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WO2025122577A1 - Dispositif fluidique refermable et son application - Google Patents

Dispositif fluidique refermable et son application Download PDF

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Publication number
WO2025122577A1
WO2025122577A1 PCT/US2024/058403 US2024058403W WO2025122577A1 WO 2025122577 A1 WO2025122577 A1 WO 2025122577A1 US 2024058403 W US2024058403 W US 2024058403W WO 2025122577 A1 WO2025122577 A1 WO 2025122577A1
Authority
WO
WIPO (PCT)
Prior art keywords
resealable
fluidic device
frame
slide
fluid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/058403
Other languages
English (en)
Inventor
Rui Zheng
Zheng XING
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Spatomics LLC
Original Assignee
Spatomics LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Spatomics LLC filed Critical Spatomics LLC
Publication of WO2025122577A1 publication Critical patent/WO2025122577A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0822—Slides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates

Definitions

  • the present invention relates to the field of devices for cellular and tissue assays. More particular, the present invention relates to a resealable fluidic device for glass slide based biological samples.
  • Biological sample microscopic analysis is a widely used method. For visualization of cells and tissues under microscopes, biological samples are collected and disposed on a glass side before being visualized under the microscopes.
  • Biological samples on glass slides such as tissue sections, microbeads, and live cell assays, are extensively utilized in both biomedical and clinical research.
  • the prevailing method for handling these biological samples involves placing a thin cover glass over the glass slide on which the biological samples disposed to contain the reaction liquid.
  • this approach offers limited control over liquid volume, making it susceptible to evaporation and temperature fluctuations.
  • direct contact between the cover glass and the biological sample can cause potential disruption of the integrity of the biological sample, and removing the cover slide without disturbing the sample presents further challenges.
  • this invention discloses a resealable fluidic device for conducting assays of a biological sample.
  • the device comprises a frame having a sample opening; and a cover slide configured to be received in the frame; wherein the frame is configured to receive a base slide on which a biological sample is configured to be disposed; wherein the cover slide and the base slide are configured to form a flow cell chamber housing the biological sample, and the flow cell chamber is configured to be aligned with the sample opening when the frame, the cover slide, and the based slide are assembled.
  • the frame comprises a fluid inlet, and a fluid outlet; wherein the fluid inlet and the fluid outlet are in fluid communication with the flow cell chamber.
  • At least one solution for assays of the biological sample is configured to be injected into the flow cell chamber via the fluid inlet.
  • the at least one solution for assays of the biological sample is configured to be removed from the flow cell chamber via the fluid outlet.
  • each of the fluid inlet and the fluid outlet has a diameter between about 0.2-2 mm.
  • the cover slide has a first fluid hole and a second fluid hole, wherein the first fluid hole is configured to be aligned with the fluid inlet on the frame and the second fluid hole is configured to be aligned with the fluid outlet on the frame when the frame, the cover slide, and the base slide is assembled.
  • each of the first and second fluid holes has a diameter between about 0.2-20 mm.
  • the cover slide has a thickness between about 0.12-0.20 mm.
  • the cover slide is made of glass.
  • the device further comprises a middle layer configured to be disposed between the cover slide and the base slide.
  • the middle layer comprises an opening which is configured to form side walls of the flow cell chamber. In one embodiment, the middle layer comprises an opening which is configured to form side walls of the flow cell chamber.
  • the middle layer has a thickness between about 10-500 um.
  • the middle layer is integrated with the cover slide.
  • the middle layer is made of silicon.
  • the frame comprises one or more magnets.
  • the device further comprises one or more retaining tabs, wherein the one or more retaining tabs removably attach to the frame to hold the cover slide and the base slide to the frame.
  • a resealable fluidic devices assembly comprises multiple resealable fluid devices, a holding plate on which the multiple resealable fluid devices are configured to be disposed.
  • the holding plate comprises multiple pairs of protrusions, each pair of protrusions is configured to be received in a pair of alignment holes on the frame of each of the multiple resealable fluid devices.
  • the holding plate comprises a heating element for heating the biological samples in the flow cell chamber.
  • Fig. 1 illustrates a schematic view of an assembled resealable fluidic device according to one embodiment of the present invention.
  • Fig. 2 illustrates an exploded view of the resealable fluidic device according to the embodiment in Fig. 1 .
