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WO2007012000A2 - Classificateur de particules presentes dans un liquide utilisant un ejecteur de gouttelettes a grand orifice - Google Patents

Classificateur de particules presentes dans un liquide utilisant un ejecteur de gouttelettes a grand orifice Download PDF

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Publication number
WO2007012000A2
WO2007012000A2 PCT/US2006/028014 US2006028014W WO2007012000A2 WO 2007012000 A2 WO2007012000 A2 WO 2007012000A2 US 2006028014 W US2006028014 W US 2006028014W WO 2007012000 A2 WO2007012000 A2 WO 2007012000A2
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WIPO (PCT)
Prior art keywords
liquid
particles
airborne
droplet
classifier
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PCT/US2006/028014
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English (en)
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WO2007012000A3 (fr
Inventor
Steven Clyde Hill
Richard Kounai Chang
Hermes Chi-Yuan Huang
Yongle Pan
Original Assignee
Steven Clyde Hill
Richard Kounai Chang
Hermes Chi-Yuan Huang
Yongle Pan
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Application filed by Steven Clyde Hill, Richard Kounai Chang, Hermes Chi-Yuan Huang, Yongle Pan filed Critical Steven Clyde Hill
Publication of WO2007012000A2 publication Critical patent/WO2007012000A2/fr
Publication of WO2007012000A3 publication Critical patent/WO2007012000A3/fr

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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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • 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
    • G01N15/1492—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties within droplets
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/019—Biological contaminants; Fouling
    • 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
    • G01N2015/1481—Optical analysis of particles within droplets
    • 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
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid

Definitions

  • This invention pertains generally to chemical analytical and immunological testing, and particularly to processes wherein samples are analyzed by using self-operated mechanisms or devices, and more particularly to processes wherein a continuously flowing stream of a sample or carrier fluid is formed and flows into and through analysis wherein the continuously flowing stream is segmented by alternately injecting a sample, reagent or any number of fluids into a common flow path.
  • a need for improved instrumentation to identify biological particles carried in air exists in industry.
  • the instrumentation should be able to run continuously and have a rapid response, but still use only a very small amount of consumable items such as liquids, reagents, or filters, so that the cost of running continuously is not prohibitive.
  • consumable items such as liquids, reagents, or filters, so that the cost of running continuously is not prohibitive.
  • such instruments may be deployed in many locations, for example, public buildings, airports, subways, and military installations. Because it would be useful to deploy many such instruments, the acquisition and operational costs should not be large.
  • a way to identify biological particles in air is to collect the particles into a water solution that contains fluorescent-tagged antibodies (F-Ab) that were chosen for their tendency to bind to a specific type of bacteria, spore, rickettsia, virus, or other particle that may be in the air sample.
  • F-Ab fluorescent-tagged antibodies
  • the particles collected from air include one or more of the specific bacteria to which the F-Ab bind preferentially, then the F-Ab diffuse to the specific bacteria and bind to them. Then if the sample's fluorescence is measured on a fine scale, the bacteria with the F-Ab bound to the surface appear as bright spots in a less bright background.
  • the water solution itself retains some background fluorescence because it takes time for the F-Ab to diffuse to the surface of the bacteria, but also because the antibodies may bind to other particles in the sample, although they should bind far less well to these particles, and because some of the fluorophore may come unbound from the F-Ab, and because the amount of F-Ab may exceed the number of sites on the bacteria to which the F-Ab may bind, and for other less important reasons.
  • the fluorescence of the fluorophore bound to the antibody does not change after a binding event. That is, whether the antibody is bound or not, the fluorescence of the fluorophore is unchanged. What changes is the local concentration of the fluorophore, so that when the liquid is broken into small droplets, the droplet containing a particle with many bound F-Ab will be brighter than droplets that contain only the background concentration of F-Ab.
  • Fluorescence microscopy and flow cytometry are two methods used to determine the numbers of F-Ab tagged bacteria in a sample. Fluorescence microscopy is relatively slow and not simple to automate for a flow-through system that runs continuously.
  • Flow cytometers are used extensively to enumerate particles, especially biological cells, in liquid samples, and especially fluorescent- labeled cells.
  • Flow cytometry is not used for routine, continuous monitoring and identification of particles collected from air samples, in part because it would be expensive to deploy and operate such instruments, even if they were automated and combined with highly efficient instruments that collect airborne particles into liquid.
  • the reagents required to continuously operate existing flow cytometers to identify of biological agents in samples collected from air are expensive. Also, there has been concern that clogging of orifices in flow cytometers may be a problem, at least in applications of flow cytometry to identification of particles collected from air.
