Resealable fluidic device and application of same

Abstract

A resealable fluidic device for conducting assays of a biological sample 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.

Description

[0001] RESEALABLE FLUIDIC DEVICE AND APPLICATION OF SAME

[0002] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0003] This application claims priority to and benefit of U.S. Provisional Application Serial No. 63 / 606,271 which is filed on December, 5, 2023, and hereby incorporated by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] 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.

[0006] BACKGROUND OF THE INVENTION

[0007] The background description provided herein is for the purpose of presenting the context of the invention. The subject matter discussed in the background of the invention section should not be assumed to be prior art merely because of its mention in the background of the invention section. Similarly, a problem mentioned in the background of the invention section or associated with the subject matter of the background of the invention section should not be assumed to have been previously recognized in the prior art. The subject matter in the background of the invention section merely represents different approaches, which in and of themselves may also be inventions. Work of the presently named inventors, to the extent it is described in the background of the invention section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the invention.

[0008] 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.

[0009] 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. However, this approach offers limited control over liquid volume, making it susceptible to evaporation and temperature fluctuations. Additionally, 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.

[0010] Therefore, heretofore unaddressed needs exist in the art to address the aforementioned deficiencies and inadequacies.

[0011] SUMMARY OF THE INVENTION

[0012] In light of the foregoing, 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.

[0013] In one embodiment, 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.

[0014] In one embodiment, at least one solution for assays of the biological sample is configured to be injected into the flow cell chamber via the fluid inlet.

[0015] In one embodiment, 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.

[0016] In one embodiment, each of the fluid inlet and the fluid outlet has a diameter between about 0.2-2 mm.

[0017] In one embodiment, 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.

[0018] In one embodiment, each of the first and second fluid holes has a diameter between about 0.2-20 mm.

[0019] In one embodiment, the cover slide has a thickness between about 0.12-0.20 mm.

[0020] In one embodiment, the cover slide is made of glass.

[0021] In one embodiment, the device further comprises a middle layer configured to be disposed between the cover slide and the base slide.

[0022] In one embodiment, 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.

[0023] In one embodiment, the middle layer has a thickness between about 10-500 um.

[0024] In one embodiment, the middle layer is integrated with the cover slide.

[0025] In one embodiment, the middle layer is made of silicon.

[0026] In one embodiment, the frame comprises one or more magnets.

[0027] In one embodiment, 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.

[0028] In another aspect of the invention, 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.

[0029] In one embodiment, 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.

[0030] In one embodiment, the holding plate comprises a heating element for heating the biological samples in the flow cell chamber.

[0031] These and other aspects of the present invention will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings illustrate one or more embodiments of the invention and together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

[0034] Fig. 1 illustrates a schematic view of an assembled resealable fluidic device according to one embodiment of the present invention.

[0035] Fig. 2 illustrates an exploded view of the resealable fluidic device according to the embodiment in Fig. 1 .

[0036] Fig. 3 illustrates an exploded view of the resealable fluidic device according to another embodiment of the present invention.

[0037] Fig. 4 illustrates a top and bottom view of a frame of the resealable fluidic device according to the embodiment in Fig. 1.

[0038] Fig. 5 illustrates photographs of the resealable fluidic device disposed on a display and in association with a microscope.

[0039] Fig. 6 illustrates a photograph of a process for fluid exchange in the resealable fluidic device.

[0040] 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.

[0041] Fig. 8 shows microscopic images of biological samples processed by the resealable fluidic device of the present invention.

[0042] Fig. 9 shows microscopic images of one particular biological sample stained for various biomarker using the resealable fluidic device.

[0043] 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.

[0044] 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.

[0045] Fig. 12 shows a workflow process of using the resealable fluidic device of the present invention for processing biological samples.

[0046] Fig. 13 shows a photograph of a heating adaptor used in association with the resealable fluidic device.

[0047] Fig. 14 shows a photograph of a vacuuming device used in association with the resealable fluidic device.

[0048] Fig. 15 shows a top schematic view of an assembled resealable fluidic device according to the embodiment of Fig. 3.

[0049] Fig. 16 shows a bottom schematic view of an assembled resealable fluidic device according to the embodiment of Fig. 3.

