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WO2022241243A1 - Techniques de détection et de quantification de bactéries vivantes et mortes dans un échantillon de fluide - Google Patents

Techniques de détection et de quantification de bactéries vivantes et mortes dans un échantillon de fluide Download PDF

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Publication number
WO2022241243A1
WO2022241243A1 PCT/US2022/029236 US2022029236W WO2022241243A1 WO 2022241243 A1 WO2022241243 A1 WO 2022241243A1 US 2022029236 W US2022029236 W US 2022029236W WO 2022241243 A1 WO2022241243 A1 WO 2022241243A1
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Prior art keywords
microorganisms
live
amount
image
stain
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Monika WEBER
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Fluid Screen Inc
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Fluid Screen Inc
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    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/90—Determination of colour characteristics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10024—Color image
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10056—Microscopic image
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30024—Cell structures in vitro; Tissue sections in vitro

Definitions

  • Detection and identification of particles e.g., bacterial and viral pathogens
  • cell containing solutions e.g., blood, urine, CSF, mammalian cell culture, CHO cell matrix, CAR-T drug product, CAR-T specimen, CAR-NK drug product, body fluids, apheresis samples or samples related to immunotherapy
  • protein containing solutions e.g., for pharmaceuticals during manufacturing, drug product, drug substance
  • analyte extraction from microbiome samples, water, sterile fluids and other fluids is possible by employing isolation on cultural media and metabolic fingerprinting methods.
  • Immunoassay and nucleic acid-based assays are now widely accepted techniques, providing more sensitive and specific detection and quantification of bacteria.
  • Dielectrophoresis relates to a force in an electric field gradient on objects having dielectric moments.
  • DEP has shown promise for particle separation, but has not yet been applied in clinical settings, pharmaceutical quality assurance settings, or immunotherapy.
  • DEP uses a natural or induced dipole to cause a net force on a particle in a region having an electric field gradient. The force depends on the Clausius-Mossotti factor associated with the particle.
  • aspects of the technology described herein relate to detection and quantification of live and dead bacterial cells or non-bacterial particles (e.g., yeast, mold). In some embodiments, the techniques are provided for distinguishing between live bacteria and dead cells.
  • aspects of the technology described herein relate to detection and quantification of live and dead microorganisms.
  • the techniques are provided for distinguishing between live microorganisms and dead microorganisms on non- microorganisms particles.
  • a hue value of an organism may be used to determine whether the organism is live or dead.
  • a hue value may be used to determine whether the organism as it appears in an image obtained in a microfluidic system is green or yellow or red. The determined color of green or yellow or red may be used to determine whether the organism is live or dead.
  • a method for selectively staining live and/or dead particles of a sample. For example, successive scans of a sample may be performed using a microfluidic system. The sample may be stained before each scan. In some embodiments, a stain may be applied to the sample before the first scan such that only dead particles are stained. Accordingly, the first scan may reflect the staining of only the dead particles. In some embodiments, a stain may be applied after the first scan and before a second scan which stains all particles. Accordingly, the second scan may reflect the staining of all particles, live and dead. The scans may be compared to distinguish between the live and dead particles of the sample.
  • Some embodiments relate to a method for quantifying live and/or dead microorganisms in a color image.
  • the method comprises receiving a color image captured by an optical system, the color image including at least one electrode and one or more microorganisms arranged on a surface of the at least one electrode, classifying each of the one or more microorganisms as being a live microorganism or a dead microorganism based, at least in part, on a hue of the microorganism in the color image, quantifying, based on the classifying, an amount of live microorganisms and/or dead microorganisms in the color image, and outputting a result of the quantifying.
  • the one or more microorganisms in the color image are unstained.
  • the color image is a red-green-blue (RGB) image.
  • classifying each of the one or more microorganisms based, at least in part, on a hue of the microorganism in the color image comprises classifying a microorganism of the one or more microorganisms as a live microorganism when the hue of the microorganism is greater than a threshold value, and classifying the microorganism of the one or more
  • the hue of the microorganism is specified as a numerical value corresponding to an angle on a red-green-blue color wheel, where red on the color wheel has a numerical value of 0, green on the color wheel has a numerical value of 120 and blue on the color wheel has a numerical value of 240.
  • the threshold value is between 60 and 120. In one aspect, the threshold value is between 70 and 90. In one aspect, the threshold value is 75.
  • outputting a result of the quantifying comprises providing an alarm when the amount of live microorganisms is above a threshold value.
  • the one or more microorganisms comprise bacteria.
  • the one or more microorganisms comprise bacteria, yeast and mold.
  • the optical system comprises a red-green-blue (RGB) camera.
  • the optical system comprises a monochrome camera having a plurality of color filters, and receiving a color image captured by an optical system comprises receiving a superposition of monochrome images captured by the monochrome camera.
  • the plurality of color filters includes a red filter and a green filter.
  • the method further comprises filtering the color image prior to classifying each of the one or more microorganisms as being a live microorganism or a dead microorganism.
  • the method is applied to determining sterility of immunotherapy samples within 8 hours. In one aspect, the method is applied to determining sterility of fluid samples within 24 hours. In one aspect, the method is applied to determining sterility of fluid samples within 24 hours in an automated method.
  • a system for quantifying live and/or dead microorganisms in a fluid sample comprises a microfluidic passage for receiving a fluid sample, the sample comprising one or more microorganisms, at least one electrode disposed in the microfluidic passage, the at least one electrode configured to immobilize, when activated, the one or more microorganisms onto a surface of the at least one electrode using dielectrophoresis, an optical system configured to capture a color image while the one or more microorganisms are immobilized on the surface of the at least one electrode, and at least one computing device.
  • the at least one computing device is configured to classify, the one or more microorganisms represented in the color image as being a live microorganism or a dead microorganism based, at least one computing device.
  • the one or more microorganisms in the color image are unstained.
  • the color image is a red-green-blue (RGB) image.
  • classifying each of the one or more microorganisms based, at least in part, on a hue of the microorganism in the color image comprises classifying a microorganism of the one or more microorganisms as a live microorganism when the hue of the microorganism is greater than a threshold value, and classifying the microorganism of the one or more microorganisms as a dead microorganism when the hue of the microorganism is less than the threshold value.
  • the hue of the microorganism is specified as a numerical value corresponding to an angle on a red-green-blue color wheel, where red on the color wheel has a numerical value of 0, green on the color wheel has a numerical value of 120 and blue on the color wheel has a numerical value of 240.
