Classifications

• • 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
  • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
  • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
  • G01N15/13—Details pertaining to apertures

Definitions

• This invention relates to an improvement in a method of separating small particles and for analysing particles in liquid suspensions.
• a filtration device which device comprises a substantially circular orifice through which liquid can pass, there being an insert mounted within the circular orifice to form an annular orifice, the insert projecting beyond the end of the circular orifice for a distance such that ratio of the width of the annular orifice to the distance the insert projects beyond the circular orifice is from 1: 10 to 1:500, preferably 1:20 to 1:250.
• the insert can be tapered and moveable in and out of the circular orifice to provide an annular orifice of variable size.
• the angle of the taper should be less than 8° and preferably 4 ° -8 °.
• angle of the taper is meant the angle formed between two lines on opposite sides of the insert projected, if necessary, beyond the end of the insert.
• the ratio of the width of the annular orifice to the length of the substantially circular orifice is less than 4:1 and can be below 1:1; in practice the orifice can be almost closed by the insertion of the insert, in which case the ratio can be as low as 1 : 10 or less.
• liquid containing the particles to be separated is passed through the device so that the liquid passes over the projecting portion of the insert before passing through the orifice and we have found unexpectedly that there is very effective prevention of particles above a specific size from passing through the annular orifice, even though these particles are smaller than the orifice and would have been expected to pass through. Also there is substantially very little blocking of the annular orifice, even though quite "sticky" organic or biological particles are present.
• the effect is such that for a given size of orifice there is a nearly complete separation of particles above a specific size.
• the end of the insert projecting out is preferably curved or smooth or of a shape which reduces the likelihood of turbulence as liquid passes over it, e.g. in the shape of a tapered round column.
• Insertion of the insert into the substantially circular orifice will decrease the width of the annular orifice and will alter the flow characteristics of liquid flowing through the annular orifice and this can enhance the filtration effect.
• the device of the invention can be made of any material which is inert to the liquid being passed through it.
• the device of the invention is useful for separating particles from bio-active systems such as cells where particles are typically in a size range of 0.1 to 30 microns and it is useful in separating and quantifying particles below a specific size.
• the device of the invention can be used in any system where it is desired to remove particles above a particular size in the appropriate range, e.g. up to about 100 microns.
• a module can be made or formed of a plurality of the devices of the invention joined together to cover a larger cross-sectional area.
• the inserts would be fixed in position and the module would act as an efficient precise filter.
• a particular application of the present invention is for separating agglutinated from non-agglutinated particles.
• the beads can be made of glass, latex, polystyrene or other material known or used for this purpose. If it were possible to determine how many beads were agglutinated this would be a measure of the concentration of the specific material in the liquid to which the coated beads are added.
• the use of the device of the present invention is an effective method for separating agglutinated from non-agglutinated particles.
• Particles of a diameter substantially below the width of the annular gap can pass through the gap relatively easily, thus it is possible to separate the agglutinated beads from the non-agglutinated beads.
• This diversion of the larger particles has a relatively reduced shearing effect compared to conventional filtration and can be used to separate agglutinated particles from non-agglutinated particles.
• a liquid is thought to contain a specific bio-active material, its concentration can be determined by adding to the liquid a known number of beads coated with an antigen or antibodies to the bio-active material to give a known concentration of coated beads.
• the agglutinated beads are separated from the non-agglutinated beads and the concentration of non-agglutinated beads are determined.
• the volume of the sample containing the coated beads should preferably be kept as low as possible as this will reduce the distance between the beads and the reactant, thereby increasing the number of collisions or contacts between the beads and the reactant in a given time and thus increasing the number of beads to which the reactant adheres.
• the liquid containing the agglutinated and the non-agglutinated beads is diluted for ease of operation e.g. with a diluant salt solution.
• Another application of the present invention is for detecting and counting live microorganisms, particularly when mixed in liquids with dead micro-organisms or other particles in the same size range. It is known that live micro-organisms can be impregnated with a substance which causes a vital stain which, when exposed to a specific waveband of light, particularly ultra-violet light will fluoresce at a second waveband, this is known as a Stokes Shift.
• an incident beam is required, such as a laser, which has an intensity which is likely to damage micro-organisms present and would cause there to be so much reflected light that it would make detection of the relatively weak fluorescence difficult.
• the insert is made of an optically transparent insert material whereby liquid flowing through the orifice can pass over the insert, and light is transmitted through liquid and fluorescence of fluorescable particles in the liquid passing over the insert can be detected.
• fluorescable particles particles which fluoresce when exposed to ultraviolet light.
• the orifice preferably has a diameter of between 60 and 200 ⁇ m, more preferably of between 70 and lOO ⁇ m.
• the orifice can be formed in any suitable inert material such as glass quartz, ruby, plastic, sapphire or other material, using known methods.
• the insert is preferably made of optical quality glass and is constructed of two different glasses of different refractive indices, so that one glass forms a sheath around the other glass in order that total internal reflection takes place for light passing down the insert.
• the light is then preferably conducted away from the insert by means of a conventional light guide.
• the insert is preferably placed as close to the orifice as possible without affecting the flow of liquid through the orifice to an unacceptable degree.