[0050] Fig. 17 shows a top and a bottom schematic view of an assembled resealable fluidic device according to another embodiment of the present invention.

[0051] 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. DETAILED DESCRIPTION OF THE INVENTION

[0052] The invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like reference numerals refer to like elements throughout.

[0053] The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and / or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated if the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

[0054] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0055] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the invention. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0056] It will be understood that, as used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and equivalents thereof known to those skilled in the art. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.

[0057] It will be understood that when an element is referred to as being “on”, “attached” to, “connected” to, “coupled” with, “contacting”, etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on”, “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0058] It will be understood that, although the terms 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.

[0059] It will be further understood that the terms “comprises” and / or “comprising”, or “includes” and / or “including”, or “has” and / or “having”, or “carry” and / or “carrying”, or “contain” and / or “containing”, or “involve” and / or “involving”, “characterized by”, and the like are to be open-ended, i.e., to mean including but not limited to. When used in this disclosure, they specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0060] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the invention, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0061] As used in the disclosure, “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.

[0062] As used in the disclosure, 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. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0063] Embodiments of the invention are illustrated in detail hereinafter with reference to accompanying drawings. The description below is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. The broad teachings of the invention can be implemented in a variety of forms. Therefore, while this invention includes particular examples, the true scope of the invention should not be so limited since other modifications will become apparent upon a study of the drawings, the specification, and the following claims. For purposes of clarity, the same reference numbers will be used in the drawings to identify similar elements. It should be understood that one or more steps within a method may be executed in different order (or concurrently) without altering the principles of the invention.

[0064] 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.

[0065] In certain embodiments, the present invention discloses a resealable fluidic device having a flow cell chamber in which the biological sample is disposed. In certain embodiments, 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.

[0066] In certain embodiments, the biological sample maintains its integrity during and after the sample processing and assays.

[0067] In certain embodiments, 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.

[0068] In certain embodiments, 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.

[0069] In certain embodiments, the present invention discloses a resealable fluidic device which is compatible with most of the fluorescent microscopes and liquid handling equipment on the market.

[0070] In certain embodiments, the present invention discloses a resealable fluidic device which has a significantly bigger imaging area (40mm*20mm) comparing to conventional base glass slide.

[0071] In certain embodiments, 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).

[0072] In certain embodiments, the present invention discloses a resealable fluidic device which has low reagent consumption for staining / processing within the flow cell chamber.

[0073] In certain embodiments, 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. As a result, 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). In certain embodiments, the present invention discloses a resealable fluidic device which can be used with Cleavable Fluorescent Probe (CFP) chemistry.

[0074] In certain embodiments, the present invention discloses a resealable fluidic device which achieves high-plex protein and RNA detections on FFPE tissue samples.

[0075] Fig. 1 shows a schematic view of an assembled resealable fluidic device 10, and Fig. 2 shows an exploded view of the resealable fluidic device 10. In certain embodiments, the assembled resealable fluidic device 10 a frame 11. In certain embodiments, the frame 11 includes one or more fluidic exchange inlets 118, and one or more fluidic exchange outlet 112. In certain embodiments, 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.

[0076] In certain embodiments, the frame 11 has a peripheral rim 111, a sample opening 113, and / or a label opening 115.

[0077] In certain embodiments, the frame is made of plastic. In certain embodiments, 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.

[0078] In certain embodiments, 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.

[0079] In certain embodiments, as shown in Figs. 2-3, 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. In certain embodiments, 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.

[0080] In certain embodiments, 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.

[0081] In certain embodiments, 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.

[0082] In certain embodiment, 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.

[0083] In certain embodiments, 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.

[0084] As shown in Figs. 2-3, the resealable fluidic device 10 includes a middle layer 15, which forms peripheral side walls of the flow cell chamber. In certain embodiments, the middle layer 15 is attached to the cover slide 13 by a fastening element. In certain embodiments, the middle layer 15 is received in and attached to the frame 11.

[0085] In certain embodiments, as shown in Fig. 2, 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.

[0086] In certain embodiments, 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.

[0087] In certain embodiments, 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.

[0088] In certain embodiments, 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.

