for moving the particles in the fluid medium relative to the light field so that the particles pass successively through the intensity variations, and means for detecting variations in light intensity caused by passage of the particles relative to the light field.

26. Apparatus according to claim 25, wherein the detector is arranged to detect variations in the intensity of light scattered by the particles.

27. The apparatus of any of claims 21 to 26, further comprising means for bringing about relative movement of the dispensing means and the substrate along a track.

28. The apparatus of any of claims 1 to 26, further comprising means for bringing about relative movement in two dimensions between the dispensing means and the substrate so that the particles are deposited in an array.

29. A dilute liquid suspension of particles for use in the method or apparatus of any of preceding claim.

30. A method of cloning microorganisms (which term includes viruses) which comprises depositing different microorganisms on a support using the method or apparatus of any preceding claim, culturing the microorganisms and retrieving one or more clones from the array.

31. A method of screening a library of different chemical compounds which have been formed by combinatorial chemistry which comprises depositing in an array on a support beads carrying the different compounds using the method or apparatus of any preceding claim, subjecting the beads to a test procedure, and on the basis of the text procedure selecting and retrieving one or more desired compounds.

32. A method of depositing different single particles in an array at different locations on a substrate which comprises relatively moving deposition apparatus and the substrate to the several locations and at each location forming a stream of fluid containing particles spaced widely along the stream, detecting the presence of a single particle in a predetermined portion of the stream and depositing said predetermined portion onto a substrate.

33. The method of claim 32, wherein said predetermined portion is held in a reservoir tube.

34. The method of claim 32 or 33, wherein other portions of the stream are recycled.

35. The method of claim 32 or 33 wherein other portions of the stream are discharged to waste.

36. The method of any of claims 32-35, wherein the single particles in the stream of fluid are of different types, and means recognises the type of the individual particles.