Cartridges for analyzing cells and methods of using them

Abstract

Aspects of the present disclosure provide an analysis cartridge that allows analyzing cellular components of a fluid sample, such as a blood sample. In certain embodiments, the analysis cartridge comprises a complete blood count (CBC) module for analyzing cells. The CBC module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable distance to produce a monolayer of cells between the panels. The CBC module also comprises a mechanism to deliver into the imaging chamber a small volume of fluid, for example, between 0.1 to 2 µl of fluid. Also provided are methods of analyzing fluid samples in the analysis cartridges provided herein.

Description

[0001] CARTRIDGES FOR ANALYZING CELLS AND METHODS OF USING THEM

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 634,315, filed April 15, 2024, which application is incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[0005] Analyses of biological samples, such as blood samples, are limited by the availability of samples. Also, analyses of biological samples, such as blood samples often involve testing cellular components. Therefore, analyses of biological samples, particularly for their cellular components, using the smallest possible amounts are desirable.

[0006] SUMMARY

[0007] In certain aspects, the disclosure provides analysis cartridges that allow analyzing small volumes of biological fluid samples. In certain embodiments, the analysis cartridges allow analyses of cellular components of biological fluid samples.

[0008] In certain aspects, the disclosure provides an analysis cartridge, comprising: an imaging chamber comprising a top panel and a bottom panel separated by a suitable gap to produce a monolayer of cells between the top and the bottom panels, the imaging chamber having a sample delivery edge, wherein the gap between the top panel and the bottom panel is accessible to a fluid sample delivered to the sample delivery edge, and a sample delivery module configured to deliver the fluid sample to the sample delivery edge thereby allowing the fluid sample to fill the imaging chamber.

[0009] In certain embodiments, the analysis cartridges disclosed herein allow a complete blood count (CBC). Certain such analysis cartridges comprise a CBC module. A CBC module may comprise an imaging chamber configured to hold a small volume of fluid samples, for example, between 0.1 pl and 2 pl of fluid samples. In some cases, the imaging chamber comprises a top panel and a bottom panel separated by a suitable gap to produce a monolayer of cells, for example, blood cells, between the top and the bottom panels. The gap between the top and bottom panels can be between 1 pm and 50 pm, such as between 3 pm and 10 pm. In some cases, the imaging chamber comprises a sample delivery edge, wherein the gap between the top panel and the bottom panel is accessible to a fluid sample delivered to the sample delivery edge.

[0010] Inaccurate loading of a fluid sample can impact the imaging functionality and accuracy of the test result. To address such problems, certain aspects of the disclosure provide accurate loading into the imaging chamber of a small volume of fluid samples, such as peripheral whole blood or a surrogate samples, such as liquid controls and calibrators. For example, the fluid sample may be delivered to an open edge of the imaging chamber, whereupon specific forces, such as capillary force or electric forces, passively or actively push the fluid sample into the imaging chamber. Such delivery is performed by sample delivery modules in the analysis cartridges disclosed herein.

[0011] In some cases, a sample delivery module that delivers a fluid sample to the imaging chamber comprises a digital microfluidics (DMF) chip that moves a fluid sample to a sample delivery edge of the imaging chamber such that the fluid sample fills the imaging chamber. In some cases, a DMF chip comprises a first substrate; a second substrate; a gap separating the first substrate from the second substrate; a plurality of electrodes that generate electrical actuation forces on the droplets between the first and the second substrates to transport the droplets.

[0012] In some cases, a sample delivery module that delivers a fluid sample to the imaging chamber comprises a sample chamber. In such embodiments, the sample chamber is filled with a fluid sample by a sample deposition member. As a sufficient volume of the fluid sample fills the sample chamber the fluid sample reaches the sample delivery edge of the imaging chamber and the fluid sample fills the imaging chamber.

[0013] In certain embodiments, a delivery module comprises a sample chamber, a sample deposition member that delivers the fluid sample into the sample chamber, and a sample drain fluidically connected to the sample chamber, wherein the sample drain receives excess fluid sample beyond the fluid sample filled into the imaging chamber.