  • Fig. 3 illustrates an exploded view of the resealable fluidic device according to another embodiment of the present invention.
  • Fig. 4 illustrates a top and bottom view of a frame of the resealable fluidic device according to the embodiment in Fig. 1.
  • Fig. 5 illustrates photographs of the resealable fluidic device disposed on a display and in association with a microscope.
  • Fig. 6 illustrates a photograph of a process for fluid exchange in the resealable fluidic device.
  • Fig. 7 shows microscopic images of stained thyroid tissue using conventional glass side (left panel) and the resealable fluidic device (right panel) of the present invention.
  • Fig. 8 shows microscopic images of biological samples processed by the resealable fluidic device of the present invention.
  • Fig. 9 shows microscopic images of one particular biological sample stained for various biomarker using the resealable fluidic device.
  • Fig. 10 shows microscopic images of 12 different proteins stained with CFTs and DAPI on the same FFPE tonsil tissue at the same location, using the resealable fluidic device of the present invention.
  • Fig. 11 shows microscopic images of H&E stained FFPE tonsil at the end of the 12 cycles of protein staining process, using the resealable fluidic device of the present invention.
  • Fig. 12 shows a workflow process of using the resealable fluidic device of the present invention for processing biological samples.
  • Fig. 13 shows a photograph of a heating adaptor used in association with the resealable fluidic device.
  • Fig. 14 shows a photograph of a vacuuming device used in association with the resealable fluidic device.
  • Fig. 15 shows a top schematic view of an assembled resealable fluidic device according to the embodiment of Fig. 3.
  • Fig. 16 shows a bottom schematic view of an assembled resealable fluidic device according to the embodiment of Fig. 3.
  • Fig. 17 shows a top and a bottom schematic view of an assembled resealable fluidic device according to another embodiment of the present invention.
  • Fig. 18 shows a top schematic view of multiple assembled resealable fluidic devices disposed on a device holder according to one embodiment of the present invention.
  • first, second, third etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the invention.
  • “around”, “about”, “approximately” or “substantially” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the terms “around”, “about”, “approximately” or “substantially” can be inferred if not expressly stated.
  • the phrase “at least one of A, B, and C” should be construed to mean a logical (A or B or C), using a non-exclusive logical OR.
  • the term “and/or” includes any and all combinations of one or more of the associated listed items.
  • the current invention introduces a resealable fluidic device, which is an easy to use, low cost, widely applicable resealable fluidic device for glass slide based biological samples.
  • the present invention discloses a resealable fluidic device having a flow cell chamber in which the biological sample is disposed.
  • the present invention discloses a resealable fluidic device having a flow cell chamber in which one or more solutions necessary for staining, processing, and/or culturing the biological sample is received and/or exchanged.
  • the one or more solutions includes antibodies, stains, and other reagents necessary for the process.
  • the one or more solutions can be injected into the flow cell chamber via a fluidic exchange inlet and thus contact the biological samples. Once the reaction is accomplished, the solutions can be removed from the flow cell chamber via a fluidic exchange outlet. This process can be repeated with different solutions necessary for different steps of staining, processing, and/or culturing the biological sample.
  • the biological sample maintains its integrity during and after the sample processing and assays.
  • the present invention discloses a resealable fluidic device in which the biological sample is not in contact with a cover slide from the top when the biological sample is disposed on a base slide for assays.
  • the present invention discloses a resealable fluidic device has a detachable design that allows users to remove a base slide on which the biological sample is disposed after testing and/or imaging, and can store them for further testing and evaluation.
  • the present invention discloses a resealable fluidic device which is compatible with most of the fluorescent microscopes and liquid handling equipment on the market.
  • the present invention discloses a resealable fluidic device which has a significantly bigger imaging area (40mm*20mm) comparing to conventional base glass slide.
  • the present invention discloses a resealable fluidic device which is made from high temperature resistant material, allowing the users to conduct antigen retrieval and antibody stripping steps that require high temperature (-100C).
  • the present invention discloses a resealable fluidic device which has low reagent consumption for staining/processing within the flow cell chamber.
  • the present invention discloses a resealable fluidic device which provides more uniform interaction between reagents and the biological samples compared to the conventional tissue staining methods.
  • the resealable fluidic device provides the users with microscopic images of stained/processed biological samples having a higher sensitivity and a better SNR (signal to noise ratio).