  • an instrument that analyzes the liquid samples into which particles from air have been collected does not need to operate at 100,000 particles per second, as some commercial flow cytometers do. A rate of 10 to 1000 particles/second may be adequate; (3) Because collection of particles from air requires significant amounts of energy, then if flow cytometry is used to identify and enumerate the particles collected from air, it would be ideal if the flow cytometer detects every particle that is collected, i.e., not miss some particles because they do not flow through the detection volume.
  • fluorescent-labeled mammalian cells have a much larger fluorescence than labeled bacteria, and can therefore be detected above the background fluorescence, i.e., the fluorescence from the unbound antibodies, in a larger volume of liquid.
  • the interrogation volume should be small. To obtain a small interrogation volume, while keeping the internal cross section of the cytometer tube not too small, in order to minimize clogging, while also minimizing the usage of sheath flow, is difficult.
  • the present invention provides a classifier for particles in liquid by enumerating particles of interest in a liquid sample according to an optical property of the particles of interest.
  • the classifier includes a large-orifice droplet ejector that holds the liquid sample and ejects droplets of the liquid sample, wherein the ejected droplets of the liquid sample are at least several times smaller than any orifice surrounding the liquid sample at the liquid-air interface, so that clogging of the orifice by particles that are in the liquid sample is minimized; and an airborne-droplet measurement subsystem that measures an optical property of each of the droplets of the liquid sample, so that the optical property of any particles of interest in the droplets of the liquid sample can be estimated from the measured optical property.
  • the present invention also provides a method of enumerating particles of interest in a liquid sample according to an optical property of the particles of interest.
  • the method includes the steps of: ejecting droplets of liquid sample using a large-orifice droplet ejector that holds the liquid sample; and measuring an optical property of each droplet of the liquid sample by an airborne-droplet measurement subsystem so that the optical property of any particles of interest in the droplets of the liquid sample is be deduced from the measured optical property.
  • FIG. 1 schematically illustrates a classifier for particles in a liquid.
  • FIG. 2 schematically illustrates a classifier for particles in a liquid with an acoustic wave liquid ejector (AWLE).
  • FIG. 3 schematically illustrates a classifier for particles which collects particles from air into a liquid.
  • FIG. 4 illustrates a classifier for particles in which the airborne particles are drawn through a nozzle and then directly impacted into the collection liquid into input well.
  • an object of the present invention is to provide a classifier for particles in liquid (CPL) 100 that can measure particles of interest (POI) 2OA in a liquid sample (LS) 10, while circumventing several problems that occur in some types of flow cytometers. That is, the CPL 100 provided: (a) does not require a liquid sheath flow, (b) examines essentially all of the particles 20 in the LS 10, (c) has a very small interrogation volume, and (d) has a relatively low probability of clogging.
  • LODE large orifice droplet ejector
  • the main subsystems of this CPL are as follows: (1 ) large orifice liquid ejector (LODE) that holds LS, and ejects droplets of the liquid sample (DLS) 30 through an orifice 220, where the orifice 220 is large relative to the size of the DLS 30 ejected, and to any particles 20 in the LS 10; and
  • ADMS airborne-droplet measurement subsystem 400 that measures an optical property of the DLS 30 ejected by the LODE 200, so that the presence of POl 20 in the DLS 30 ejected can be determined from this measured optical property.
  • the liquid sample (LS) 10 contains particles (20). Some of these particles 20 may be particles of interest (POI) 2OA.
  • the POI 2OA have at least one optical property that is different from the particles 20 that are not POI 2OA. In a preferred embodiment that optical property is the fluorescence in some wavelength band, and the POI 2OA have a greater fluorescence in this wavelength band than do the particles 20 that are not POI 2OA. In another preferred embodiment the POI 2OA have a greater fluorescence in both of two different wavelength bands than do the particles 20 that are not POI 2OA. There is no size restriction on the POI 2OA.
  • a POI 2OA may be 0.5 micrometers or greater
  • a POI 2OA can be as small as a single molecule that contains a single fluorescent molecule, as in the single-molecule in droplets work of Michael Barnes and Michael Ramsey et al., described above, or it may be a single quantum dot. It is the optical property, not the physical size of the particle 20, that determines whether a particle 20 is a POI 2OA.
  • particle-size can be a part of the criterion for what is or is not a POi 2OA, because size can sometimes be estimated from the light scattering abilities of the particle 20 but it is not necessary in general.
  • the LS 10 can contain multiple types of POI 2OA where each of these multiple types of POI 2OA differ from each other based on at least one optical property.