[0014] In some cases, a sample delivery module further comprises a monitor that monitors the front edge of the fluid sample as the fluid sample fills the imaging chamber. When the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism causes the sample delivery module to stop delivering any additional fluid sample to the sample delivery edge of the imaging chamber and, optionally, move any excess fluid sample away from the sample delivery edge. The predetermined location can be determined based on a desired volume of fluid sample to be analyzed and the dimensions of the imaging chamber.

[0015] For example, the sample delivery module comprises a DMF chip, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the DMF chip to move away from the sample delivery edge any fluid sample that has not filled into the imaging chamber. In some cases, the sample delivery module comprises a sample deposition member that delivers a fluid sample into the sample chamber, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample deposition member to move away from the sample delivery edge any fluid sample that has not filled the imaging chamber.

[0016] In some cases, an analysis cartridge disclosed herein comprises a top panel and a bottom panel and a double sided adhesive material holding together the top and bottom panels and also providing a gap between the top and bottom panels. In some cases, the top panel and the bottom panel comprise a polyethylene terephthalate (PET) film. Methods for producing such cartridges are also provided.

[0017] Also provided herein are methods of analyzing a fluid sample, such as a blood sample, in the analysis cartridges provided herein.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1A depicts an exploded view of an exemplary CBC module. This module comprises a sample chamber, an imaging chamber, and two sample drains for draining excess sample.

[0020] FIG. 1 B depicts the CBC module shown in FIG. 1 A with a cover, which may be a film or flexible or rigid plastic.

[0021] FIG. 1C depicts the CBC module shown in FIG. 1A covered with a with a cover, which may be a film or flexible or hard plastic to hold blood and seal as shown in FIG. 1 B and further covered with a top cover, which may be a film or flexible or rigid plastic.

[0022] FIG. 1 D illustrates a sample loading port to load a fluid sample into the microfluidic slide.

[0023] FIG. 1 E illustrates the top view of an exemplary CBC module comprising a sample delivery edge. FIG. 2A depicts an exemplary method for extending drain filling times for CBC module by lengthening drain and adjusting other dimensions proportionately.

[0024] FIG. 2B provides a relationship between certain parameters that affect drain fill times.

[0025] FIG. 2C provides the top view of an exemplary imaging chamber as it is filled with a fluid sample.

[0026] FIG. 3 shows an imaging chamber having one edge interacting with a sample chamber and the other open edge.

[0027] FIG. 4 shows an exemplary digital microfluidics chip integrating DMF and imaging chamber.

[0028] FIG. 5 shows imaging of a surfactant deposited reservoir, electrodes, and loading of a 4 pl droplet into an imaging chamber.

[0029] FIGS. 6A-6B show an exemplary cartridge comprising a CBC chamber and a multiple-electrode DMF chamber.

[0030] FIGS. 7A-7B show an exemplary imaging chamber with a number of compartments that could be filled with blood as well as reference frames used for optical calibration.

[0031] DETAILED DESCRIPTION

[0032] Certain aspects of the present disclosure provide analysis cartridges that allow analyzing cellular components of biological fluid samples, such as blood samples. In certain embodiments, an analysis cartridge disclosed herein comprises a CBC module for analyzing cells.

[0033] In some cases, the CBC module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable distance to produce a monolayer of cells between the panels. The CBC module also comprises a mechanism to deliver into the imaging chamber a small volume of fluid sample, for example, between 0.1 and 2 pl of fluid sample. Also provided herein are methods of analyzing a fluid sample, such as a blood sample in the analysis cartridges provided herein.

[0034] Before the present devices and methods are described in greater detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the devices and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the devices and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the devices and methods.

[0035] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0037] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0038] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0039] The term “comprising” is used herein as requiring the presence of the named component and allowing the presence of other components. The term “comprising” should be construed to include the term “consisting essentially of” and “consisting of”. The “consisting essentially of” allows the presence of the named component(s), along with other components which do not change the function / structure of the named component(s). The “consisting of” allows the presence of the named component(s), along with any adhesives or other bonding means for attaching the listed component(s).