  • the threshold value is between 60 and 120. In one aspect, the threshold value is between 70 and 90. In one aspect, the threshold value is 75.
  • outputting a result of the quantifying comprises providing an alarm when the amount of live microorganisms is above a threshold value.
  • the one or more microorganisms comprise bacteria.
  • the plurality of microorganisms comprise bacteria, yeast and mold.
  • the optical system comprises a red-green-blue (RGB) camera.
  • the at least one computing device is further configured to filter the color image prior to classifying each of the one or more microorganisms as being a live microorganism or a dead microorganism.
  • the optical system comprises a monochrome camera having a plurality of color filters, and capturing a color image comprises capturing a plurality of monochrome images, wherein the color image is a superposition of the captured monochrome images.
  • the plurality of color filters includes a red filter and a green filter.
  • a method for quantifying live and/or dead microorganisms in a fluid sample comprises receiving a first image and a second image captured by an optical system during processing of the fluid sample, each of the first and second images including at least one electrode of the microfluidic system and one or more
  • the one or more microorganisms in the first image are stained using a first stain and the one or more microorganisms in the second image are stained using a second stain, wherein the first stain is configured to selectively stain live microorganisms or dead organisms, quantifying a first amount of live microorganisms or dead microorganisms in the first image, and quantifying a second amount of live microorganisms and/or dead microorganisms in the second image, quantifying an amount of live and/or dead microorganisms in the fluid sample based on the first amount and the second amount, and outputting the amount of live and/or dead microorganisms in the fluid sample.
  • the first stain is configured to selectively stain dead microorganisms.
  • the first stain comprises a metabolic stain.
  • the second stain is configured to stain all microorganisms. In one aspect, the second stain is configured to stain only live microorganisms.
  • quantifying the amount of live and/or dead microorganisms in the fluid sample comprises subtracting the first amount from the second amount.
  • the method further comprises determining first positions of the one or more microorganisms in the first image, and determining second positions of the one or more microorganisms in the second image, wherein quantifying the amount of live and/or dead microorganisms in the fluid sample is further based, at least in part, on the first positions and the second positions.
  • outputting the amount of live and/or dead microorganisms in the fluid sample comprises outputting an indication of the first amount and the second amount. In one aspect, outputting the amount of live and/or dead microorganisms in the fluid sample comprises outputting the amount of live and/or dead microorganisms in the fluid sample only when the amount of live microorganisms is above a threshold value.
  • the method further comprises receiving a third image captured by the optical system during processing of the fluid sample, the third image including the at least one electrode of the microfluidic system and one or more microorganisms arranged on a surface of the at least one electrode, wherein the one or more microorganisms in the third image are stained using a third stain different from the first and second stains, quantifying a third amount of live microorganisms and/or dead microorganisms in the third image, and quantifying an amount of live and/or dead microorganisms in the fluid sample further based on the third amount.
  • outputting the amount of live and/or dead microorganisms in the fluid sample comprises outputting an indication of the first amount, the second amount, and the third amount.
  • the first stain comprises propidium iodide and the second stain comprises SYTO 9.
  • a system for quantifying live and/or dead microorganisms in a fluid sample comprises a microfluidic passage for receiving a fluid sample, the sample comprising microorganisms, at least one electrode disposed in the microfluidic passage, the at least one electrode configured to immobilize, when activated, the microorganisms onto a surface of the at least one electrode using dielectrophoresis, an optical system configured to capture a first image and a second image while the microorganisms are immobilized on the surface of the at least one electrode, wherein microorganisms in the first image are stained using a first stain and the microorganisms in the second image are stained using a second stain, wherein the first stain is configured to selectively stain live microorganisms or dead organisms, and at least one computing device.
  • the at least one computing device is configured to quantify a first amount of live microorganisms or dead microorganisms in the first image, quantify a second amount of live microorganisms and/or dead microorganisms in the second image, quantify an amount of live and/or dead microorganisms in the fluid sample based on the first amount and the second amount, and output the amount of live and/or dead microorganisms in the fluid sample.
  • the first stain is configured to stain all particles and the second stain is configured to stain only microorganisms. In one aspect, the first stain is configured to selectively stain dead microorganisms. In one aspect, the first stain comprises a metabolic stain. In one aspect, the second stain is configured to stain all microorganisms. In one aspect, quantifying the amount of live and/or dead microorganisms in the fluid sample comprises subtracting the first amount from the second amount.
  • the at least one computing device is further configured to determine first positions of the one or more microorganisms in the first image, and determine second positions of the one or more microorganisms in the second image, wherein quantifying the amount of live and/or dead microorganisms in the fluid sample is further based, at least in part, on the first positions and the second positions.
  • outputting the amount of live and/or dead microorganisms in the fluid sample comprises outputting an indication of the first amount and the second amount. In one aspect, outputting the amount of live and/or dead microorganisms in the fluid sample comprises outputting the amount of live and/or dead microorganisms in the fluid sample only when the amount of live microorganisms is above a threshold value.
  • the optical system is further configured to capture a third image, the third image including the at least one electrode of the microfluidic system and one or more microorganisms arranged on a surface of the at least one electrode, the one or more microorganisms in the third image are stained using a third stain different from the first and second stains, and the at least one computing device is further configured to quantify a third amount of live microorganisms and/or dead microorganisms in the third image, and quantify an amount of live and/or dead microorganisms in the fluid sample further based on the third amount.
  • outputting the amount of live and/or dead microorganisms in the fluid sample comprises outputting an indication of the first amount, the second amount, and the third amount.
  • the first stain comprises propidium iodide and the second stain comprises SYTO 9.
  • a method of operating a microfluidic system to quantify live and/or dead microorganisms in a fluid sample comprises passing the fluid sample through a microfluidic passage of the microfluidic system, the microfluidic passage including at least one electrode, wherein microorganisms in the fluid sample are captured on a surface of the at least one electrode using dielectrophoresis as the fluid sample is passed through the microfluidic passage, passing a first fluid through the microfluidic passage while the microorganisms remain captured on the surface of the at least one electrode, the first fluid including a first stain configured to selectively stain live microorganisms or dead microorganisms, capturing, with an optical system, a first image of the surface of the at least one electrode, passing a second fluid through the microfluidic passage while the microorganisms remain captured on the surface of the at least one electrode, the second fluid including a second stain different from the first stain, capturing, with the optical
  • the method further comprises applying a high voltage signal to the at least one electrode prior to passing the fluid sample through the microfluidic passage, wherein
  • the high voltage signal is sufficient to kill all microorganisms on the surface of the at least one electrode.