  • the objective for the CPL 100 is to enumerate each type of POI 2OA in the LS 10, that is, to classify the different types of POI 2OA. For simplicity we describe this invention primarily in terms of enumerating a single type of POI 2OA in one sample.
  • An example of the LS 10 is a water mixture which contains: (i) water, (ii) particles collected from air, and (iii) fluorescent-tagged antibodies (F-Ab) that were chosen for their tendency to bind to a specific type of bacteria that may be present in the air sample. If the specific type of bacteria is in the water mixture, the F-Ab diffuse to and bind the surface of the specific bacteria. Once a sufficient number of F-Ab bind to the a particle, this particle becomes sufficiently more fluorescent than a similar volume of the LS 10 that does not contain that specific bacteria, and so this bacteria becomes a POI 2OA.
  • F-Ab fluorescent-tagged antibodies
  • the POI 2OA could be as small as a single fluorescent molecule as in the single-molecule in droplets detection work described above.
  • concentration of the specific bacteria in the LS 10 is extremely high, most of the DLS 30 ejected contain no bacterial particles, but only contain unbound F-Ab. This unbound F-Ab gives a background signal.
  • the DLS 30 that contain the specific bacteria also contain unbound F-Ab, but in addition they contain the F-Ab that has bound to the specific bacteria, and so these DLS 30 have a significantly greater fluorescence.
  • the concentration of the specific bacteria in the sample is determined from the number of droplets that have this significantly greater fluorescence.
  • the DLS 30 By choosing the DLS 30 to be small, for example 4 to 8 micrometers in diameter, the background signal from the unbound F-Ab, relative to the F-Ab bound to the specific bacteria, can be minimized, and so the detection sensitivity can be increased.
  • a key advantage of the present invention is that it can eject small DLS 30, and thereby achieve high detection sensitivity, and it does this without a comparably small orifice 220.
  • the antibodies can be chosen to bind to specific spores, viruses, rickettsia, or other materials.
  • the recognition elements need not be antibodies, but may be antibody fragments, aptamers, or other biomolecules, or may be dyes that bind to specific types of molecules, such as acridine orange which binds to DNA and can indicate whether a cell is alive or dead.
  • the LS 10 is not obtained by collecting particles directly from air, but is obtained by washing a surface that may be contaminated, or it may be from waste water or a blood sample.
  • the CPL 100 has two main subsystems: (a) a large-orifice droplet ejector (LODE) 200 that holds the LS 10, and ejects droplets of the LS (DLS) 30 through an orifice 220, where the orifice 220 is large relative to the size of the DLS 30 ejected and to any particles in the LS 10, and (b) an airborne-droplet measurement subsystem (ADMS) 400 that measures an optical property of the DLS 30 ejected by the LODE 200, so that the presence of POI 20 in the DLS 30 ejected can be determined from this measured optical property.
  • LODE large-orifice droplet ejector
  • ADMS airborne-droplet measurement subsystem
  • the LODE 200 and the ADMS 400 may be connected with a gas-tight connection, but need not be. For example, when some types of commercially available or off- the-shelf ADMS 400 are used, it may be most convenient to not use a gas- tight-connection of the ADMS 400 to the LODE 200.
  • the humidity is high enough and the time before measurement is short enough that the DLS 30 evaporates relatively little before its fluorescence or other optical property is measured.
  • An advantage of this embodiment is that the ejected DLS 30 will tend to move on the same trajectory of the other DLS 30 because all the DLS 30 ejected have a somewhat constant mass independent of the size of particles contained in the DLS 30.
  • the humidity is low enough that the liquid in the DLS 30 evaporates rapidly and so, at the time the DLS 30 is measured the DLS 30 may be primarily solid and, possibly very small.
  • An advantage of this embodiment is that the there is no additional liquid to complicate measurement of the particles that remain after the liquid evaporates.
  • the orifice 220 is large relative to the size of the DLS 30 ejected, and to any particles 20 in the LS 10, so that clogging of the orifice is unlikely even if particles that are larger than the DLS 30 are in the LS 10, while at the same time, small DLS still can be ejected and analyzed, so that the measured optical properties of DLS that do not contain POI are not such that they cannot be distinguished from DLS that do contain POI.
  • An advantage of the LODE is that it can repeatedly eject very small DLS without requiring a small orifice.
  • one type of LODE is the acoustic wave liquid ejector (AWLE) as described by Dia Huang and Eun Sok Kim, “Micromachined Acoustic-Wave Liquid Ejector," Journal of Electromechanical Systems, 10, 442-449 (2001 ), herein incorporated by reference.