[0040] Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0041] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0042] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 grams to 10 grams” is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0043] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 1 1%, and “about 1 ” may mean from 0.9-1.1. It should be noted that many of the terms used herein are relative terms. For example, the terms “upper” and “lower” are relative to each other in location, i.e., an upper component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the component is flipped. The terms “inlet” and “outlet” are relative to a fluid flowing through them with respect to a given structure, e.g., a fluid flows through the inlet into the structure and flows through the outlet out of the structure.

[0044] The terms “horizontal” and “vertical” are used to indicate direction relative to an absolute reference, i.e., ground level. However, these terms should not be construed to require structures to be absolutely parallel or absolutely perpendicular to each other. For example, the first vertical structure and the second vertical structure are not necessarily parallel to each other. The terms “top” and “bottom” are used to refer to surfaces where the top is always higher than the bottom relative to an absolute reference, i.e., the surface of the earth. The terms “upwards” and “downwards” are also relative to an absolute reference; upwards is always against the gravity of the earth while downwards is always towards the gravity of the earth.

[0045] The term “parallel” should be construed in its lay sense of two surfaces that maintain a generally constant distance between them, and not in the strict mathematical sense that such surfaces will never intersect when extended to infinity.

[0046] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0047] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present devices and methods. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0048] DEVICES

[0049] As summarized above, certain aspects of the present disclosure provide analysis cartridges for analyzing cellular components of biological fluid samples, such as blood samples. Also, certain aspects of the disclosure provide analysis cartridges that allow analysis of small volumes of fluid samples.

[0050] A suitable fluid sample analyzed in the analysis cartridges disclosed herein can be blood, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, or the like. Additional fluid samples that could be analyzed in the analysis cartridges disclosed herein can be readily identified by a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0051] Certain biological fluid samples to be analyzed in the analysis cartridges disclosed herein include venous blood, capillary blood, serum, or plasma.

[0052] “Subject” as used herein refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In some embodiments, the subject is a human. The subject or patient may be undergoing other forms of treatment.

[0053] CBC Module

[0054] In certain embodiments, the analysis cartridge comprises a CBC module. In some cases, a CBC module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable gap to produce a monolayer of cells between the top and the bottom panels. In some cases, the imaging chamber has a sample delivery edge, wherein the gap between the top panel and the bottom panel is accessible to a fluid sample delivered to the sample delivery edge. The analysis cartridge can further comprise a sample delivery module configured to deliver a fluid sample to the sample delivery edge thereby allowing the fluid sample to fill the imaging chamber.

[0055] In some cases, the sample delivery module is configured to deliver to the imaging chamber a fluid sample in a small volume, for example, a fluid sample between 0.1 pl and 2 pl, such as 0.1 pl, 0.2 pl, 0.3 pl, 0.4 pl, 0.5 pl, 0.6 pl, 0.7 pl, 0.8 pl, 0.9 pl, 1 .0 pl, 1 .1 pl, 1 .2 pl, 1 .3 pl, 1 .4 pl, 1 .5 pl, 1 .6 pl, 1 .7 pl, 1 .8 pl, 1 .9 pl, or 2.0 pl.

[0056] Exemplary embodiments of analysis cartridges comprising a sample delivery module is provided in FIG. 1 A to FIG. 1 E and FIG. 2. In certain such embodiments, a sample delivery module comprises a sample chamber 101 , a sample deposition member (not shown) that delivers the fluid sample into the sample chamber, and a sample drain 102 fluidically connected to the sample chamber. The sample drain receives excess fluid sample beyond the fluid sample filled into the imaging chamber. The sample drain may contain one or more channels fluidically connected to the sample chamber such that the one or more channels of sample drain receive excess fluid sample beyond the fluid sample filled into the imaging chamber. For example, the sample drain may contain one, two, three, four, five, or more than five fluid channels. In the embodiments described in FIGS. 1 A to 1 C, the sample drain comprises two channels, one on either side of the sample chamber.

[0057] The analysis cartridge 100 provided in FIG. 1A comprises the sample chamber 101 and the sample loading port 105. The sample loading port is fluidically connected to the sample delivery edge 104 of the imaging chamber 103. The sample chamber is fluidically connected to the sample drain 102. As shown in FIG. 1 A, the sample drain 102 comprises two channels, each fluidically connected to the sample chamber.