  • the high voltage signal has an amplitude of at least 20 volts.
  • the method further comprises applying a low voltage signal to the at least one electrode while passing the sample through the microfluidic passage, wherein the low voltage signal does not alter the viability of microorganisms captured on the surface of the at least one electrode.
  • the low voltage signal has an amplitude less than 20 volts.
  • the method further comprises passing a third fluid through the microfluidic passage prior to passing the sample through the microfluidic passage, the third fluid comprising a control solution without the first stain or the second stain.
  • the method further comprises passing the third fluid through the microfluidic passage after passing the first fluid through the microfluidic passage, and before capturing the first image.
  • the method further comprises passing the third fluid through the microfluidic passage after passing the second fluid through the microfluidic passage, and before capturing the second image.
  • the method further comprises passing a third fluid through the microfluidic passage after passing the first fluid through the microfluidic passage, and before capturing the first image, the third fluid comprising a control solution without the first stain or the second stain. In one aspect, the method further comprises passing the third fluid through the microfluidic passage after passing the second fluid through the microfluidic passage, and before capturing the second image.
  • the method further comprises passing a third fluid through the microfluidic passage after capturing the second image, wherein the third fluid includes a third stain different from the first stain and the second stain, and capturing, with the optical system, a third image of the surface of the at least one electrode.
  • the method further comprises passing a fourth fluid through the microfluidic passage after passing the third fluid through the microfluidic passage, and before capturing the third image, the fourth fluid comprising a control solution without the first stain, the second stain, or the third stain.
  • FIG. 1 schematically illustrates a system for detection and quantification of live and dead bacteria in a sample, according to some embodiments of the present technology
  • FIG. 2 illustrates a microfluidic system for detection and quantification of live and dead bacteria in a sample, according to some embodiments of the present technology
  • FIG. 3 illustrates a static system for detection and quantification of live and dead bacteria in a sample, according to some embodiments of the present technology
  • FIG. 4 is a flowchart of a process for quantifying an amount of live and/or dead microorganisms in a color image, according to some embodiments of the present technology
  • FIG. 5 illustrates values for a color wheel that may be used to label live and/or dead microorganisms in a color image, according to some embodiments of the present technology
  • FIG. 6 schematically illustrates a microfluidic system for use with some embodiments of the present technology
  • FIG. 7 schematically illustrates a microfluidic system for use with some embodiments of the present technology
  • FIG. 8 is a flowchart of a process for processing a sample to determine an amount of live and/or dead microorganisms in the sample, according to some embodiments of the present technology
  • FIG. 9 is a flowchart of a process for quantifying an amount of live and/or dead microorganisms in a sample, according to some embodiments of the present technology.
  • FIGS. 10A-10C illustrate plots describing outputs of a process for quantifying an amount of live and/or dead microorganisms in a sample, according to some embodiments of the present technology.
  • aspects of the technology described herein relate to an apparatus and methods for detecting and/or quantifying biological organisms (e.g., bacteria) present in a fluid sample.
  • biological organisms e.g., bacteria
  • the technology described herein provides techniques for detection and/or
  • microfluidic system comprising one or more electrodes configured to generate dielectrophoretic forces that act on the sample.
  • Microbial contamination is a serious and growing global threat to human health and economic development, including pharmaceutical manufacturing and immunotherapy manufacturing and treatment.
  • An example of a conventional technique to assess the presence and degree of microbial contamination in a sample is the Plate Counting Method (PCM).
  • PCM typically includes at least four steps. In step 1, a sample to be analyzed is manually placed in each of multiple test tubes, and the sample in each test tube is diluted to a desired concentration using a buffer solution. In step 2, the diluted samples are plated onto petri dishes containing agar media. Petri dishes including dilution media only (i.e., without the sample) are also plated for use as controls for comparison against the plated diluted samples.
  • step 3 the plurality of plated samples, the dilution media plates, and empty agar plates are cultured for 24 hours to 14 days to enable microbial particles to grow on the media within the petri dishes.
  • step 4 the number of bacterial colonies on each of the plates cultured in step 3 is determined, for example, using a microscope.
  • PCM is routinely used in medical, pharmacological and food industries to identify bacterial contamination.
  • PCM is slow, only moderately sensitive, labor intensive and prone to human errors.
  • the entire PCM process takes 1-14 days, includes several manual steps in which human intervention is needed, and requires a large number of plated samples at different dilutions and controls. There is therefore a need for new technologies that allow for faster, more sensitive and/or more reliable assessment of microbial contamination in fluid samples.
  • DEP Dielectrophoresis
  • Some embodiments of the technology described herein relate to a novel DEP bacterial capture and separation technique (also referred to herein as “Fluid- Screen” or “FS”) that addresses at least some of the limitations of prior DEP techniques.
  • FS Fluid- Screen
  • a fluid sample containing bacteria is processed in a microfluidic system that includes a microfluidic device.
  • the sample may be subjected to DEP forces to enable separation, detection, enrichment and/or quantification of microorganisms in the fluid sample.
  • Examples of a microfluidic system suitable for use in accordance with the techniques described herein include the Fluid-Screen Microfluidic System, aspects of which are described in U.S. Patent Application No. 16/093,883 titled “ANALYTE DETECTION METHODS AND APPARATUS USING
  • FIG. 1 illustrates an example system 100 for detecting bacteria in a sample, in accordance with some embodiments.
  • the system 100 comprises a microfluidic device 104 in communication with a computing device 110.
  • microfluidic device 104 may be any suitable device, examples of which are provided herein.
  • microfluidic device 104 is implemented as a microfluidic chip having one or more passages (e.g., microfluidic channels or chambers) through which a fluid sample 102 is provided for analysis.
  • passages e.g., microfluidic channels or chambers
  • microfluidic channel or simply “channel” is used herein to describe a passage through which fluid flows through microfluidic device 104, it should be appreciated that a fluid passage having any suitable dimensions may be used as said channel, and embodiments are not limited in this respect.
  • Microfluidic device 104 may comprise a single channel or multiple channels configured to receive a single sample 102 (e.g., to perform different analyses on the sample) or multiple channels configured to receive different samples for analysis. In embodiments having multiple channels, the microfluidic device may be configured to process the single sample or multiple samples in parallel (e.g., at the same or substantially the same time).
  • sample 102 may include any fluid containing bacteria or other microorganism of interest.