  • AWLE acoustic wave liquid ejector
  • droplets as small as 5 micrometers in diameter were ejected, even though the opening, which can be considered to be the "orifice" is a square with 1600 micrometer sides, which is many times larger than typical droplet sizes that would be useful for measuring optical properties of particles.
  • the ejected droplets have a diameter that is larger than the orifice.
  • a vibrating orifice droplet generator or a typical piezoelectric droplet generator the ejected droplets have a diameter that is larger than the orifice.
  • repeatedly ejecting droplets that have diameters less than 25 micrometers and also contain significant amounts of bacteria or other particles that are one micrometer and larger is very difficult because the orifices tend to clog.
  • the solutions must be filtered in order to keep particles in the solution from clogging the orifice, but the filtering removes the particles.
  • the AWLE 240 in this present invention is not restricted to the type of "acoustic wave liquid ejector" described by Huang and Kim, or Kim and Kwan incorporated above. Any of a variety of acoustic wave liquid ejectors that do not need a small orifice to restrict the size of the ejected DLS 30 can be used for the present invention.
  • One such acoustic wave liquid ejector is the "acoustic ejection device" as described by Richard N. Ellson, Mitchell W. Mutz, and Richard Michael Caprioli, US Patent Application Publication, 2005/0121537 A1 , Publication Date, June 9, 2005, herein incorporated by reference, in which they also refer to several of their prior patents and patent applications using such devices.
  • the volume of the ejected droplet is less than about 1 pL, e.g., in the range of about 0.025 pL to about 1 pL" (paragraph 15 of their application).
  • the DLS 30 diameters are in the range 3 to 10 micrometers, a range that is useful for our primary goal in this main embodiment, that of measuring small numbers of POl 2OA that contain F-Ab tagged bacteria or other biological particles.
  • AWLE 240 Another such example of an AWLE 240 is the "liquid ejector" described by Hiroshi Fukumoto, Jyunichi Aizawa, and Hiromu Narumiya, US Patent, 6154,235, "Acoustic Liquid Ejector and Printer Apparatus Incorporating the Ejector," herein incorporated by reference.
  • the LODE 200 can also be the "piezoelectric droplet ejector" with “flextensional transducer” as described by Gokhan Percin and Butrus T. Khuri-Yakub, "Piezoelectric Droplet Ejector for Ink-jet Printing of Fluids and Solid Particles," Review of Scientific Instruments, 74, 1120-1127 (2003), herein incorporated by reference.
  • This device requires an orifice 220, which in the paper by Percin and Khuri-Yakub, is 50 to 200 , micrometers in diameter, a size that is large compared to the sizes of the particles 20 and DLS 30 that would be useful for many applications, for example, 5 or 10 micrometers in diameter. With this device, Percin and Khuri-Yakub ejected droplets with diameters as small as 5 micrometers.
  • the LODE 200 is the "Ultrafine Fluid Jet Apparatus," similar to that described by Kazuhiro Murata, Tsukuba-shi, and Ibaraki-ken, US Patent Application Publication US 2005/0116069 A1 , herein incorporated by reference.
  • the key difference is that in the present invention there is provided a hole in the counter electrode, positioned so that the ejected DLS 30 moves through this hole (instead of colliding with the counter electrode as in the Murata et al., invention), so that the optical properties of the DLS 30 can be measured by the ADMS 400.
  • the purpose of the ADMS 400 is to measure the desired optical property of the droplets in some embodiments and particles in some embodiments that enter it.
  • the ADMS 400 is an example of a type of commercially available ADMS 400 that would be useful for measurement of the particles 20, including the POI 2OA, that could remain after a droplet evaporated, for cases where the droplet does evaporate, the TSI Aerodynamic Particle Sizer (for example, the 3320) or any of a number of similar instruments that have been sold by TSI for the past 20 years, would be useful.
  • TSI Aerodynamic Particle Sizer for example, the 3320
  • Measurement of fluorescence, spontaneous Raman emission, lasing, stimulated Brillouin scattering, stimulated Raman scattering, sum-frequency generation, and other emission from airborne droplets are reviewed by Steven C. Hill and Richard K.
  • This CPL 100 might be termed a "flow cytometer with acoustic- wave generation of droplets," and that would fit with a broad definition of flow cytometer.