[0058] As shown in FIGS. 1 B and 1 C, the analysis cartridge of FIG. 1A can further comprise a cover 107 on top of the cartridge to hold the fluid sample and seal it. The cover may be a film or flexible or rigid plastic. The port 105 is open to the top through the opening 106 in the cover 107.

[0059] FIG. 1 D shows an exemplary embodiment of how the sample chamber is fluidically connected to the imaging chamber. Particularly, the port 105 provides a fluidic connection between the sample chamber and the imaging chamber such that when the sample chamber is sufficiently filled with a sample, the sample is delivered to the sample delivery edge of the imaging chamber via the port 105. FIG. 1 E and FIG. 2A provide additional exemplary embodiments of analysis cartridges comprising a sample delivery module. FIG. 1 E comprises area 108 that can be fabricated / molded to contain inlet, sample drain, and optionally, a thumb holder to facilitate gripping of the analysis cartridge by a user. The imaging chamber can be covered with a protective plastic cover slide. A sample flows from the sample chamber into the imaging chamber through a sample loading port 109. The cartridge also comprises the sample drain 1 1 1 fluidically connected to the sample chamber 1 10.

[0060] FIGS. 2A-2B provide certain parameters of a sample drain and its effects on the sample delivery time. Various parameters of the sample drain can be adjusted. For example, the depth of the sample drain can be between 0.1 mm and 1 mm, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 .0 mm. Also, the length of the sample drain can be between 10 mm and 100 mm, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 mm. The width of the sample drain can be between 0.5 and 1 .5 mm, such as 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1 .0, 1 .05, 1.1 , 1.15, 1 .2, 1 .25, 1 .3, 1 .35, 1 .4, 1 .45, or 1 .5 mm.

[0061] A sample deposition member can deposit a fluid sample into a sample chamber. A sample deposition member can deposit between 0.1 pl and 10 pl, such as 0.5 pl, 1.0 pl, 1 .5 pl, 2.0 pl, 2.5 pl, 3.0 pl, 3.5 pl, 4.0 pl, 4.5 pl, 5.0 pl, 5.5 pl, 6.0 pl, 6.5 pl, 7.0 pl, 7.5 pl, 8.0 pl, 8.5 pl, 9.0 pl, 9.5 pl, or 10.0 pl of fluid sample into the sample chamber.

[0062] The dimensions of the imaging chamber are adjusted according to the volume of the fluid sample to be analyzed. Accordingly, the imaging chamber can have a gap between the top panel and the bottom panel between 1 pm and 50 pm, such as between 3 pm and 10 pm. For example, in some cases, such gap is between 1 pm and 10 pm, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 pm. The imaging chamber can be a quadrilateral, such as a square or a rectangle. The imaging chamber can also be a circle, ellipse, triangle, or any other appropriate shape suitable for imaging a monolayer of cells. The surface area of the imaging chamber can be between 50 mm2to 200 mm2. A person of ordinary skill in the art can determined appropriate dimensions and / or the shape for the imaging chamber so that the imaging chamber can hold a desirable volume of a fluid sample.

[0063] When the fluid sample reaches the sample delivery edge of the imaging chamber, the fluid sample flows and fills the imaging chamber. Such fluid movement can be facilitated by capillary action or active positive or negative pressure. Also, such fluid movement can be facilitated if the bottom surface facing the sample chamber of the top panel 107 is hydrophobic.

[0064] As used herein the term “hydrophobic”, such as in reference to a “hydrophobic material” (e.g., membrane, cover, film, etc.) refers to those materials having a water contact angle greater than about 80 degrees.

[0065] Further, when the fluid sample flows and reaches the sample delivery edge of the imaging chamber, the fluid sample fills in the gap between the top panel and the bottom panel of the imaging chamber. Capillary action can facilitate such filling of the imaging chamber. In some cases, the dimensions of the imaging chamber are such that when the imaging chamber is substantially filled with the fluid sample, the back-pressure from the fluid sample avoids further movement of the fluid sample into the imaging chamber. The excess fluid then drains into the fluidically connected sample drains. Draining of the fluid sample into the sample drains can also be facilitated by providing that the bottom surface facing the sample chamber of the top panel 107 is hydrophobic.