  • the sample comprises a biological fluid such as saliva, urine, blood, water, any other fluid such as an environmental sample or potentially contaminated fluid, protein matrices, mammalian cell culture, immunotherapy drug product, cell and gene therapy drug product, cell and gene therapy drug sample, bacterial culture, growth media, active pharmaceutical ingredients, enzyme products, or substances used in biomanufacturing, etc.
  • microfluidic device 104 includes at least one electrode 106.
  • the at least one electrode 106 may be configured to receive one or more voltages to generate positive and/or negative dielectrophoresis (DEP) force(s) that act on a sample arranged proximate to the at least one electrode.
  • the at least one electrode 106 may be configured to receive one or more voltages (e.g., one or more AC voltages) to generate at least one dielectrophoresis force that acts on the sample.
  • the at least one DEP force may cause certain components of the sample to move relative to (e.g., be ahracted to or repulsed from) a surface of the at least one
  • the small size of bacteria presents an obstacle to optical observation and quantification of bacteria in the sample.
  • the inventors have recognized that activation of the at least one electrode 106 results in an electric field that may be used to selectively trap bacteria on the surface of the electrode(s).
  • capturing bacteria on the surface of the electrode(s) may prevent the bacteria from moving in and out of focus of the optical system to enable real-time bacteria detection and quantification, a process referred to herein as “on-chip quantification.”
  • the electric field used to capture the bacteria concentrates the bacteria, which enables imaging with fluorescence microcopy or another optical detection technique. Accordingly, bacterial capture using the techniques described herein allows for detection and quantification of bacteria at significantly lower limits compared to some conventional methods, such as the PCM technique described above.
  • the ability to detect and/or quantify bacteria in a sample, even in small amounts, may be useful in applications including, but not limited to, biomanufacturing, gene therapy, analysis of patient samples, vaccine development and/or biothreat detection, antibiotic susceptibility testing.
  • the at least one DEP force acting on the sample may cause bacteria (or certain bacteria) to separate from other components of the sample (e.g., via positive DEP). Bacteria in the sample may be attracted to the surface of the at least one electrode 106 allowing for enhanced detection and/or quantification, despite the small size and/or small amount of the bacteria in the sample.
  • microfluidic device 104 is illustrated as having a single electrode, it should be understood that in some embodiments, microfluidic device 104 comprises multiple electrodes arranged in any suitable configuration.
  • System 100 may further comprise a computing device 110 configured to control one or more aspects of microfluidic device 104.
  • computing device 110 may be configured to direct the sample 102 into a channel of the microfluidic device.
  • computing device 110 is configured to control the at least one electrode 106 to generate the at least one DEP force acting on the sample 102.
  • computing device 110 may cause one or more components of an optical system (not shown) to perform one
  • Non-limiting examples of a computing device 110 that may be used in accordance with some embodiments are further described herein.
  • FIG. 2 illustrates an example microfluidic system 200 for detecting the presence of microorganisms (e.g., bacteria) in a sample, in accordance with some embodiments.
  • System 200 includes microfluidic device 208 (e.g., indicated in FIG. 2 as a microfluidic chip) that includes one or more electrodes for generating DEP forces that act on a sample 204 provided as input to the system.
  • Sample 204 may contain microorganisms for which separation, detection, enrichment, and/or quantification may be performed.
  • the one or more electrodes may be arranged in any suitable configuration within the microfluidic device 208.
  • the electrodes may be arranged in one-dimension along the flow direction of the fluid, perpendicular to fluid flow direction or on a diagonal relative to the fluid flow direction.
  • a multidimensional (e.g., 2- dimensional, 3 -dimensional) array of electrodes may be used.
  • a dense array of electrodes arranged both along the direction of fluid flow and perpendicular to the direction of fluid flow may be used.
  • a flow system 202 is provided.
  • the flow system 202 may provide a solution for transporting the sample 204 to the microfluidic device 208.
  • a first pump 206 may be used to pump the solution and the sample 204 to the microfluidic device 208 at a predetermined flow rate.
  • First pump 206 may be of any suitable type. In some embodiments, first pump 206 is omitted and sample 204 is manually loaded (e.g., using a pipette or capillary flow) as input to one or more channels of microfluidic device 208.
  • Microfluidic device 208 is configured to receive sample 204 for processing.
  • Microfluidic device 208 may include one or more passages through which the sample 204 flows.
  • the one or more passages may include at least one electrode formed therein or adjacent thereto.
  • the at least one electrode may be formed within a passage.
  • the at least one electrode when activated, is configured to generate an electric field that acts on the sample 204 as it flows through the one or more passages.
  • An electrical system 212 e.g., a signal generator or controller
  • Microfluidic system 200 may include an optical system 210 to facilitate analysis of the sample 204 by performing on-chip quantification.
  • optical system 210 may comprise one or more optical sensors (e.g., a red-green-blue camera) for viewing and/or imaging the sample.
  • the optical sensor(s) may provide for enhanced detection and/or quantification of the captured microorganisms and/or the other components of the sample 204 relative to detection and quantification techniques that require separate culturing of captured microorganisms or an effluent sample from the device. Any suitable optical sensor(s) may be used.
  • the optical sensor(s) comprises a digital camera.
  • the optical sensor(s) includes a monochrome camera having a plurality of color filters.
  • the monochrome camera may be configured to capture a plurality of monochrome images with different color filters, and a color image may be formed based on superposition of the plurality of monochrome images.
  • the different color filters may include a red filter and a green filter, and the color image may be a superposition of a monochrome image captured with the red filter and a monochrome image captured with the green filter.
  • the optical sensor(s) comprises electronic sensors including CMOS compatible technology.
  • the optical sensor(s) comprise fiber optics.
  • bacteria in the sample are stained (e.g., with a fluorescent dye) and the optical system 210 is configured to perform microscopy (e.g., fluorescence microscopy) of captured stained bacteria.
  • optical system 210 is configured to capture one or more images (e.g., color images) of the at least one electrode while the sample is flowing through the microfluidic device 208.
  • the detector comprises nanowire and/or nanoribbon sensors.
  • Microfluidic system 200 also includes computer 230 configured to control an operation of optical system 210 and/or to receive images from optical system 210 and to perform processing on the received images (e.g., to count a number of microorganisms trapped by the microfluidic device 208). In some embodiments, the received images are analyzed to determine the number of microorganisms captured by the at least one electrode. For instance,
  • microorganisms may be identified in the received images as spots (e.g., fluorescent spots) located on the edges of the electrodes.
  • spots e.g., fluorescent spots
  • a captured target microorganism species may be differentiated from other components in the sample that are not captured and may appear as floating above the at least one electrode or located between electrodes.