  • Gucker's airborne-particle counter can be used as a flow cytometer (see Howard Shapiro, Practical Flow Cytometry, 4 th edition (John Wiley, 2003), p. 10), and our fluorescence particle spectrum analyzer (Richard K. Chang et al., US Patent 6,532,067, and Richard Chang et al., US Patent Application Publication 2004/0125371 A1 , July 1 , 2004) could be termed a flow cytometer.
  • This CPL has similarities to the droplet-based single-molecule detection (SMD) methods in which the liquid sample is broken into a series of droplets, each of which contains either zero or one (or in the unlikely cases two or three) single molecules, as described by N. Lermer, M. D. Barnes, C. Y. Kung, W. B. Whitten, and J. M. Ramsey, "High efficiency molecular counting in solution: Single-molecule detection in electrodynamically focused microdroplet streams," Analytical Chemistry, 69, 2115-2121 (1997), and by C. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, and J. M.
  • SMD droplet-based single-molecule detection
  • the CPL 100 uses an LODE that can eject droplets of a size that is desirable for measurement, and so the droplet's fluorescence can be measured relatively soon after it is ejected, and this feature, along with the ability to eject droplets in a repeatable direction (several of the example LODE were developed in part for inkjet printing) mean that no linear quadrupole for focusing is needed, and that no low-vapor-pressure liquid is needed in the liquid sample, requirements that could be restrictive for cases in which the speed of reaction, for example between antibodies and particles, is important, and where it is not desirable to use additional fluids such as glycerol.
  • FIG. 2 schematically illustrates a classifier for particles in a liquid with an acoustic wave liquid ejector (AWLE).
  • the LODE 200 is the acoustic-wave liquid ejector (AWLE) 240 as described by Dia Huang and Eun Sok Kim, "Micromachined Acoustic-Wave Liquid Ejector," Journal of Electromechanical Systems, 10, 442-449 (2001 ), which is incorporated herein by reference as fully set forth, where the AWLE 240 is illustrated in Figures 1 , 3 and 11 , of that paper and where Figures 15, 16 and 18 of that paper show photographs of ejected droplets.
  • AWLE acoustic-wave liquid ejector
  • the AWLE 240 further includes a "solid plate having a circular hole” (SPCH) 250 as described by Huang and Kim on p. 448.
  • SPCH circular hole
  • the "circular hole" of the SPCH 250 defines the orifice 220.
  • This SPCH 250 provides a means to keep the level of the LS 10 in the CPL 100 constant, so that the size of the DLS 30 ejected does not vary with time. Without the SPCH 250 the LS 10 level in the AWLE 240 may vary with time if nothing else is done to keep it constant, and if it does vary the size of the DLS 30 ejected may vary, or may not be ejected at all.
  • the orifice 220 may be comparable in size to one side of the AWLE 240, and theoretically there is no upper limit to that dimension.
  • 5- micrometer-diameter DLS 30 could be ejected at a rate of over 10 kHz.
  • This problem of detecting an increase in fluorescence in a volume of LS 10 that contains specific bacteria as compared with a volume that does not contain the specific bacteria also occurs with flow cytometers where the cells or particles are carried in a continuously flowing liquid. By minimizing the volume that is detected at any one time, it is possible to maximize the signal-to-background ratio, and the ability to clearly measure the specific bacteria desired.
  • a significant volume of sheath fluids are required to focus this sample volume into the small region that is interrogated, and/or not all of the LS 10 is measured, and/or the cross section of the capillary through which the LS 10 flows is so small that clogging can be a problem.
  • the ADMS 400 employs a laser 410 that emits light having a wavelength tuned to excite fluorescence in the fluorophore of the F-Ab.
  • the fluorescence emitted from the DLS 30 is collected by a lens 420, then filtered by an optical filter 422 that blocks the excitation light and passes the fluorescence, and is then detected by a photodetector 424.
  • the photodetector 424 is a photodiode. In another embodiment it is a photomultiplier tube.
  • a second photodetector measures the light that is elastically scattered from the droplet. From this scattered light intensity the size of the DLS 30 is estimated.
  • the DLS 30 is ejected downward. In other embodiments the DLS 30 is ejected in other directions. For embodiments in which the orifice 220 is so large that the LS 10 is not held in the LODE 200 by capillary forces, the DLS 30 is ejected upward or somewhat upward and the ADMS is positioned substantially above the LODE 200.
  • a means is included to generate and control an airflow that entrains the droplets ejected from the LODE 200 and to carry these droplets to the region where the optical property is measured.
  • optical properties are also measured, usually in addition to the fluorescence, in some embodiments of the CPL 100.
  • Other such optical properties are fluorescence lifetime, fluorescence polarization, multiphoton-excited fluorescence, elastic scattering, two-dimensional angular optical scattering, absorption, laser-induced breakdown spectroscopy, and Raman emission.