  • the present invention discloses a resealable fluidic device which can be used with Cleavable Fluorescent Probe (CFP) chemistry.
  • CFP Cleavable Fluorescent Probe
  • the present invention discloses a resealable fluidic device which achieves high-plex protein and RNA detections on FFPE tissue samples.
  • Fig. 1 shows a schematic view of an assembled resealable fluidic device 10
  • Fig. 2 shows an exploded view of the resealable fluidic device 10.
  • the assembled resealable fluidic device 10 a frame 11.
  • the frame 11 includes one or more fluidic exchange inlets 118, and one or more fluidic exchange outlet 112.
  • the frame 11 also includes a glass slide retainer for attaching the glass slide with the frame so as to prevent any movement between the frame and the glass slide.
  • the frame 11 has a peripheral rim 111, a sample opening 113, and/or a label opening 115.
  • the frame is made of plastic.
  • the frame is made of one or more of bioplastics, polylactic acid (PLA), cellulose-based plastics, Delrin (acetal), Ultem (Polyetherimide), and PEEK (Polyether ether ketone), PMMA, silicone, natural rubber, starch-based materials, glass, metal, and wood composite.
  • the frame is rigid. In certain embodiments, the frame is elastic for adapting to minor shape variance of the glass slide, and/or easy assembling of the resealable fluidic device.
  • the resealable fluidic device 10 includes a cover slide 13.
  • the cover slide 13 is received inside the frame 11 when the resealable fluidic device 10 is assemble.
  • the cover slide 13 is removably attached to the resealable fluidic device 10.
  • the cover slide 13 forms a top surface of a flow cell chamber, which receives a biological sample and any fluids necessary for the sample processing.
  • the cover slide 13 has one or more of cover slide holes 131. In certain embodiments, the cover slide 13 has one cover slide hole 131, which is aligned to the fluidic exchange inlet 118 on the frame 11 when the resealable fluidic device 10 is assembled. In certain embodiments, the cover slide 13 has two cover slide hole 131, one of which is aligned to the fluidic exchange inlets 118 on the frame 11, and the other is aligned to fluidic exchange outlet 112 on the frame 11 when the resealable fluidic device 10 is assembled.
  • the cover slide 13 is made of glass/optical glass. In certain embodiments, the cover slide 13 is made of one or more of glass, plexiglass, polycarbonate, sapphire, fused silica, polyethylene terephthalate (PET or PETG), PMMA, Cycle Olefin Polymers, cellulose acetate, silicon, etc.
  • the cover slide has a thickness between 0.16-0.18 mm. In certain embodiment, the cover slide has a thickness between 0.18-0.20 mm. In certain embodiment, the cover slide has a thickness between 0.20-0.22 mm. In certain embodiment, the cover slide has a thickness between 0.14-0.16 mm. In certain embodiment, the cover slide has a thickness between 0.12-0.14 mm.
  • the cover slide hole has a diameter between 0.2-0.4 mm. In certain embodiments, the cover slide hole has a diameter between 0.4-0.6 mm. In certain embodiments, the cover slide hole has a diameter between 0.6-0.8 mm. In certain embodiments, the cover slide hole has a diameter between 0.8-1.0 mm. In certain embodiments, the cover slide hole has a diameter between 1.0-1.2 mm. In certain embodiments, the cover slide hole has a diameter between 1.2-1.4 mm. In certain embodiments, the cover slide hole has a diameter between 1.4-1.6 mm. In certain embodiments, the cover slide hole has a diameter between 1.6- 1.8 mm. In certain embodiments, the cover slide hole has a diameter between 1.8-2.0 mm.
  • the resealable fluidic device 10 includes a middle layer 15, which forms peripheral side walls of the flow cell chamber.
  • the middle layer 15 is attached to the cover slide 13 by a fastening element.
  • the middle layer 15 is received in and attached to the frame 11.
  • the middle layer 15 is independent of the cover slide 13. In certain embodiments, as shown in Fig. 3, the middle layer 15 is integrated with the cover slide 13 to form one part.
  • the middle layer 15 has a thickness between 10-100 um. In certain embodiments, the middle layer 15 has a thickness between 100-200 um. In certain embodiments, the middle layer 15 has a thickness between 200-300 um. In certain embodiments, the middle layer 15 has a thickness between 300-400 um. In certain embodiments, the middle layer 15 has a thickness between 400-500 um.
