WO2019204333A1 - Dispositif de tri de particules pour la séparation, l'isolement et l'enrichissement de particules à ultra-faible concentration - Google Patents

Dispositif de tri de particules pour la séparation, l'isolement et l'enrichissement de particules à ultra-faible concentration Download PDF

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WO2019204333A1
WO2019204333A1 PCT/US2019/027717 US2019027717W WO2019204333A1 WO 2019204333 A1 WO2019204333 A1 WO 2019204333A1 US 2019027717 W US2019027717 W US 2019027717W WO 2019204333 A1 WO2019204333 A1 WO 2019204333A1
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particle
particles
cells
sorter
sorting
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PCT/US2019/027717
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English (en)
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Enrico Gratton
Weian Zhao
Margaux BOUZIN
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The Regents Of The University Of California
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Publication of WO2019204333A1 publication Critical patent/WO2019204333A1/fr

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    • 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
    • G01N15/1429—Signal processing
    • 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
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • 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
    • 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
    • G01N2015/0038—Investigating nanoparticles
    • 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
    • G01N2015/0042—Investigating dispersion of solids
    • G01N2015/0053—Investigating dispersion of solids in liquids, e.g. trouble
    • 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
    • G01N2015/1006—Investigating individual particles for cytology
    • 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
    • G01N2015/1486—Counting the particles

Definitions

  • the invention relates to particle sorting, sample processing, biological assays, biotechnology, and diagnostics.
  • the same concentration limit applies to a variety of micro fluidic-based sorting devices [6], ranging from active systems exploiting acoustic, electric, optical or magnetic forces, to passive systems relying on inertial forces or immobilization procedures [3].
  • the typical adopted flow rate ( ' -pL/min) provides droplet-based micro fluidic sorting systems with low (- «2 kHz) throughput, and the small ( ⁇ mL) total handled sample volume is not suitable for fast diagnostic assays on low-abundant pathogens, which require exploration of a 0.2-10 mL clinical sample volume just to identify sufficient targets for meaningful downstream analysis [6,7].
  • flow cytometry and amplification-based methods including PCR (Polymerase Chain Reaction) are capable of reducing the assay time, but do not always meet the sensitivity and specificity required to target low-abundant bacteria in a pool of heterogeneous non-target species (for example, red blood cells in unprocessed blood samples) [12,13].
  • Standard methods often require complex and lengthy sample pre-processing, are unsuitable for turbid media, suffer from low throughput or simply cannot handle a large (>mL) total sample volume.
  • all these techniques fail in enabling the sorting and isolation of the detected pathogens for subsequent processing and analyses.
  • the device In combination with droplet blood microencapsulation and DNAzyme sensor fluorescence technology (i.e., in our recently developed Integrated Comprehensive Droplet Digital Detection - IC3D- platform [13]), the device has already been successfully applied to the detection of rare E. Coli (1-10000 units/mL) in spiked blood samples with a single, culture-free, specific and fast (1 to 4 hours) reaction [13].
  • This invention provides a particle-sorting unit, component or device that can be complemented and integrated with a particle counter, to enable separation, isolation and/or enrichment of target particles.
  • sorted particles are subject to downstream analyses and processing including sequencing for genetic profiling and diagnosis.
  • the said particles are nano- and micro-sized spheres, emulsions, droplets, pathogens and cells with various sizes and shapes.
  • the automated, integrated, user-friendly particle detection and sorting unit described here can be applied to the isolation of a variety of rare food-, water- and blood-bome target cell populations, including, e.g., low-abundant (antibiotic resistant) bacteria, circulating tumor or fetal cells, and hematopoietic stem cells in fluid samples.
  • low-abundant bacteria antibiotic resistant bacteria
  • circulating tumor or fetal cells hematopoietic stem cells
  • the particle-sorter comprises a real-time particle identification procedure, a receiver for the detected particles, and a micro-injector or an electrically controlled pneumatic, solenoid, or piezo valve system that generates negative pressure to direct the detected particles into the receiver.