  • the sample 204 may be removed from the microfluidic device 208.
  • a second pump 216 may be provided for pumping the sample 204 out of the microfluidic device 208.
  • the second pump 216 may be of any suitable type.
  • microfluidic system 200 comprises a flow sensor 214 for measuring a flow rate at which the sample 204 is removed from the microfluidic device 208.
  • the flow sensor 214 and the second pump 216 may be in communication to control a flow rate at which the sample 204 is removed from the microfluidic device 208.
  • microfluidic system 200 may be used for separating bacteria from other components (or for separating certain bacteria from other bacteria) in sample 204.
  • Microfluidic system 200 comprises a waste region 218 arranged to receive other components of the sample 204 which have been separated from the bacteria by the microfluidic device 208 and subsequently removed from the sample 204, for example, using the second pump 216.
  • analysis of the fluid collected in waste region 218 may be referred to as analysis of the “effluent sample.”
  • Microfluidic system 200 may further include effluent region 220 for receiving a purified version of sample 204 containing substantially only target microorganisms that were captured using microfluidic device 208.
  • an amount of time needed to process a sample using microfluidic system 200 is substantially less than an amount of time required to process a sample using a conventional PCM sample processing system.
  • processing a sample using system 200 may include at least three steps.
  • a sample is provided as input to microfluidic system 208 and bacteria are captured from the sample in the presence of an applied electric field.
  • automated on-chip quantification is performed, for example, using computer 230 to analyze one or more images recorded by optical system 210.
  • step 270 further analysis may be performed on waste 218 and/or effluent sample 220, as desired.
  • 16 may take on the order of minutes or an hour to a few hours, which is substantially faster than the multiple days (e.g., 1 to 14 days) typically required to process samples using PCM.
  • sample 204 may be manually provided as input to microfluidic device 208 for analysis.
  • one or more droplets of sample 204 may be provided as input to microfluidic device 208 using a pipette or other suitable technique.
  • the sample is analyzed in a “static” condition rather than in a condition in which microorganisms are captured by the at least one electrode as the sample flows past the electrode(s) (e.g., as in the case of microfluidic system 200 as shown in FIG. 2).
  • FIG. 3 illustrates a microfluidic system 300 for detecting microorganisms in a sample, according to some embodiments.
  • microfluidic system 300 may include many of the same components as microfluidic system 200, but may omit certain components of the microfluidic system 200, such as the first pump 206, which are not needed when the sample is manually provided as input to the microfluidic device 308 (indicated in FIG. 3 as a static microfluidic chip).
  • Microfluidic systems used in accordance with some embodiments of the present technology provide a precise and rapid system for qualitative and/or quantitative differentiation between live and dead bacteria and other organisms (e.g., spores) and other particles.
  • This differentiation may be based on measurements obtained from a single organism (e.g., bacteria, virus, fungi, yeast, etc.) or from a mix of organisms.
  • Such measurements may provide information useful in quality assurance, product sterility and biomanufacturing processes, among others.
  • engineered tissues and organs require high quality and sterility assurance before they are implanted in patients. Similar to drugs with a short shelf life or immunotherapy drugs, engineered tissues require careful handling, are prone to contamination, and have a short shelf life before they must be implanted into patients.
  • HAIs Hospital Acquired Infections
  • Several pathogens can persist in the environment for extended periods and serve as vehicles of transmission and dissemination in the hospital setting. Cross-transmission of these pathogens can occur via hands of healthcare workers, who become contaminated directly from patient contact or indirectly by touching contaminated environmental surfaces. Less commonly, a patient could become colonized by direct contact with a contaminated environmental surface.
  • Rapidly detecting the presence of live microorganisms may be important to verify whether water treatment, environmental surface treatment or sterilization of pharmaceutical substances or other sterile equipment was effective. If the treatment was successful, all microorganisms present in the sample should be dead.
  • Existing techniques for detecting bacteria in fluid samples may be inefficient in several ways including, but not limited to, their inability to detect low levels of contaminant and/or their inability to culture certain types of microorganisms.
  • existing detection methods may take days to provide results. While faster methods such as quantitative polymerase chain reaction (qPCR) can reduce the response time to a few hours, such methods require complex sample preparation, high costs, have limited portability, and cannot be used for process streamlining. Other techniques such as staining may be prone to subjective biases.
  • qPCR quantitative polymerase chain reaction
  • a shortcoming of existing methods is the inability to accurately differentiate viability from a mix of strains.
  • Live/Dead stains such as SYTO 9 and propidium iodine included in the LIVE/DEAD BacLight Bacterial Viability kit have limited accuracy.
  • some embodiments described herein relate to techniques for differentiating viability of bacteria from a mix of strains.
  • the techniques described herein may be implemented using a microfluidic system (e.g., the microfluidic systems described in FIGS. 1-3).
  • the microfluidic system may control particle motion in a fluid by dielectrophoresis (DEP), which describes the motion of all particles in a non-uniform electric field gradient.
  • DEP dielectrophoresis
  • bacteria and other cells can be captured on a surface of one or more electrodes used to generate an electric field having particular characteristics.
  • the capture can be universal, capturing all particles within a range of sizes, or selective for a singular particle type, depending on the tuning of the electric field applied.
  • the electrode(s) of the microfluidic system may be specially designed to maximize bacterial response to the electric field.
  • bacterial viability may be detected from a mix of strains using their unique fingerprints.
  • the strains may be unlabeled.
  • the techniques may be automated. In some embodiments, the techniques may be performed rapidly (e.g., 30 minutes or less).
  • Some embodiments are directed to techniques for quantifying amounts of live and/or dead organisms based on the hue of organisms in a color image captured when cells are attracted to the surface of one or more electrodes using a microfluidic system as described herein.
  • Such an implementation provides for an automated determination of whether the captured organism is live or dead.
  • Hue may be defined as an angle around the red, green, blue (RGB) color wheel.
  • RGB red, green, blue
  • a hue threshold may be set to automatically facilitate determining whether an organism is green (e.g., having a hue greater than the threshold) or yellow (e.g., having a hue less than the threshold). Accordingly, the color of the organisms as determined by the hue threshold may assist in determining whether the organism is live or dead, without requiring manual inspection of the organisms.
  • a hue threshold of 75 may be set, for example, requiring organisms captured by the microfluidic system to have a hue angle greater than the threshold in order to be categorized as a live cell. Accordingly, the techniques described herein may provide for an automated process for distinguishing between live and dead cells without requiring an experienced microbiologist to distinguish between live and dead cells subjectively, which is typically required using existing techniques.