• the insert has a substantially flat end so that light emitted from the particles as they pass the insert can pass into the end of the insert, in some cases, the particles may hit the end of the insert.
• the light can pass down the insert into the liquid and the fluorescent light emitted by the particles can then pass back up the insert.
• the insert By suitably positioning the insert in relation to the size of the orifice, substantially all the particles in the liquid are caused to pass near enough to the end of the insert so that they receive light at a strength sufficient to cause the fluorescable particles to fluoresce.
• the light from a narrow bandpass source can pass across the end of the insert and the fluorescent light then passes into the insert.
• the fluorescent light emitted by the fluorescable particles passes up through the insert via filters to remove any reflected ultra-violet light before going to a detector and counter.
• Conventional detectors and counters can be used such as a single photon counting module, e.g. model SPCM- 1 00-PQ, made by General Electric Canada Inc. Modulation of the incident light will also assist in the ability to read the low level output of the fluorescent particles.
• Live particles such as cells or micro-organisms, fluoresce at a different wavelength to dead particles so, by counting the number of particles which fluoresce at different wavelengths, the ratio of dead to live particles can also be obtained.
• the present invention enables particles to be counted accurately as they pass through the orifice and so it enables the concentration of the fluorescable particles in the liquid to be obtained, even when mixed with other particles in the same size range.
• the outputs from the light detector can be fed to a recording device or into a computer for further processing.
• Fig. 1 is a side view of a device of the invention
• Fig. 2 is a crosssectional view of the device viewed along the insert.
• Fig. 3 shows the light path from orifice to detector and light source
• Fig. 4 is a side view of the device in solution
• Fig. 5 is a side view of the orifice and input light source
• a plate (1) has a circular orifice (3) of diameter (d) which is 70 microns, a tapered insert (2) fits within the orifice (3) to leave an annular orifice (4) of width (a).
• the insert (2) projects a distance (b) beyond plate (1).
• the angle ⁇ is the angle of taper of the insert.
• the orifice was formed in a synthetic ruby and the insert (A) was formed in quartz glass.
• the length of the circular orifice (3) shown as (e) was 25 microns and a was 5 degrees.
• a glass insert (12) of diameter 30-100 ⁇ m is positioned adjacent to the orifice (11) with a gap between the end of the orifice and the plate (17) of a range of 2 - 400 ⁇ m, so that liquid can pass over the orifice.
• Light guide (13) made of glass cable is attached to the insert (12) so that light will pass from the insert down the light guide and vice versa.
• the light guide (13) bifurcates to form two arms (14) and (15).
• the arm (14) goes to a source of ultraviolet light (25) and the arm (15) goes to a filter (16) which filters out reflected ultraviolet light and then bifurcates again into two arms (27) and (28).
• the arm (27) has a filter to remove red light and the arm (28) has a filter (20) to remove green light.
• Arms (27) and (28) then go to light counters (21) and (22) respectively, which are single photon counting modules No. SPCM- 100 PQ made by General Electric Canada Inc.
• liquid containing particles go through orifice (11) into and over insert (12).
• Ultra-violet light passes down light guide (14), strikes the particles and reflected fluorescent light and passes back down insert (12). Some of this reflected light passes along light guide (15) where ultra-violet light is filtered out by filter (16). Some of this light passes down light guide (27) and through filter (19) which only allows green light to go to counter (21). Light passing down light-guide (28) passes through filter (20) which only allows red light to go to counter (22).
• Live particles will fluoresce in the red part of the spectrum and the light they emit will go to counter (22), which counts such particles as a pulse of red light.
• Dead particles will fluoresce in the green/yellow part of the spectrum and the light they emit will go to counter (21), which counts such particles as a pulse of green/yellow light.
• This counting is done automatically by counters (21) and (22) and, if required, their output can be fed directly into a recording device, either as separate counts or as a ratio.
• a single filter which can be rotated to filter out sequentially red and green light, can be used. In this case, only one photon counting module is required.
• a container (33) contains liquid in which there are the particles which are to be detected and counted.
• a holder (32) consists of a vessel which has an orifice (23) of about 70 ⁇ m diameter in its bottom. There is a "spear” (24) made of glass which has a diameter of its end (26) of 60 ⁇ m. The spear (24) can be moved nearer and further from the orifice.
• Light collimator or guide (29) is able to pass light from light source (27) through filter (28) across the orifice (23) as shown by the arrows.
• the spear (24) can conduct light reflected by particles passing across orifice (23) through filter (31) to detector and counter (32).
• Conduit (25) is connected to a vacuum pump so that liquid can be drawn through the orifice (23).
• liquid containing the particles or organisms it is desired to detect and count is drawn through the orifice (23) by the action of the vacuum pump connected to conduit (25).
• the orifice bounded by the two sides (42a) and (42b) and liquid containing particles is drawn through the orifice.
• Light passes down the light guide (41) as shown by the arrows and emerges to illuminate the particles in the liquid.
• this light strikes an organism as shown by (44) which fluoresces the fluorescent light formed passes down the end of the probe or spear (43) as shown by the arrows.

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• Chemical & Material Sciences (AREA)
• Dispersion Chemistry (AREA)
• Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Separation Of Solids By Using Liquids Or Pneumatic Power (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Applications Claiming Priority (5)

Publications (1)

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• 2000
  • 2000-09-18 WO PCT/GB2000/003559 patent/WO2001022059A2/en not_active Application Discontinuation
  • 2000-09-18 AU AU74327/00A patent/AU7432700A/en not_active Abandoned
  • 2000-09-18 CA CA002384897A patent/CA2384897A1/en not_active Abandoned
  • 2000-09-18 EP EP00962679A patent/EP1238259A2/de not_active Withdrawn

Non-Patent Citations (1)

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