  • the receiver is a needle or a capillary for particles that are nano- or micro-sized.
  • particles are micro-sized spheres, emulsions, droplets, pathogens or cells with various sizes and shapes, where the emulsions and droplets can comprise a cell, a plurality of cells.
  • a switchable valve can be provided whereby extracted particles can be dispensed into one or multiple containers after extraction.
  • the sorted particles are subject to downstream analyses and processing.
  • analyses and processing includes sequencing for genetic profiling and diagnosis.
  • the particle sorter is applied to the isolation of a variety of rare food-, water- and blood-bome target cell populations.
  • the target populations include low-abundant (antibiotic resistant) bacteria, viruses, circulating tumor cells, fetal cells, and/or hematopoietic stem cells, in fluid samples.
  • the particle sorter can be applied to infectious diseases, antibiotic resistance, cancer research, stem cell therapy, virus diagnostics, prenatal diagnostics, biotechnology industry, agriculture and/or environmental applications.
  • Figure 1A is a schematic of the particle-counter optical path
  • Figure IB shows the scanned volume in the IC3D particle counter
  • Figure 2A shows an exemplarary time trace
  • Figure 2B exemplifies the pattern-recognition algorithm adopted for the particle- counter data analysis for analyzing the time-trace of Figre 2A;
  • Figure 3 is a schematic of the particle-sorter prototype
  • Figure 4 shows fluorescence intensity time traces collected with the particle counter showing combatibility of the proposed sorting mechanism with droplet microfluidics;
  • Figure 5 A shows results (number of counted particles) of the pattern-recognition- based analysis to exclude turbulence effects induced by the needle of the particle sorter;
  • Figure 5B shows results (optimal standard deviation for the Gaussian recognition filter) of the pattern-recognition-based analysis to exclude turbulence effects induced by the needle of the particle sorter;
  • Figure 6 shows frames from a time-lapse image sequence demonstrating successful operation of the particle sorter
  • Figure 7 shows frames from a time-lapse image sequence acquired under the same experimental conditions of Figure 6 demonstrating control of the sorting mechanism (non target particles do not get sorted).
  • Figure 8A shows the prototype of the particle-sorter.
  • Figure 8B shows the cross-correlation of the signals between the detection and positioning volume.
  • Figure 8C shows an image of the extracted particle in the capillary used for extraction.
  • Figure 9A shows how particles can be collected during extraction.
  • Figure 9B shows how particles collected in Figure 9A can be dispensed into one or several containers.
  • the particle counter developed at the LFD [20-22] (Laboratory for Fluorescence Dynamics, University of California-Irvine, Irvine, CA, USA) is an essential building block of the claimed particle sorter. We therefore start by summarizing its working principles.
  • the employed particle counter generally consists in a portable, low-cost, horizontal-geometry fluorescence confocal microscope ( Figure 1A; Dl and D2, dichroic mirrors; Ll, objective lens; L2 and L3, lenses; F, emission filter; PMT, Photo- Multiplier Tube; and ADC, Analog-to-Digital converter).
  • Japan or from other appropriate vendors for the acquisition of a fluorescence intensity time- trace and for subsequent data-analysis via a pattern-recognition algorithm.
  • Particle-counter data analysis pattern recognition algorithm.
  • the raw data of a particle-counting experiment consist of a temporal fluorescence intensity trace, with peaks of fluorescence intensity (produced by target particles when crossing the excitation volume) overlaid to an ideally zero, constant background (an exemplary time-trace is shown in Figure 2A).
  • Such a time-trace is analyzed by the pattern-recognition algorithm we describe in Figure 2B.
  • a fit to a predetermined profile here assumed to be Gaussian, as assigned by the shape of the excitation volume
  • variable amplitude A and fixed standard deviation s is performed: if the chi-square of the fit is below a pre-required value and the fit amplitude exceeds a minimum intensity threshold T a hit is counted .
  • High-intensity spikes of different shape e.g., those arising from cuvette impurities
  • the absolute target concentration is quantified by simply dividing the total number of counted particles by the scanned volume Vobs, which can be either precisely computed (Figure IB) or pre-calibrated by successive dilutions of a reference sample of known concentration.