[0089] In certain embodiments, the frame 11, the cover slide 13 and the middle layer 15 form the resealable fluidic device 10. In certain embodiments, the frame 11, the cover slide 13, the middle layer 15 and a base slide 17 form the resealable fluidic device 10.

[0090] As shown in Figs. 2-3, in certain embodiments, the base slide 17 is a commonly used slide on which the biological sample is disclosed. In certain embodiments, the base slide 17 is a slide specialized in being use with the resealable fluidic device 10. In certain embodiments, the base slide 17 has a sample disposing section 171 and a labeling section 173.

[0091] In certain embodiments, 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. 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.

[0092] In certain embodiments, when the resealable fluidic device 10 is assembled, the sample disposing section 171 is aligned with the sample opening 113, and the labeling section 173 is aligned with the labeling opening 115.

[0093] In certain embodiments, 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.

[0094] As shown in Fig. 3, in certain embodiments, 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. In certain embodiments, 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.

[0095] Figs. 15-16 show a top and bottom view of the resealable fluidic device 20 of Fig. 3 in an assembled state. In particular, 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.

[0096] As shown in Fig. 4, in certain embodiments, a frame 31 has one or more alignment holes 310 for aligning the frame 31 with the cover slide and the base slide.

[0097] In certain embodiments, the frame 31 has one or more magnets 314 for holding the flow cells down. In particular, 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.

[0098] In certain embodiments, the magnets are disposed on the four comers of the frame 31.

[0099] In certain embodiments, 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. In certain embodiments, 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.

[0100] In certain embodiments, 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.

[0101] Fig. 17 shows an alternative embodiment of the present invention. In particular, 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. In certain embodiments, the base slide 67 is slidably received in the two grooves 612. In certain embodiments, however, the frame 61 only has a sample viewing window 613 but not a label viewing window.

[0102] Figs. 5-6 show photographs of a resealable fluidic device 40 disposed on a device holding base 49 and under the microscope.

[0103] As shown in Fig. 5, a device holding base 49 has a holding peripheral frame 49 forming an opening for receiving one or more resealable fluidic devices 40. In certain embodiments, the holding peripheral frame 49 has an upper edge, a lower edge, and two side edges forming the opening.

[0104] In certain embodiments, the holding peripheral frame 49 have one or more holding protrusions 491 for being aligned with the alignment holes 310 of the frame 31. In certain embodiments, 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.

[0105] As shown in Fig. 6, the holding peripheral frame 49 receives and holds multiple resealable fluidic devices.

[0106] Fig. 18 shows another embodiment in which multiple resealable fluidic devices 50 are received and hold by a holding tab 59. In certain embodiments, the resealable fluidic devices 50 has the same embodiment as shown in Fig. 17. In certain embodiments, 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.

[0107] Fig. 13 shows a heating adaptor which can be used in association with the resealable fluidic device. As shown in Fig. 13, a heating adaptor 59 can be use in association with one resealable fluidic device or multiple resealable fluidic devices. In certain embodiments, 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. In certain embodiments, the heating sections 592 are a raised section which is in direct contact with the bottom surface of the sample section 171. In certain embodiments, 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. In certain embodiments, 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.

[0108] The injection of any solution into the flow cell chamber 316 via the fluidic exchange inlet 318 can be accomplished manually or automatically. The removal of any solution from the flow cell chamber 316 via the fluidic exchange outlet 312 can be accomplished manually or automatically. 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.

[0109] In certain embodiments, 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.

[0110] In certain embodiments, when the biological assays are accomplished, the base slide 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.

[0111] In certain embodiments, if needed, the base slide can also be reinstalled to the resealable fluidic device 10 for multi-round applications.

[0112] 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. In particular, histone H3 in the thyroid tissue is stained in bright yellow. As it can be seen from Fig. 7, 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. In particular, comparing to the conventional tissue staining methods in which the liquid sits stagnant during the workflow, 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. As it can be seen in Fig. 7, 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.

[0113] 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. In particular, 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.

[0114] 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.

[0115] In certain embodiments, 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.