  • FIG. 4 illustrates a process 400 for quantifying live and/or dead microorganisms
  • a color image of a surface of at least one electrode is received.
  • the color image may be a red-green- blue (RGB) image captured by a color camera of the optical system or a superposition of multiple monochrome images captured by a monochrome camera having different color filters.
  • RGB red-green- blue
  • the one or more microorganisms in the color image may be unstained.
  • Process 400 then proceeds to act 412, where the one or more microorganisms are classified as being live or dead based on a hue of the microorganism in the color image.
  • the hue of the microorganism in the color image may be compared to a threshold value and the classification may be made based on whether the hue is above or below the threshold value.
  • hue may be represented in some embodiments as a numerical value on a color wheel, an example of which is shown in FIG. 5.
  • a red hue may have a value of 0 on the color wheel
  • a yellow hue may have a value of 60 on the color wheel
  • a green hue may have a value of 120 on the color wheel
  • a blue hue may have a value of 240 on the color wheel.
  • live microorganisms appear green in the color
  • a threshold value corresponding to a hue value that distinguishes green objects from yellow objects may be used to distinguish live microorganisms from dead microorganisms or other particles.
  • the threshold value is within the range 60-120 on the color wheel. In some embodiments, the threshold value is within the range 70-90 on the color wheel. In some embodiments, the threshold value is 75 on the color wheel. In such embodiments, when the hue of a detected microorganism is less than the threshold value (e.g., 75), the microorganism is classified as dead and when the hue of the detected microorganism is greater than the threshold value, the microorganism is classified as live.
  • the color image received in act 410 is processed prior to performing classification in act 412.
  • Process 400 then proceeds to act 414, where an amount of live and/or dead organisms in the color image is quantified based on the classification of microorganisms in act 412. Quantifying the number of live and/or dead organisms may be performed in any suitable way. For instance, all organisms characterized as live organisms in act 412 may be counted in act 414.
  • FIG. 6 schematically illustrates an example system with a microfluidic device that may be used to detect and/or quantify live and/or dead microorganisms (e.g., bacteria) in accordance with some embodiments.
  • the system includes a container 3 having a sample disposed therein.
  • the sample may include bacteria (e.g., E.
  • the system also includes container 2 having a controlled solution disposed therein.
  • the controlled solution include deionized water, water, a buffer solution, and a sterile buffer solution.
  • the system also includes one or more containers having disposed therein a stain.
  • container 4 A may contain a first stain (e.g., propidium iodine) and container 4B may contain a second stain (e.g., SYTO 9).
  • any suitable number of stains may alternatively be included in the system.
  • one or more of containers 4 A and 4B contain multiple stains.
  • one or more of the containers 2, 3, 4 A and 4B may be replaced with a fluid flow line (e.g., a pipe, tube, etc.). Fluids from containers 2, 3, 4A and 4B are provided as input to an inlet 5 of microfluidic passage 6 of the microfluidic device. A cross-section of the microfluidic passage 6 is shown. The fluid flows through the microfluidic passage in the direction indicated by arrow 14 and exits the microfluidic passage via outlet 15 where it is collected in effluent sample container 16.
  • Controller 20 Disposed within microfluidic passage 6 is a plurality of electrodes 7, 8, 9 coupled to a controller 20 via contact 13. Controller 20 is configured to provide a signal having a voltage with amplitude VI and a frequency fl. In some embodiments, the signal provided by the controller is applied to electrodes of opposite polarity as +V1, fl and -VI, fl or as VI, fl and 0V. In some embodiments, multiple signals are applied to one or more of the electrodes 7, 8, 9. In response to applying a voltage to electrodes 7, 8, 9, a corresponding electric field 10, 11, 12 is generated within the microfluidic passage 6. The lines shown in FIG.
  • the system shown in FIG. 6 also includes a light source 17, such as a light emitting diode (LED) configured to facilitate imaging by optical system 18.
  • a light source 17 such as a light emitting diode (LED) configured to facilitate imaging by optical system 18.
  • one or more particles captured by the electrodes 7, 8, 9 may be labeled with fluorophores and the light source 17 may be configured to excite the fluorophores prior to imaging the electrode surface with optical system 18.
  • the particles may not be labeled, and the light source 17 may be configured to provide light for bright field imaging using optical system 18 to image the unlabeled particles.
  • Optical system 18 may include an optical sensor and a detector configured to capture one or more images of a surface of electrodes 7, 8, 9.
  • the detector is a fluorescent light detector included within a fluorescent microscope.
  • the optical system 18 includes an LED light source 17 with an objective and a color image detector (e.g., a color digital camera).
  • Optical system 18 may be configured to image the electrode system and capture an image.
  • Optical system 18 may be configured to image multiple parts of the electrodes 7, 8, 9.
  • the microfluidics device may be placed on a stage that enables movement in the x and/or y directions.
  • Optical system 18 is operatively connected to a computer 19 that includes a hardware computer processor configured to analyze the one or more images (e.g., fluorescent or bright field images) captured by the optical system 18.
  • the computer is programmed to count bacteria present in the image(s) to quantify an amount of bacteria in the sample provided as input to the microfluidic device.
  • the computer is a network-connected device.
  • the network-connected device may be configured to transmit quality control information to a server adapted to store and analyze trends involving product production from multiple locations. Such a system may allow for tracking of the spread of contamination, for example.
  • the network-connected device may be configured to transmit sterility assurance information to a server adapted to store and analyze trends involving product production from multiple locations. Such a system may allow for tracking of the spread of contamination, for example.
  • FIG. 7 shows a simplified version of the system shown in FIG. 6. As shown in FIG. 7,
  • FIG. 8 illustrates a process for rapidly detecting viable bacteria in a fluid sample with reference to the systems shown in FIGS. 6 and 7, in accordance with some embodiments. In act
  • the sample from container 3 is passed through the microfluidic passage 6 to capture microorganisms (e.g., bacteria) in the sample on the surface of one or more electrodes (e.g., electrodes 7, 8, 9).
  • microorganisms e.g., bacteria
  • the signal from the controller 20 may be on to generate the electric fields 10, 11, 12 to capture at least some of the microorganisms on the surface of the electrodes.
  • an entire volume of the sample in container 3 may be passed through the microfluidic passage.
  • the remaining sample volume may be preserved in container 3 for other purposes.
  • a controlled solution fluid from container 2 may be passed through the microfluidic passage.
  • the signal from the controller 20 may be on to generate the electric fields 10, 11, 12.