[0066] As mentioned above, various parameters of a sample drain can be adjusted to control the flow of a fluid sample into the imaging chamber and into the sample drain. An example of various forces at work is provided in FIG. 2C. In FIG. 2C, 201 indicates the inlet meniscus and 202 indicates the imaging chamber meniscus. A fluid sample would flow in channel until forces are balanced between the imaging chamber meniscus and inlet meniscus. This schematic does not consider adhesive forces at walls that will lower overall force due to meniscus in the imaging chamber. The inlet channel can be between 400 pm and 1000 pm deep.

[0067] For example, the meniscus in the imaging chamber can be used to calculate force on fluid, according to the Young-Laplace equation. Similar calculations can be used to determine different parameters of an imaging chamber, sample chamber, and sample drain.

[0068] In some cases, a sample deposition member can deposit a fluid sample into a sample chamber. Any suitable fluid movement mechanism can be used to deposit a fluid sample into a sample chamber. For example, appropriate positive or negative pressure can be applied to a fluid sample from other parts of the analysis cartridge into a sample chamber. For example, a fluid sample may be moved into a sample chamber via diffusion, convection, pumping, applied pressure, gravity-driven flow, density gradients, temperature gradients, chemical gradients, pressure gradients (positive or negative), pneumatic pressure, gas-producing chemical reactions, centrifugal flow, capillary pressure, wicking, electric field-mediated, electrode-mediated, electrophoresis, dielectrophoresis, magnetophoresis, magnetic fields, magnetically driven flow, optical force, chemotaxis, phototaxis, surface tension gradient driven flow, Marangoni stresses, hermos-capillary convection, surface energy gradients, acoustophoresis, surface acoustic waves, electroosmotic flow, thermophoresis, electrowetting, opto-electrowetting, a pipette, a peristaltic pump, syringe pump, a pressure-drive flow control pump, or the like.

[0069] In some cases, a sample delivery module comprises a sample chamber having one edge interacting with an imaging chamber. The edge interacting with an imaging chamber could be used to deliver a fluid sample into an imaging chamber. The other edge of the imaging chamber can be open to air. An exemplary CBC module for such embodiments is provided in FIG. 3. In this embodiment, the CBC module 300 comprises the sample chamber 301 . The sample chamber can be filled with a fluid sample using a sample deposition member 304, which, for exemplification is indicated in FIG. 3 to be a pipette. However, any other suitable sample deposition member could be used.

[0070] A fluid sample once deposited into the sample chamber in a sufficient amount would interact with the sample delivery edge 302 of the imaging chamber 305. The fluid sample enters the imaging chamber through the sample delivery edge via capillary force. The imaging chamber has another edge that is open to air at position 303. As the fluid sample fills the imaging chamber it pushes out air until it completely fills the imaging chamber. The dimensions of the imaging chamber are designed so that the surface tension of the fluid sample at the open edge 303 does not allow spilling of the fluid sample outside of the imaging chamber. The surface tension also provides sufficient back pressure such that no more fluid sample enters the imaging chamber 305. This provides a balanced filling of the imaging chamber where, depending on a desired volume of a sample to be measured and, accordingly, selected dimensions of the imaging chamber, a specific volume of a fluid sample can be loaded and analyzed.

[0071] Thus, a fluid sample is deposited into a sample chamber using a sample deposition member. When a sufficient sample is filled in the sample chamber, the fluid sample interacts with the sample delivery edge of the imaging chamber. The fluid sample then fills the imaging chamber, for example, under the capillary force. The dimensions of the imaging chamber are such that when the leading edge of the fluid sample reaches the open edge of the imaging chamber, the surface tension at the open edge prevent any further movement of the fluid sample into the sample chamber. Thus, the imaging chamber is filled with a fluid sample having a predetermined volume as controlled by the dimensions of the imaging chamber.