[0116] Example: FFPE Hi-Plex Protein Protocol

[0117] Table 1 shows materials and equipment for FFPE Hi-Plex Protein Protocol

[0118] 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. As shown in Fig. 12, in certain embodiments, the workflow includes a sample preparing stage 91, a staining / processing stage 93, and optionally a recycle stage 95. During the sample preparing stage 91, 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. Once the sample preparing stage 91 is accomplished, 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.

[0119] Optionally, in certain embodiments, once the staining / processing stage 93 is accomplished, 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.

[0120] Optionally, in certain embodiments, a H&E staining / imaging stage 97 is conducted after the staining / processing stage 93.

[0121] Table 2. Test procedures using the resealable fluidic device. ,

[0122] : 3. Pretreatment 1 ; a. Set hot plate at 95°C and wait to reach the set temperature.

[0123] : b. Inject 200ul of PSI and incubate for 30mins@95° C

[0124] ; c. Remove flow cell from hot plate. Wait until it cools down to Room Temperature ( RT)

[0125] ; d. Rinse with wash solution: 200ul, 3 times

[0126] : 4. Pretreatment 2 : a. Inject 200ul of lx PS2 and incubate for 5min@RT i b. Rinse with wash solution: 200ul, 3 times

[0127] ; 5. Pretreatment 3 i a. Inject 200ul PS3 and incubate for l hr@,RT lution: 200ul, 3 times Stain Imaging Apply the same image setting of ‘step 6’, Scan and save images (Zeiss

[0128] Axio Observer ; d. Rinse with DI water: 200ul, 3 times

[0129] : e. Rinse with Ethanol: 200ul, 1 time f. Apply vacuum to the flowcell port to dry the tissue : g. Inject Eosin Y solution and incubate for 2-3mins@ h. Rinse with Ethanol: 200ul, 3 times

[0130] 14. H&E imaging : Image with bright field

[0131] Table 3. Flow Cell Installation

[0132] The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

[0133] The embodiments were chosen and described to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

[0134] Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and / or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A resealable fluidic device for conducting assays of a biological sample, the device comprising: 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.

2. The resealable fluidic device according to claim 1, wherein 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.

3. The resealable fluidic device according to claim 2, wherein at least one solution for assays of the biological sample is configured to be injected into the flow cell chamber via the fluid inlet.

4. The resealable fluidic device according to claim 3, wherein 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.

5. The resealable fluidic device according to claim 2, wherein each of the fluid inlet and the fluid outlet has a diameter between about 0.2-2 mm.

6. The resealable fluidic device according to claim 2, wherein the cover slide has a first fluid hole and a second fluid hole, wherein the first fluid hole is configured to be alignedwith 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.

7. The resealable fluidic device according to claim 6, wherein each of the first and second fluid holes has a diameter between about 0.2-20 mm.

8. The resealable fluidic device according to claim 6, wherein the cover slide has a thickness between about 0.12-0.20 mm.

9. The resealable fluidic device according to claim 8, wherein the cover slide is made of glass.

10. The resealable fluidic device according to claim 2 further comprising a middle layer configured to be disposed between the cover slide and the base slide.

11. The resealable fluidic device according to claim 10, wherein the middle layer comprises an opening which is configured to form side walls of the flow cell chamber.

12. The resealable fluidic device according to claim 11, wherein the middle layer comprises an opening which is configured to form side walls of the flow cell chamber.

13. The resealable fluidic device according to claim 12, wherein the middle layer has a thickness between about 10-500 um.

14. The resealable fluidic device according to claim 11, wherein the middle layer is integrated with the cover slide.

15. The resealable fluidic device according to claim 14, wherein the middle layer is made of silicon.

16. The resealable fluidic device according to claim 2, wherein the frame comprises one ormore magnets.

17. The resealable fluidic device according to claim 2 further comprising 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.

18. A resealable fluidic devices assembly, the assembly comprising: multiple resealable fluid devices according to claim 1, a holding plate on which the multiple resealable fluid devices are configured to be disposed.

19. The resealable fluidic devices assembly according to claim 18, wherein 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.

20. The resealable fluidic devices assembly according to claim 18, wherein the holding plate comprises a heating element for heating the biological samples in the flow cell chamber.

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