  • the light scattered at multiple excitation wavelengths is measured and used to estimate the absorption spectrum of the particle at the excitation wavelengths. Such scattering, absorption and emission throughout the optical range, from terahertz, to infrared through visible to ultraviolet.
  • upconverting phosphors are used to label the antibodies (see, for example, J. Hampl, et al., "Upconverting phosphor reporters in immunochromatographic assays," Analytical Biochemistry, 288 (2), 176-187, January 15 (2001 ), herein incorporated by reference).
  • upconverting phosphors are used to label the aptamers.
  • upconverting phosphors are used to lable RNA probes which bind to selected RNA or DNA molecules.
  • quantum dots are used to label the antibodies. In other embodiments quantum dots are used to label the aptamers.
  • the liquid evaporates from the DLS 30 and then scattering is measured by several detectors and used to estimate the size of the DLS 30 that remains after the liquid has evaporated, and in another embodiment, the two-dimensional angular optical scattering by the particle is measured so it can provide information about the shape of the DLS 30 that remains, as described in US Patent Application Publication 2003/0223063, by Steven Clyde Hill, Ronald Gene Pinnick, Yong-Le Pan, Kevin Aptowicz, Kristan P. Gurton, and Richard Kounai Chang, titled, "Method and Instrumentation for Determining Absorption and Morphology of Individual Airborne Particles," herein incorporated by reference.
  • the optical property of the DLS 30 and its residue is measured at multiple positions as the DLS 30 moves, so that these multiple measurements can be used to reduce uncertainties in the measured optical properties for each DLS 30.
  • FIG. 3 a classifier for particles which collects particles from air into a liquid is schematically illustrated.
  • the input port 210 of the LODE 200 is connected to the output of an airborne-particle into liquid collector (APLC) 500, which collects particles from air into a liquid.
  • APLC airborne-particle into liquid collector
  • the APLC is an impaction-based device, such as the one described by H. N. Phan and A. R. McFarland, "Aerosol- to- Hydrosol transfer stages for use in bioaerosol sampling,” Aerosol Science and Technology, 38, 300-310, 2004, herein incorporated by reference.
  • the APLC is an impaction based APLC similar to that described by D. A. Masquelier, F. P. Milanovich and K. Willeke, "High Air Volume to Low Liquid Volume Aerosol Collector," US Patent 6,520,034 B1 , herein incorporated by reference.
  • the collection liquid is in an input well of a microfluidic chip that is similar to one of the cell buffer wells of the microfluidic chips that are supplied in the "Cell Fluorescence LabChip Kit” sold by Agilent for their Bioanalyzer 2100.
  • FIG. 4 schematically illustrates a classifier for particles in which the airborne particles are drawn through a nozzle and then directly impacted into the collection liquid into input well.
  • This input well is directly connected through a microfluidic tube to the input port 210 of the LODE 200 of the CPL 100.
  • this APLC also includes a means to deposit an additional droplet of liquid onto the position where the particles collide with the collection liquid so that will be drawn into the collection liquid and be analyzed.
  • any of a number of means to replenish the liquid in this microfluidic input well can be used.
  • impaction-based APLC There are many more examples of impaction-based APLC.
  • air- based puffer can impart the impaction force selectively onto particles flowing in a stream based on other criteria, allowing for presorting of the particles before processing by the CPL.
  • the collection liquid held in the APLC includes labeled molecular recognition elements, which can be fluorescent-tagged antibodies, fluorescent-tagged antibody fragments, fluorescent-tagged aptamers or other biorecognition molecules which can bind to selected particles, so that the collection liquid into which the airborne particles impact, becomes an LS 10, and need not later be mixed with labeled molecular recognition elements to become the LS 10.
  • labeled molecular recognition elements which can be fluorescent-tagged antibodies, fluorescent-tagged antibody fragments, fluorescent-tagged aptamers or other biorecognition molecules which can bind to selected particles, so that the collection liquid into which the airborne particles impact, becomes an LS 10, and need not later be mixed with labeled molecular recognition elements to become the LS 10.
  • the collection liquid does not contain all the needed labeled molecular recognition elements, but contains other substances which prepare the collected airborne particles so that they can react with the labeled molecular recognition elements, which are added later.
  • the collection liquid can contain a protein that can bind nonspecifically to a variety of materials, and thereby block some of these non-specific binding sites, so that the biorecognition molecules that are added later do not bind nonspecifically to these sites.