  • the signal of the controller 20 may be configured to apply a voltage to the electrode(s) sufficient to
  • a high voltage signal e.g., at least 20V
  • a lower voltage signal e.g., 20V or less
  • the controlled solution fluid from container 2 (or other reference or controlled fluid from another container) may be passed through the microfluidic passage.
  • the signal from controller 20 may remain on to ensure that the microorganisms captured on the electrode surfaces remain captured.
  • Process 800 then proceeds to act 812, where a first fluid with a first stain (e.g., from container 4A) is passed though the microfluidic passage while the microorganisms remain captured on the surface of the electrodes.
  • the first stain may be configured to selectively stain live microorganisms or dead microorganisms.
  • the first stain is configured to selectively stain dead microorganisms.
  • the first stain is a metabolic stain configured to selectively stain live microorganisms.
  • Process 800 then proceeds to act 814, where a first image of the surface of the one or more electrodes is captured using the optical system 18.
  • multiple first images may be captured as a stage on which the microfluidic device is positioned is moved to image different portions of an electrode surface and/or different electrodes.
  • a series of first images of captured microorganisms may be recorded, in such a way that each microorganism is imaged multiple times. The series of first images may then be analyzed and live microorganisms identified based on a displayed shift in positions in the series of images over time.
  • the controlled solution from container 2 may be passed through the microfluidic passage to rinse the electrode surface.
  • the signal from the controller 20 may remain on to ensure that the microorganisms captured on the electrodes remain captured.
  • Process 800 then proceeds to act 816, where a second fluid containing a second stain
  • the second stain may be configured to
  • Process 800 then proceeds to act 818, where a second image of the surface of the one or more electrodes is captured.
  • a second image of the surface of the one or more electrodes may be captured as a stage on which the microfluidic device is positioned is moved to image different portions of an electrode surface and/or different electrodes.
  • the controlled solution from container 2 may be passed through the microfluidic passage to rinse the electrode surface. During the rinse, the signal from the controller 20 may remain on to ensure that the microorganisms captured on the electrodes remain captured.
  • Process 800 then proceeds to act 820, where the first image(s) and the second image(s) are analyzed to quantify an amount of live and/or dead microorganisms in the sample.
  • FIG. 9 illustrates a process 900 for quantifying an amount of live and/or dead microorganisms based on a first image and a second image, in accordance with some embodiments.
  • act 910 first and second images, each having different staining for live and/or dead microorganisms are received.
  • a first image may have been captured after staining with a first stain configured to stain only dead microorganisms or live microorganisms
  • the second image may have been captured after staining with a second stain configured to stain all microorganisms regardless of viability.
  • Process 900 then proceeds to act 912, where a first quantity of live and/or dead microorganisms is determined in the first image.
  • all dead microorganisms in the first image may be labeled with the first stain, and a number of labeled microorganisms in the first image may be automatically detected and counted to determine the quantity of dead microorganisms in the sample.
  • Process 900 then proceeds to act 914, where a second quantity of live and/or dead microorganisms in the second image is determined. For instance, all microorganisms regardless of viability may be labeled with the second stain, and a number of labeled microorganisms in the second image may be automatically detected and counted to determine the quantity of microorganisms (live and dead) in the sample and/or other particles.
  • Process 900 then proceeds to act 916, where the quantity of live and/or dead microorganisms in the sample is determined based on the first amount and the second amount.
  • the first amount may be subtracted from the second amount to determine the quantity of live microorganisms in the sample.
  • the positions of the labeled microorganisms in the first and second images may be determined, and the quantity of live and/or dead microorganisms in the sample may be determined, at least in part, on the determined positions of the labeled microorganisms.
  • each of the labeled microorganisms in the first image may be associated with a first position and each of the labeled microorganisms in the second image may be associated with a second position.
  • some embodiments use the determined positions for the labeled microorganisms in the first and second images to determine which labeled microorganisms are present in both images. Based on that determination, a more accurate count of the live and/or dead microorganisms in the sample may be more precisely determined. For instance, only labeled microorganisms that are present in a similar position in both images may be considered as dead (or live) microorganisms, depending on the selective staining.
  • Process 900 then proceeds to act 918, where an amount of live and/or dead microorganisms in the sample is output.
  • Outputting the amount of live and/or dead microorganisms may be performed in any suitable way. For example, in some embodiments an indication of the first amount of live and/or dead microorganisms identified in the first image and an indication of the second amount of live and/or dead microorganisms identified in the second image may be output. In some embodiments, outputting the amount of live and/or dead microorganisms in the sample may only be performed when the amount of live microorganisms is above a threshold value.
  • a process for rapidly monitoring viable bacteria is provided. Such a process may be used, for example, to monitor bacterial viability in microbiome drug manufacturing, or to monitor effectiveness of cleaning and or sterilization.
  • a third fluid having a third stain may be passed through the microfluidic passage of the microfluidic device after capturing the second image.
  • the third stain may be different than the first stain and the second stain.
  • the third stain may be configured to selectively stain live microorganisms.
  • the signal from the controller 20 may remain on throughout the process of passing the third fluid through the microfluidic passage to ensure that any microorganisms remain captured on the surface of the
  • a third image of the surface of the one or more electrodes may be captured.
  • multiple third images may be captured as a stage on which the microfluidic device is positioned is moved to image different portions of an electrode surface and/or different electrodes.
  • a controlled solution from container 2 or another container may be passed through the microfluidic passage to rinse the electrode surface. During the rinse, the signal from the controller 20 may remain on to ensure that the microorganisms captured on the electrodes remain captured.
  • three numbers may be reported, each of which represents a number of quantified microorganisms for each of the three stains.
  • FIG. 10A shows the quantified number of bacteria for three stains analyzed for Sample 1
  • FIG. 10B shows the quantified number of bacteria for three stains analyzed for Sample 2.
  • Each number, 1, 2, and 3 represents a quantified result for each of three stains.
  • the result for Sample 2 is substantially different from the result for Sample 1 (control sample), which indicates that there may be an error in the process and the number of viable cells is not as expected in a correctly running process.
  • all three numbers may be reported to ensure consistency of the process.
  • the ratios and or trends between the reported numbers may also be reported, an example of which is shown in FIG. IOC. When the ratios or trends start varying along the multiple steps of the same process, it serves as an indicator that there is a problem in the manufacturing process and corrective action should be taken.
  • Applications of the technology described herein including, but are not limited to, detecting live legionella bacteria and other bacteria from a panel of problematic pathogens that cause infections in humans (e.g., from hospital water systems).