  • the pattern-recognition analysis is performed upon completion of the data collection.
  • the analysis of a typical 2 minutes-long time trace (6 million data points for a 50 kHz fluorescence sampling frequency) can be performed in a few seconds (SimFCS software, Laboratory for Fluorescence Dynamics, CA, USA).
  • Particle-sorting unit structure and operation overview.
  • the particle-sorting device invented here can be integrated with the particle counter described above and, in principle, with any particle detecting system.
  • the setup simultaneously performs the acquisition and analysis of the experimental data.
  • the pattern-recognition based analysis of the raw temporal fluorescence intensity trace collected by the particle counter is preserved but implemented for real-time performance: fluorescence intensity spikes are fit to the theoretically expected predetermined (Gaussian) profile during the acquisition of the intensity time-trace, so that a sorting event can be triggered immediately upon the identification of a positive particle. Sorting is performed by a detection-and-suction mechanism.
  • a system of (multiple) pneumatic valves connected to a needle positioned inside the cylindrical cuvette containing the sample, is exploited to generate the vacuum pressure required to suck the detected particle inside a connection tube.
  • a system of (multiple) pneumatic valves connected to a needle positioned inside the cylindrical cuvette containing the sample, is exploited to generate the vacuum pressure required to suck the detected particle inside a connection tube.
  • DaqBoard/3000USB, IoTech Inc., OH, USA or from other appropriate vendors is operated in a specific data- streaming mode.
  • Data acquisition proceeds indefinitely upon the start signal until the accumulation of the pre-specified number of requested data points; during the acquisition, data get transferred to the computer buffer and processed by the pattern-recognition algorithm (Figure 2B) in a slot-wise fashion.
  • the exact coordinate of the detected particle within the 4096 data slot must be recorded and taken into account when defining the time-delay between detection and sorting (i.e., when triggering the generation of a sorting event). This task is easily accomplished by a position-sensitive array-based storage of the raw intensity values provided by the data- acquisition board.
  • the chosen temporal delay between detection and sorting is set as experimental parameter in the data-acquisition software, together with the fit chi-square and amplitude thresholds for the patter-recognition data analysis.
  • Sorting mechanism Provided the real-time analysis of the raw temporal fluorescence intensity trace, a sorting event must be initiated after the identification of each target particle.
  • our particle sorter takes advantage of a needle positioned downstream of the excitation volume and connected via high-pressure-resistant tubing to a system of (multiple) programmable pneumatic valves. Negative vacuum pressure is generated inside the needle by triggering the valves opening, thereby inducing suction of each detected target into the tubing. Subsequent re-balancing of the pressure between successive sorting events keeps the sucked particles and prevents them from re-entering the solution. Only at the end of the sorting procedure, the sorted targets are ejected and transferred into a separate cuvette for subsequent analysis.
  • Needle position Accurate positioning of the needle, with respect to the particle- counter excitation volume and along the particles circular trajectory inside the cuvette, is crucial for the success of the sorting procedure.
  • the needle Given a desired time delay t between detection and sorting, the needle must be positioned at the expected particle coordinate at the very same time t, as assigned by the user-defined rotational frequency /and the vertical translation speed of the cuvette motion.
  • the needle is positioned inside such a second excitation volume: to this aim, the needle tip is fluorescently labeled with a non- water-soluble dye (Rhodamine 6G in our case), and the detected signal is maximized by real-time monitoring of the fluorescence counts.
  • the second excitation laser beam can be switched off and the entire sorting procedure can be carried out at the right position with the simple one-channel configuration described in Figure 1.
  • Particle-sorter prototype Following this design, an instrument prototype has been constructed and demonstrated (a schematic of the setup is shown in Figure 3; note that the needle and the top and side view of the cuvette are shown not to scale).