  • the middle layer 15 is made of silicon. In certain embodiments, the middle layer 15 is made of one or more of silicon, acrylic, polyurethan, epoxy resins, etc.
  • the middle layer 15 has reusable viscous materials. In certain embodiments, the middle layer 15 has adhesive materials, such that the middle layer could be removed from a base glass slide on which the biological sample is disposed, and resealed to the same or another base glass slide at a later time point.
  • the frame 11, the cover slide 13 and the middle layer 15 form the resealable fluidic device 10.
  • the frame 11, the cover slide 13, the middle layer 15 and a base slide 17 form the resealable fluidic device 10.
  • the base slide 17 is a commonly used slide on which the biological sample is disclosed.
  • the base slide 17 is a slide specialized in being use with the resealable fluidic device 10.
  • the base slide 17 has a sample disposing section 171 and a labeling section 173.
  • the base slide 17 is received by the frame 11. In certain embodiments, the base slide 17 is attached to the frame 11 via a fastening mean.
  • the base slide When the base slide is installed onto the frame with the cover slide 13, the middle layer 15, the base slide 17 forms a bottom surface of the flow cell chamber. The flow cell chamber is enclosed with in the cover slide 13, the middle layer 15, and the base slide 17.
  • the sample disposing section 171 is aligned with the sample opening 113, and the labeling section 173 is aligned with the labeling opening 115.
  • the base slide 17 is made of glass/optical glass. In certain embodiments, the base slide 17 is made of one or more of glass, plexiglass, polycarbonate, sapphire, fused silica, polyethylene terephthalate (PET or PETG), cellulose acetate, silicon, etc.
  • the resealable fluidic device 10 also includes one or more retaining tabs 29.
  • the retaining tabs 29 hold the base slide 27 and the cover slide 23 to the frame 21.
  • the frame 21 has one or more slots matching the retaining tabs 29 such that the retaining tabs 29 are removably clicked onto the frame 21.
  • Figs. 15-16 show a top and bottom view of the resealable fluidic device 20 of Fig. 3 in an assembled state.
  • the retaining tabs 29 hold the base slide 2 and cover slide 23.
  • a fluidic chamber is formed by and between the base slide 2 and cover slide 23.
  • the frame 21 has a labeling viewing window 215 and a sample viewing window 213.
  • a frame 31 has one or more alignment holes 310 for aligning the frame 31 with the cover slide and the base slide.
  • the frame 31 has one or more magnets 314 for holding the flow cells down.
  • the one or more magnets 314 on frame 31 will be attracted to the magnets on the sample holder 49, or magnets on the heating adapter 59. As such, the flow cells are held in place.
  • the magnets are disposed on the four comers of the frame 31.
  • the frame 31 has a fluidic exchange inlet 318 in fluid communication with the flow cell chamber 316.
  • a user of the resealable fluidic device can inject one or more solutions into the flow cell chamber 316 via the fluidic exchange inlet 318.
  • the frame 31 also has a fluidic exchange outlet 312 in fluid communication with the flow cell chamber 316. The liquid in the flow cell chamber can be pumped out via the fluidic exchange outlet 312.
  • the frame 31 has multiple fluidic exchange inlets 318 in fluid communication with the flow cell chamber 316. In certain embodiments, the fluidic exchange inlet 318 is aligned with one cover slide hole 131 permitting the solutions entering the flow cell chamber 316. In certain embodiments, the frame 31 also has multiple fluidic exchange outlets 312 in fluid communication with the flow cell chamber 316. In certain embodiments, the fluidic exchange inlet 318 is aligned with one cover slide hole 131 permitting the solutions exiting the flow cell chamber 316.
  • Fig. 17 shows an alternative embodiment of the present invention.
  • a frame 61 has two groove 612 matched to two side edges of the base slide 17 and therefore holding the base slide 17 and cover slide 65 to the frame 61.
  • the base slide 67 is slidably received in the two grooves 612.
  • the frame 61 only has a sample viewing window 613 but not a label viewing window.
  • Figs. 5-6 show photographs of a resealable fluidic device 40 disposed on a device holding base 49 and under the microscope.
  • a device holding base 49 has a holding peripheral frame 49 forming an opening for receiving one or more resealable fluidic devices 40.