  • the techniques described herein may be used to enable lowering hospital acquired infections from environmental factors, especially due to the ability to receive rapid results. For example, from all bacteria captured from a sample taken from a treated surface using the microfluidic device, there may be some problematic pathogen bacteria. If all of the captured bacteria are determined to be dead, it may be determined that the surface treatment was effective, and specific bacterial identification is not necessary. However, if it is determined that the captured bacteria include live bacteria that
  • the surface still includes live bacteria from the pathogen group, since pathogen group bacteria are hard to kill.
  • the technology described herein may be applied to testing patient samples for infections and/or for assessing effectivity of treatments. In some embodiments, the technology described herein may be applied to testing patient samples for assessing antibiotic resistance and antibiotic susceptibility.
  • scanning the surfaces of the electrodes by the optical system may comprise scanning only the front of the electrode area.
  • the result of such a scan is approximate statistical quantification of the captured bacteria.
  • Statistical quantification is based on the tail gradient of bacterial distribution on the microfluidic device, and may be applied, for example, for high concentration microbiome samples.
  • the sample may be provided to flow through multiple microfluidic devices, and the electrode(s) in each of the microfluidic devices may be scanned consecutively.
  • the system continuously scans and performs on chip staining at defined time intervals.
  • samples are loaded anaerobically to preserve viability of anaerobic organisms.
  • tubing and the microfluidic system may be flushed with Nitrogen or other gas to displace ambient air.
  • one or more valves may be provided in the system to prevent gas backflow.
  • the techniques described herein may be used to test a cellular response to antibiotics.
  • isolation of particular bacteria, viruses, analytes, microscale components, etc. may be performed.
  • the isolated microorganism may then be subjected to particular antibiotics, and the cell lysis products may be analyzed by a microwire array sensor or optically.
  • the techniques described herein may be used to separate viable bacteria from nonviable bacteria by capturing all bacteria on a surface of one or more electrodes and selectively releasing the captured live bacteria from the electrode(s) by adjusting the signal parameters (e.g., frequency) provided by the controller.
  • the signal parameters e.g., frequency
  • the techniques described herein may be used to implement a database for tracking bacterial resistance. For instance, a particular patient's bacterial fingerprint may be sensed and compared to a bacterial resistance database allowing for treatment with
  • inventions include, but are not limited to, use in an inline sensor.
  • inline sensors for intravenous (IV) lines may be used, e.g., for early detection of infection and/or monitoring of bacteria, viruses, and analytes.
  • IV lines intravenous
  • Such applications are suitable, for example, for inline glucose sensors, which may be prone to bacterial contamination.
  • implementation in inline sensors for biomanufacturing may be used, e.g., for early detection of contamination and/or monitoring of bacteria, viruses, and analytes.
  • bioreactors used for drug manufacturing and vaccine manufacturing which may be prone to bacterial contamination.
  • implementation in inline sensors for food and beverage safety may be used, e.g., for early detection of contamination and/or monitoring of bacteria, viruses, and analytes.
  • implementation in inline sensors for recreational water monitoring may be used, e.g., for early detection of contamination and/or monitoring of bacteria, viruses, and analytes.
  • the techniques described herein may be used in research and development (R&D) and/or manufacture of one or more of microbiome drug development, veterinary products, cosmetic and personal hygiene products, agricultural products and chemicals, or viral delivery platforms.
  • the techniques described herein may be used to test sterility in drugs (e.g., drugs with short shelf life, immunotherapy drugs, CAR-T drugs, CAR-NK drugs etc.)
  • drugs e.g., drugs with short shelf life, immunotherapy drugs, CAR-T drugs, CAR-NK drugs etc.
  • the techniques described herein may replace a laboratory- based sterility test.
  • the techniques described herein may replace a laboratory- based sterility test with a microfluidic system integrated with the processing equipment (e.g., at an immunotherapy center, apheresis).
  • the techniques described herein may replace a laboratory- based sterility test with a microfluidic system integrated with the processing equipment and the manufacturing line.
  • glass with magnifying properties is used in the microfluidic device.
  • the techniques described herein may be used to implement a sterility test where the sample includes dead microorganisms or no microorganisms, and as such can be considered sterile. In some embodiments, the techniques described herein may be used to implement a sterility test where only a single live (viable) microorganism can be detected in the sample, result in a failed sterility test.
  • 29 medium (or multiple computer readable storage media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement one or more of the various embodiments described above.
  • the computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various ones of the aspects described above.
  • computer readable media may be non-transitory media.
  • the above-described embodiments of the present technology can be implemented in any of numerous ways.
  • the embodiments may be implemented using hardware, software or a combination thereof.
  • the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers.
  • any component or collection of components that perform the functions described above can be generically considered as a controller that controls the above-described function.
  • a controller can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processor) that is programmed using microcode or software to perform the functions recited above, and may be implemented in a combination of ways when the controller corresponds to multiple components of a system.
  • a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer, as non-limiting examples. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smartphone or any other suitable portable or fixed electronic device.
  • PDA Personal Digital Assistant
  • a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing
  • a computer may receive input information through speech recognition or in other audible formats.
  • Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet.
  • networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.
  • some aspects may be embodied as one or more methods.
  • the acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • the terms “substantially”, “approximately”, and “about” may be used to mean within ⁇ 20% of a target value in some embodiments, within ⁇ 10% of a target value in some embodiments, within ⁇ 5% of a target value in some embodiments, within ⁇ 2% of a target value in some embodiments.
  • the terms “approximately” and “about” may include the target value.

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Abstract

L'invention concerne des systèmes et des procédés permettant de quantifier des micro-organismes vivants et/ou morts dans un échantillon. Le procédé consiste à recevoir une image en couleur capturée par un système optique, l'image en couleur comprenant au moins une électrode du système microfluidique et un ou plusieurs micro-organismes disposés sur une surface de ladite au moins une électrode, classifier chacun du ou des micro-organismes comme étant un micro-organisme vivant ou un micro-organisme mort sur la base, au moins en partie, d'une teinte du micro-organisme dans l'image en couleur, quantifier, sur la base de la classification, une quantité de micro-organismes vivants et/ou de micro-organismes morts dans l'image en couleur, et délivrer en sortie un résultat de la quantification.
PCT/US2022/029236 2021-05-13 2022-05-13 Techniques de détection et de quantification de bactéries vivantes et mortes dans un échantillon de fluide Ceased WO2022241243A1 (fr)

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