  • the APLC includes the aerosol deflection system (ADS) described by Richard K. Chang, Jean-Pierre Wolf, Veronique Boutou, and Yongle Pan, "Systems and Methods for Sorting Aerosols," US 2005/0028577 A1 , published, February 10, 2005, and incorporated herein by reference.
  • the ADS as claimed by Chang et al. deflects selected airborne particles into a "collection zone comprising one or more pathogen identification devices.”
  • the particles may be selected based on their laser-induced fluorescence being similar to that of biological aerosols, or based on other criteria.
  • the deflected airborne particles (DAP) that were deflected by the ADS of Chang et al. are collected into the collection liquid by impacting them into the collection liquid.
  • the deflected particles are impacted into the input well of a microfluidic chip which is connected by a short microfluidic channel to the input port 210 of the LODE 200.
  • any means for collecting the deflected airborne particles into a collection liquid can be used.
  • the APLC is a shooting-droplet airbome- particle collector having: (a) an airborne-particle detector, and (b) a droplet generator that shoots a collection droplet in a direction such that it collides with the airborne particle. Typically the collection droplet continues on a trajectory such that it moves into some type of system where it can be analyzed.
  • This shooting-droplet airborne-particle collector is described in a patent application by Steven Clyde Hill, Richard Kounai Chang and Jean-Pierre Wolf, "Aerosol Particle Analyzer for Measuring the Amount of Analyte in Airborne Particles," USPTO Number, 11/126,515, submitted May 9, 2005, notice of allowance April 18, 2006, herein incorporated by reference.
  • the droplets in the shooting-droplet airborne-particle collector are shot only at particles that have laser-induced fluorescence and/or other properties that are similar to those of biological particles or other types of particles as desired.
  • the liquid in the droplet that is shot at the airborne particle can include fluorescent-tagged antibodies or other labeled molecular recognition elements (biorecognition molecules) that can bind to specific particles.
  • labeled molecular recognition elements biorecognition molecules
  • MABs were emphasized as the labeled molecular recognition elements. In the present invention this emphasis on MABs is not needed.
  • the APLC includes of a means to impart a charge to the airborne particles drawn into it, and an oppositely charged volume of liquid held on the end of a capillary, as described in a patent application by Steven Clyde Hill and Horn-Bond Lin, "Aerosol into Liquid Collector for Depositing Particles from a Large Volume of Gas into a Small Volume of Liquid," submitted to the USPTO November 18, 2004, incorporated herein by reference, where the liquid held on the end of the capillary typically includes fluorescent-tagged antibodies or other biorecognition molecules. Other types of electrostatic-field enhanced aerosol-particle collectors may be used in other embodiments.
  • the CPL 100 also includes a container to collect the DLS 30 that DLS 30 that have had their optical property measured can be stored for later analysis by other means.
  • the CPL 100 also includes means to sorting the DLS 30 according the optical property measured, by applying a charge to the ejected DLS 30 and then deflecting these DLS 30 using voltage controlled electrodes toward one container or another.
  • means are as described in H. M. Shapiro, Practical Flow Cytometry, 4 th edition, herein incorporated by reference.
  • more than one LODE 200 eject DLS 30 so that the sample rate can be increased. That is, arrays of LODE 200 and lasers 410 can be used to process samples in parallel for increased speed and capacity. For some of these embodiments, only one laser 410 is used but multiple photodetectors 424 are used.
  • the laser 410 is a diode laser. In some embodiments the laser 410 is replaced by a light emitting diode (LED).
  • LED light emitting diode
  • the present invention also provides a method of enumerating particles of interest in a liquid sample according to an optical property of the particles of interest.
  • the method includes the steps of: ejecting droplets of liquid sample a large-orifice droplet ejector that holds the liquid sample; and measuring an optical property of each of droplets of the liquid sample by an airborne-droplet measurement subsystem so that the optical property of any particles of interest in the droplets of the liquid sample is be estimated from the measured optical property.
  • the ejected droplets of the liquid sample are at least several times smaller than any orifice surrounding the liquid sample at the liquid-air interface to minimize clogging of the orifice by any particles that may be present in the liquid sample.
  • the large-orifice droplet ejector can be an acoustic wave liquid ejector or a piezoelectric droplet ejector with flextensional transducer.
  • the acoustic wave liquid ejector may include a solid plate having a circular hole to reduce evaporation from the liquid surface and to provide for capillary forces to draw liquid into the reservoir.
  • the large-orifice droplet ejector is positioned such that the droplets are ejected in a substantially downward direction so that the particles that are denser than the liquid are preferentially ejected.