  • the holding peripheral frame 49 has an upper edge, a lower edge, and two side edges forming the opening.
  • the holding peripheral frame 49 have one or more holding protrusions 491 for being aligned with the alignment holes 310 of the frame 31.
  • the holding peripheral frame 49 has multiple pairs of the holding protrusions 491. One holding protrusion 491 of each pair is arranged on the upper edge of the holding peripheral frame 49; and the other holding protrusion 491 of each pair is arranged on the lower edge of the holding peripheral frame 49. Each pair of the holding protrusions 491 is matched with and received in the pair of alignment holes 310 on the frame 31.
  • the holding peripheral frame 49 receives and holds multiple resealable fluidic devices.
  • Fig. 18 shows another embodiment in which multiple resealable fluidic devices 50 are received and hold by a holding tab 59.
  • the resealable fluidic devices 50 has the same embodiment as shown in Fig. 17.
  • the base slide 57 has its labeling section 573 exposed out of the frame 51, so that it can be compatible with the existing holding tab 59 from the most used microscopes (Zeiss, Leica, Olympus, Nikon, etc). Therefore, the user can perform fluorescent imaging with the devices of the present invention without any modification to his or her microscopes.
  • Fig. 13 shows a heating adaptor which can be used in association with the resealable fluidic device.
  • a heating adaptor 59 can be use in association with one resealable fluidic device or multiple resealable fluidic devices.
  • the heating adaptor 59 has one or more heating sections 592 matched with the sample section 171 of the base slide 17 when the base slide 17 is assembled with the device.
  • the heating sections 592 are a raised section which is in direct contact with the bottom surface of the sample section 171.
  • the heating adaptor 59 has multiple holding magnets 594 which are matched with the magnets 314 on the frame 31 of the resealable fluidic device.
  • the heating adaptor 59 has one or more heating adaptor matching holes 591 which is matched with the alignment holes 310 on the frame 31 of the resealable fluidic device.
  • Fig. 14 shows an embodiment of a vacuuming device in connection with a vacuuming pump and a vacuuming tip. When using the vacuuming device removing the solutions from the flow cell chamber 316, the vacuuming tip is matched with the fluidic exchange outlet 312. Once the vacuuming pump is turned on, the solutions are pumped out of the flow cell chamber 316.
  • the injection of the solution and the removal of the solution are conducted simultaneously. In certain embodiments, the injection of the solution and the removal of the solution are conducted at different times. In certain embodiments, the assembled resealable fluidic device is ready for running biological assays, i.e., liquid exchange, incubation, imaging of the biological samples disposed on the base glass slide.
  • the base slide when the biological assays are accomplished, can be released from the resealable fluidic device 10, (or the frame 11 and the cover slide 31) for storage or for further downstream applications.
  • the base slide can also be reinstalled to the resealable fluidic device 10 for multi-round applications.
  • Fig. 7 shows microscopic photographs of stained thyroid tissue using conventional glass side (left panel) and the resealable fluidic device (right panel) of the present invention.
  • histone H3 in the thyroid tissue is stained in bright yellow.
  • the resealable fluidic device of the present invention provides a stained biological sample with better signal intensity and improved signal-to-noise ratio.
  • Fig. 7 also shows that the resealable fluidic device of the present invention provides signals having better distribution, which is essential for recognizing different types of single cells in the biological sample.
  • the device of the present invention e.g.
  • the flowcell chamber allows active reagents flow and exchange therewithin, thus creates more interaction between reagents and biomarkers on the biological samples. This increased interaction helps with the biomarker fluorescent labeling and provides higher contrast and better SNR ratio.
  • the top image using the flowcell staining of the present invention has a stronger and higher contrast signal (yellow color) compared to the bottom image, which is stained using conventional staining method.
  • Figs. 8-11 shows staining results of various biological samples with different biomarkers using the resealable fluidic device of the present invention, including multiplexed IHC, ISH, live cell assays, and NGS.
  • Fig. 8 panel (a) shows a microscopic image of 3-Plex IHC staining.
  • Fig. 8 panel (b) shows a microscopic image of 4-Plex ISH staining.
  • Fig. 8 panel (c) shows a microscopic image of 4-plex Live Cell Assay staining.
  • Fig. 8 panel (A) shows a microscopic image of Bead based DNA Sequencing (NGS).