  • a pneumatic programmable micro-injector operated in suction mode connected to a suitable compressed-gas source (a 60-psi compressed- air wall outlet, in our case) and to a thin (30-gauge) needle inside the cuvette containing the sample, the micro-injector generates the negative pressure required to suck the target particle inside the needle and the connection tubing.
  • the electrically controlled micro-injector we have employed (IM-300, Narishige, Japan) allows the pneumatic -valve activation time (i.e., the time span of the suction event) to be tuned in the broad range 10-300 ms.
  • the suction pressure can be conveniently regulated from 1 to 60 psi.
  • the pressure used to counter-balance the capillary effect can be set down to ⁇ 0.1 psi according to the diameter of the employed needle.
  • a tiny (0.1 pL) volume can be isolated in a single sorting event with a 30- gauge needle by simply regulating a 60-psi suction pressure and a 20-ms valve activation time.
  • a 0.1 -pL suction volume defines a maximum achievable concentration of 10 4 particles/mL for the sorted sample.
  • step-like trigger signals responsible for the collection of the temporal fluorescence intensity trace has been programmed to generate step-like trigger signals with the following features: (i) 5-V amplitude, (ii) a start point assigned by the exact time-point of the particle transit through the counter excitation volume (i.e., the exact particle coordinate in the previously mentioned 4096 data-points slot) and by the desired delay between a detection and suction event.
  • a time span equal to the desired pneumatic-valve activation time is usually selected, even though such activation time is separately pre-set on the micro-injector.
  • an array-based approach has been exploited to transfer the desired signal to the FIFO buffer of the IOTech board.
  • the sorter prototype based on the pneumatic micro-injector has been employed to run the first proof-of-principle sorting experiments on fluorescent micro-heads at ultra- low concentration in water solutions (see section Examples).
  • a drawback of the setup prototype consists in a long ( ⁇ 500 ms) and non-constant time delay between the software generation of a trigger signal and the actual activation of the desired pressure difference inside the needle. This can be ascribed to both a non-constant response time of the micro-injector, and to the long ( ⁇ lm) length of the air-filled tube connecting the micro-injector valve to the needle.
  • the micro-injector-based system is replaced with electrically controlled pneumatic valves, to be positioned closer to the needle with a water- filled connecting channel. Specifications of the valves are again being selected based on the minimum volume that can be displaced in a single sorting event and the minimum valve activation and response time.
  • the target particles include, but are not limited to, nano- and micro-sized spheres or beads, emulsions, droplets, microorganisms, pathogens (such as bacteria, viruses, fungi), cells (such as mammalian cells, human cells, animal cells, cancer cells, immune cells, stem cells, progenitor cells, differentiated cells, prokaryote cells, yeast cells, fungal cells, bacteria, eukaryotic cells, plant cells, breeding cells, engineered cells, fused cells, hybrid cells), embryos, multicellular organoids, protoplasts, lipid vesicles (such as liposomes), extracellular vesicles (such as exosomes, micro vesicles, and apoptotic bodies).
  • pathogens such as bacteria, viruses, fungi
  • cells such as mammalian cells, human cells, animal cells, cancer cells, immune cells, stem cells, progenitor cells, differentiated cells, prokaryote cells, yeast cells, fungal cells, bacteria, e
  • the claimed particle sorter can sort particles with various sizes ranging from ⁇ l0 nanometers (nm) to ⁇ 200 micrometers (pm).
  • the said particles can have different shapes or morphologies such as spherical and rod-like.
  • the claimed particle sorter operates with a typical initial target concentration ranging from ⁇ l to 1000 particles/mL and as low as 1-10
  • the claimed particle sorter is amenable to be integrated, coupled or used in conjunction of down-stream, established in the art, techniques for target or biomarker processing and analysis including, for example, PCR, immunostaining, sequencing and single-cell sequencing.
  • repeatability of the particle sorter can be optimized and improved by modulating the time span of the suction event, the applied sorting pressure, the transit speed of target particles across the counter excitation volume, the time delay between detection and suction events and the initial target concentration.