[0072] In some cases, a sample delivery module comprises a DMF chip that delivers the fluid sample to the sample delivery edge. As well-known in the art, a DMF chip comprises: a first substrate; a second substrate; a gap separating the first substrate from the second substrate; a plurality of electrodes that generate electrical actuation forces on sample droplets between the first and the second substrates to transport the sample droplets.

[0073] In certain embodiments, a DMF chip includes a first substrate and a second substrate, where the second substrate is positioned over the first substrate and separated from the first substrate by a gap. The first or the second substrate may include a plurality of DMF electrodes. The plurality of DMF electrodes may be an array or a series of electrodes that are individually controllable for activation and deactivation. The plurality of DMF electrodes may be overlayed with an insulating material to electrically isolate the DMF electrodes. In certain embodiments, the space / gap between the first and second substrates may be filled with air or with an inert fluid, such as oil. In exemplary embodiments, a series of DMF electrodes may be disposed on the first substrate and a single electrode disposed on the second substrate in a facing configuration with the series of electrodes on the first substrate. The series of electrodes and the single electrode may be covered with an insulating layer. In other cases, the series or plurality of electrodes on the first substrate may be configured as co-planar electrodes and the second substrate may not include an electrode. Various configurations of DMF electrodes are known in the art and are described, for example, in United States Patent No. 11 ,016,053, which is incorporated herein in its entirety. Any of these configurations of DMF electrodes can be present in the analysis cartridges disclosed here.

[0074] FIG. 4 provides an exemplary analysis cartridge 400 comprising the DMF chip 401 for moving the sample into the imaging chamber 404. The sample may be provided to the DMF chip from sample chamber 402. The DMF chip moves the sample from the sample chamber to the sample delivery edge 405 of the imaging chamber 404. The optional wicking pads 403 may absorb excess sample that does not enter the imaging chamber 404. In some cases, the DMF chip comprises one or more dried reagents that facilitates movement of a fluid sample over the DMF electrodes and / or staining of cells in a fluid sample. FIG. 5 provides an exemplary embodiment, where the electrodes are coated with a surfactant, namely, ethylenediamine tetrakis(ethoxylate-block-propoxylate) tetrol (90r4™ or Tetronic 904™). As evidenced in FIG. 5, Tetronic 904™ facilitates better movement of the droplets compared to electrodes without it. Any suitable surfactant can be used to facilitate such fluid movement. In some cases, the electrodes comprise EDTA, which avoids coagulation of a blood sample when manipulated in the DMF chip.

[0075] In some cases, one or more dried reagents comprise dyes used to stain cells, for example, dyes used to stain blood cells that distinguish amongst blood cells. Certain such dyes include aniline dyes, basic dyes, and acidic dyes that differentially stain nuclei and cytoplasm of different blood cells.

[0076] FIGS. 6A and 6B provide exemplary embodiments of analysis cartridges of the disclosure. FIG. 6B provides certain dimensions of various parts of the CBC module of an analysis cartridges. The DMF chamber may comprise one or more stains, for example, fluorescent dyes, to stain the blood sample before it is loaded into the CBC chamber.

[0077] As noted above, the analysis cartridges described herein provide for accurate loading of imaging chamber, particularly, accurate loading of small volumes of fluid samples, such as between 0.1 and 2 pl. For example, a fluid sample between 0.1 pl and 2 pl, such as 0.1 pl, 0.2 pl, 0.3 pl, 0.4 pl, 0.5 pl, 0.6 pl, 0.7 pl, 0.8 pl, 0.9 pl, 1 .0 pl, 1 .1 pl, 1 .2 pl, 1 .3 pl, 1 .4 pl, 1 .5 pl, 1 .6 pl, 1 .7 pl, 1 .8 pl, 1 .9 pl, or 2.0 pl can be accurately loaded into an imaging chamber disclosed herein. To that end, certain aspects of the disclosure provide analysis cartridges having a sample delivery module comprising a monitor that monitors the volume of the fluid sample as the fluid sample fills the imaging chamber. In some cases, the front edge of the fluid sample as the fluid sample fills the imaging chamber is used as a proxy to calculate and monitor the volume of the loaded fluid sample.