  • Fig. 9 shows microscopic staining images of 28-Plex FFPE Sample Spatial Protein.
  • Fig. 10 shows microscopic photographs of 12 different proteins stained with CFTs and DAPI on the same FFPE tonsil tissue at the same location, using the resealable fluidic device of the present invention.
  • Fig. 11 shows microscopic photographs of H&E stained FFPE tonsil at the end of the 12 cycles of protein staining process, using the resealable fluidic device of the present invention.
  • the resealable fluidic device of the present invention can be used for currently highest plex spatial protein assay due to its resealable and non-sample contacting features.
  • Table 1 shows materials and equipment for FFPE Hi-Plex Protein Protocol
  • Fig. 12 shows a workflow process of using the resealable fluidic device of the present invention for processing biological samples.
  • Table 2 shows detailed test procedures using the resealable fluidic device following the workflow of Fig. 12.
  • the workflow includes a sample preparing stage 91, a staining/processing stage 93, and optionally a recycle stage 95.
  • a biological sample is disposed in an assembled resealable fluidic device following a baking step, a dewaxing step, a device assemble step, and 3 pretreatment steps.
  • a background imaging is conducted, during which the processed sample in the flow chamber of the resealable fluidic device is scanned by a microscope, and the image is saved. The background signal is checked at selected color channel. Then the staining/processing stage 93 is conducted, in which the samples is treated with primary and secondary antibodies and one or more staining is conducted. During this stage, the type and time of the staining can be customized to a user’s needs.
  • a user can conduct the recycle stage 95 for recycle the samples for a second round of the staining/processing stage 93.
  • the recycle stage 95 has an antibody stripping step and a fluorescent cleaving step.
  • a H&E staining/imaging stage 97 is conducted after the staining/processing stage 93.
  • Pretreatment 1 a. Set hot plate at 95°C and wait to reach the set temperature.
  • Pretreatment 2 a. Inject 200ul of lx PS2 and incubate for 5min@RT i b. Rinse with wash solution: 200ul, 3 times

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Dispersion Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

L'invention concerne un dispositif fluidique refermable destiné à réaliser des dosages d'un échantillon biologique qui comprend un cadre comportant une ouverture d'échantillon ; et une lamelle couvre-objet conçue pour être reçue dans le cadre ; le cadre étant conçu pour recevoir une lamelle de base sur laquelle un échantillon biologique est conçu pour être disposé ; la lamelle couvre-objet et la lamelle de base étant conçues pour former une chambre de cellule d'écoulement logeant l'échantillon biologique, et la chambre de cellule d'écoulement étant conçue pour être alignée avec l'ouverture d'échantillon lorsque le cadre, la lamelle couvre-objet, et la lamelle de base sont assemblés.
PCT/US2024/058403 2023-12-05 2024-12-04 Dispositif fluidique refermable et son application Pending WO2025122577A1 (fr)

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US20180364270A1 (en) * 2015-07-07 2018-12-20 University Of Washington Systems, methods, and devices for self-digitization of samples
US10441952B2 (en) * 2010-04-23 2019-10-15 Biomerieux, S.A. Device for preparing and/or treating a biological sample
US11033897B2 (en) * 2008-11-26 2021-06-15 Ucl Business Plc Microfluidic device
CN114487401B (zh) * 2022-04-18 2022-06-21 中国农业大学 一种微生物检测的双适配体功能核酸恒温微流控芯片传感器
US20220291246A1 (en) * 2019-09-27 2022-09-15 Lunaphore Technologies Sa Biological sample processing system and microfluidic cartridge therefor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11033897B2 (en) * 2008-11-26 2021-06-15 Ucl Business Plc Microfluidic device
US10441952B2 (en) * 2010-04-23 2019-10-15 Biomerieux, S.A. Device for preparing and/or treating a biological sample
US20180364270A1 (en) * 2015-07-07 2018-12-20 University Of Washington Systems, methods, and devices for self-digitization of samples
US20220291246A1 (en) * 2019-09-27 2022-09-15 Lunaphore Technologies Sa Biological sample processing system and microfluidic cartridge therefor
CN114487401B (zh) * 2022-04-18 2022-06-21 中国农业大学 一种微生物检测的双适配体功能核酸恒温微流控芯片传感器

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