  • Our particle sorter possesses the following performance properties, including (i) fast sorting rate and high ( -0.5 mL/min) throughput, (ii) suitability for native or complex biological fluids, (iii) capability to handle large sample volumes (100 pLs to several mLs) and highly diluted targets, (iv) simple operational procedure in the framework of a fully integrated and automated system, (v) low cost and portability.
  • the droplets comprise a live cell.
  • the particle- sorting device is used for sorting of droplets that contain live cells, wherein the fluorescent particle in the said droplet is a live cell, wherein the fluorescent signal of the said cell is or is derived from a group consisting of a fluorescent protein expressed by the said cell, a fluorescent-dye-conjugated affinity agent that labels the said cell’s surface, a fluorescent cell tracking dye that stains the said cell, and a fluorogenic substrate provided to the said cell which directly or indirectly coverts the said fluorogenic substrate into a fluorescent signal.
  • a plurality of droplets co-encapsulated with one, two or more types of live cells are provided for sorting by the particle-sorting device, wherein at least one type of the said cells is fluorescent.
  • the said one or more types of live cells may produce fluorescent signal of a same, a similar or a distinct excitation/emission spectrum.
  • a single live cell may comprise two or more types of fluorophores characterized by similar but different excitation/emission spectrums.
  • the said cell refers to a mammalian cell, an animal cell, a yeast cell, a fungal cell, a bacterium, or a derived or an engineered form of any of the above-said cell.
  • the fluorescent protein refers to a violet, blue, green, orange, yellow, red, or far-red fluorescent protein that is represented by green fluorescent protein (GFP), EGFP, ZsGreen, mCherry, yellow fluorescent protein (YFP), and red fluorescent protein (RFP).
  • the affinity reagent refers to an antibody, a target-binding protein, a target-binding peptide, a ligand, an aptamer, a RNA fragment, a DNA fragment, a derivative or an engineered or a combination form of any the-above listed reagents.
  • the particle-sorting device is used for sorting of droplets that contain live cells, wherein the fluorescent particle in the said droplet is a fluorescent bead that is co-encapsulated with the said live cell, wherein the fluorescent bead serves as a qualitative or quantitative assay readout of a biological molecule that is secreted by the said cell.
  • the fluorescent bead is a nanobead or microbead or particle or polymer with a size ranging from about 30 nm to about 30 pm, from about 100 nm to about 15 pm, or from about 200 nm to about 10 pm.
  • the fluorescent bead is a particle coated with an affinity reagent to capture biological molecule produced and secreted by the said co-encapsulated cell.
  • the said biological molecule anchored on the beads further capture a fluorophore-containing antibody or probe.
  • the biological molecule that is secreted by the said cell is a protein, an antibody, a ligand, a receptor, a cytokine, a chemokine, a metabolite, a sugar, a lipid, a complex lipid, or a combination of any of the above-listed biological molecules.
  • the particle-sorting device is used to separate, isolate, or enrich cells with one or more characteristics.
  • the cells to be sorted are B cells, T cells, hybridoma cells, cancer cells, stem cells, engineered cells, fused cells, yeast cells or bacterium cells.
  • B cells B cells, T cells, hybridoma cells, cancer cells, stem cells, engineered cells, fused cells, yeast cells or bacterium cells.
  • a plurality of B cells are encapsulated in droplets which are then sorted by the particle-sorting device on the basis of antigen- specific binding or functional activity of the antibodies secreted by the B cells.
  • a plurality of T cells are co-encapsulated with a target-antigen presenting cell in droplets which are then sorted by the particle- sorting device on the basis of the cognate interaction between a T cell and the target-antigen presenting cell, wherein the positive interaction leads to the production of a fluorescent protein that serves as an assay readout for an activated T cell receptor in a said droplet.
  • the sorted droplets from the particle-sorting device are dispensed individually into a collection device for downstream processes such as single cell cloning, cell culturing and growth, single-cell reverse transcription, and polymerase chain reaction (PCR).
  • the said collection device is a 96- well, 384- well plate, a multi-well plate, a multi-well device, or a multi-tube array.
  • the sorted droplets from the particle-sorting device are collected as a pool.