  • the optical property can be fluorescence, fluorescence lifetime, fluorescence polarization, multiphoton-excited fluorescence, elastic scattering, including two-dimensional angular optical scattering, absorption, laser-induced breakdown spectroscopy, or Raman emission.
  • the optical property of the droplet and its residue are measured at multiple positions as the droplet moves to provide multiple measurements for improved accuracy of the measured optical property.
  • the method can further include one or more of the following steps: (1 ) collecting airborne particles into a collection liquid through an airborne-particle into liquid collector (APLC) connected to the input of the classifier for particles in liquid; the collection liquid can contain labeled molecular recognition elements which selectively bind to specific particles collected from air into the liquid, whereby the specific particles become labeled and acquire different optical properties than particles that do not bind to the labeled molecular recognition elements to become particles of interest;
  • APLC airborne-particle into liquid collector
  • the airborne-particle into liquid collector is an impaction based aerosol-particle collector and includes an aerosol deflection system (ADS), which deflects selected airborne particles into a collection liquid and a means for holding the collection liquid until it is drawn into the classifier for particles in liquid.
  • ADS aerosol deflection system
  • the airborne-particle into liquid collector can further include means for depositing additional droplets of liquid onto where the particles collide with the collection liquid so that any particles resting on the surface of the collection liquid without entering the collection liquid because of surface tension are drawn into the collection liquid and analyzed.
  • the labeled molecular recognition element is selected from fluorescent-labeled antibodies and fluorescent-labeled aptamers.

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Abstract

L'invention concerne un classificateur de particules présentes dans un liquide, qui permet de classer les particules étudiées d'un échantillon liquide selon une propriété optique desdites particules. Le classificateur comprend: (a) un éjecteur de gouttelettes à grand orifice qui contient l'échantillon liquide et éjecte des gouttelettes de celui-ci, les gouttelettes éjectées étant plusieurs fois inférieures à tout orifice entourant l'échantillon, à l'interface liquide-air, de sorte que le risque d'obturation de l'orifice par les particules présentes dans l'échantillon liquide est réduit; et (b) un sous-système de mesure de gouttelettes en suspension dans l'air, qui permet de mesurer une propriété optique de chacune des gouttelettes de l'échantillon liquide, la propriété optique de n'importe quelle particule étudiée dans les gouttelettes de l'échantillon pouvant être estimée à partir de cette mesure. Les gouttelettes de l'échantillon liquide pouvant être très petites, ce système permet de mesurer avec une grande sensibilité les particules étudiées, l'une après l'autre, comme en cytométrie de flux, avec peu de risques de boucher le dispositif, même si on n'utilise pas d'écoulement en gaine.
PCT/US2006/028014 2005-07-19 2006-07-19 Classificateur de particules presentes dans un liquide utilisant un ejecteur de gouttelettes a grand orifice WO2007012000A2 (fr)

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Publication number Priority date Publication date Assignee Title
JPWO2017130707A1 (ja) * 2016-01-26 2018-12-27 株式会社リコー 液滴形成装置、分注装置、方法

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US4230031A (en) * 1978-04-26 1980-10-28 Coulter Electronics, Inc. Biohazard containment apparatus and method
US5032381A (en) * 1988-12-20 1991-07-16 Tropix, Inc. Chemiluminescence-based static and flow cytometry
US5292499A (en) * 1990-09-11 1994-03-08 University Of Wales College Of Cardiff Method of preparing medical aerosol formulations including drug dissolved in reverse micelles
US5698397A (en) * 1995-06-07 1997-12-16 Sri International Up-converting reporters for biological and other assays using laser excitation techniques
US6827287B2 (en) * 2002-12-24 2004-12-07 Palo Alto Research Center, Incorporated High throughput method and apparatus for introducing biological samples into analytical instruments
US20050127206A1 (en) * 2003-12-10 2005-06-16 Xerox Corporation Device and system for dispensing fluids into the atmosphere

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2017130707A1 (ja) * 2016-01-26 2018-12-27 株式会社リコー 液滴形成装置、分注装置、方法
EP3409377A4 (fr) * 2016-01-26 2019-06-26 Ricoh Company, Ltd. Dispositif de formation de gouttelettes et dispositif distributeur
US10732087B2 (en) 2016-01-26 2020-08-04 Ricoh Company, Ltd. Liquid droplet forming device, dispensing device, and method of preparing base material
AU2020202476B2 (en) * 2016-01-26 2021-05-06 Ricoh Company, Ltd. Droplet Forming Device And Dispensing Device

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