  • the optical path of the particle counter has been duplicated at 90°: two laser sources with equal specifications and two identical Photomultiplier Tubes (PMTs) have been accommodated to provide fluorescence excitation and detection in the particle-counter channel (hereafter referred to as channel #1) and in the newly added channel #2.
  • the two excitation volumes have been positioned at the same height inside the sample cuvette, 90° apart along the particles circular trajectory.
  • Such a configuration has been achieved by keeping the first excitation volume fixed, by mounting the entire optical path of channel #2 on a three-axis stage micrometer (lO-pm accuracy along each axis), and by continuously adjusting the position of the second excitation volume while maximizing the cross-correlation function of the intensity time-traces collected in the two channels.
  • the second excitation laser beam could in principle be switched off once the needle has been placed (i.e., it is not strictly required for the particle sorter to operate). However, we have decided to further take advantage of the second fluorescence detection channel to provide direct inspection of the success of sorting events.
  • the needle tip stained with Rhodamine 6G appears as a saturated fluorescent spot at the center of the field of view: a particle enters the imaging area and, as soon as the pressure difference is activated, it disappears when getting sucked into the needle.
  • Direct visualization of the trigger signal sent to the micro-injector has been achieved by positioning a green LED close to the camera sensitive chip, and by turning the LED on and off via the data- acquisition software through the very same 5V signal used to activate the micro-injector (i.e., when the LED turns on, the micro-injector receives the electric signal to activate the valve).
  • the LED on-time (frames 85-123) can be visualized in Figure 6G, which shows the average intensity per frame versus time.
  • the time difference between the activation of the LED and the time-point where the particle is sorted by the needle matches with the separately measured time delay required for the pressure difference to actually appear inside the needle. This delay has been taken into account when defining the trigger- signal time on the sorter software.
  • the LED on-time is pre-set on the software too but has no practical consequence on the pneumatic valve activation time (which is directly programmed on the micro-injector).
  • FIG. 8B shows the cross correlation of the fluorescence signals detected from a solution containing fluorescent particles (15 pm diameter yellow-green fluorescent microspheres, Invitrogen, USA) in both arms. The cross-correlation amplitude peak indicates that particles detected in the first laser focus appear in the second laser focus at 60 ms delay.
  • FIG. 9 The concept of particle dispensing is shown in Figure 9.
  • the dispensing port is closed ( Figure 9A).
  • a vacuum is applied to suck the particles into a reservoir, which in the simplest case can be the tubing itself.
  • the port connecting to the extraction needle/capillary is closed, the port to the dispenser opened, and positive pressure is applied to eject the particles into one or multiple containers such as a multi-well plate ( Figure 9B).

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Abstract

Un trieur de particules qui peut être complété et intégré à une installation de comptage de particules, pour permettre la séparation, l'isolement et/ou l'enrichissement de particules cibles, comprend un compteur de particules, un récepteur pour les particules détectées, et un système de vannes pneumatiques commandées électriquement pour générer la pression négative requise pour diriger les particules détectées dans le récepteur.
PCT/US2019/027717 2018-04-17 2019-04-16 Dispositif de tri de particules pour la séparation, l'isolement et l'enrichissement de particules à ultra-faible concentration WO2019204333A1 (fr)

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WO2021138212A1 (fr) * 2020-01-03 2021-07-08 Bioloomics, Inc. Systèmes et procédés de tri de cellules et d'extraction de cellules
EP4097243A4 (fr) * 2020-01-31 2024-02-28 Astrin Biosciences, Inc. Système de filtration de fluide biologique

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WO2021138212A1 (fr) * 2020-01-03 2021-07-08 Bioloomics, Inc. Systèmes et procédés de tri de cellules et d'extraction de cellules
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US11873475B2 (en) 2020-01-03 2024-01-16 Bioloomics, Inc. Systems and methods for cell sorting and cell extraction
EP4097243A4 (fr) * 2020-01-31 2024-02-28 Astrin Biosciences, Inc. Système de filtration de fluide biologique

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