[0078] In some cases, the gap between the top panel and the bottom panel of an imaging chamber is between 1 pm and 10 pm, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 pm. Also, the area of the imaging chamber can be between 40 mm2and 200 mm2, such as 50 mm2, 60 mm2, 70 mm2, 880 mm2, 90 mm2, 100 mm2, 110 mm2, 120 mm2, 130 mm2, 140 mm2, 150 mm2, 160 mm2, 170 mm2, 180 mm2, 190 mm2, and 200 mm2. To provide an area between 50 mm2and 200 mm2the imaging chamber can be a square of between 7 mm and 16 mm, a circle having a radius between 4 mm and 8 mm, or a rectangle having width between 5 and 10 mm and length between 10 and 20 mm. Any other suitable shape can be used with appropriate dimensions to provide a desired surface area.

[0079] For example, in one embodiment, an imaging chamber is a square of 10 mm, thus providing a surface area of 100 mm2. The gap between the top and the bottom panels is about 10 pm thereby producing an imaging chamber having the total volume of 1 pl. A monitor can be designed to monitor that the volume of the fluid sample filled in the imaging chamber is between 0.1 and 0.9 pl, such as 0.2 pl, 0.3 pl, 0.4 pl, 0.5 pl, 0.6 pl, 0.7 pl, 0.8 pl, or 0.9 pl. A person of ordinary skill in the art can determine appropriate dimensions of the imaging chamber and a desired volume to be loaded into the Imaging chamber and such embodiments are within the purview of the disclosure.

[0080] Depending on the dimensions of the imaging chamber, loading a fluid sample so that the leading edge of the fluid sample at a certain location of the imaging chamber indicates that the loaded fluid has certain volume. Therefore, monitoring the leading edge of the loaded fluid sample could be used to assess the volume of the fluid sample loaded into the imaging chamber.

[0081] For example, as shown in FIGS. 7A-7B, depending on how much of the imaging chamber is filled with a fluid sample, one can accurately determine the volume of the fluid sample loaded into the imaging chamber. An exemplary embodiment is provided in FIG. 7A, where the desirable leading edge is indicated by the dotted line. When the leading of the sample matches the desirable leading edge, the imaging chamber contains a certain predetermined volume of a fluid sample. This predetermined volume could be used in the calculations of blood cell counts. FIG. 7B shows loading of 0.332 pl of blood sample loaded into the imaging chamber.

[0082] In certain embodiments, the monitor is configured to detect that the front edge of the fluid sample crosses a predetermined location in the imaging chamber. Any suitable apparatus could be used to monitor the front edge of the fluid sample in an imaging chamber. In some cases, monitoring comprises observing the leading edge of the loaded fluid sample with video, changes in light, or reflection. In one such example, the apparatus is a video camera. In one such embodiment an illuminator of 400 nm to 430 nm Soret band of oxidized hemoglobin is used to monitor the edge of the fluid sample. In some cases, a monitor can comprise a camera that tracks the front edge of the fluid sample and compares it to a predetermined location on the imaging chamber to determine whether the front edge of the fluid sample has crossed the predetermined location.

[0083] Alternatively, certain electrodes implemented at a predetermined location of the imaging chamber can monitor that the front edge of the fluid sample crossed the predetermined location. For example, an LED and a photodiode implemented at a predetermined location of the imaging chamber can monitor that the front edge of the fluid sample crossed the predetermined location.

[0084] Additional apparatuses suitable for monitoring the front edge of the fluid sample are well-known to a person of ordinary skill in the art and use of such apparatuses is within the purview of the disclosure.

[0085] In some cases, when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample delivery module to stop delivering any additional fluid sample to the sample delivery edge of the imaging chamber and, optionally, move any excess fluid sample away from the sample delivery edge.

[0086] For example, if the sample delivery module comprises a DMF chip, and the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, then the monitor activates a mechanism that causes the DMF chip to move away from the sample delivery edge any fluid sample that has not filled into the imaging chamber.

[0087] Alternatively, when the sample delivery module comprises a sample deposition member for depositing the fluid sample into the sample chamber and the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample deposition member to move away from the sample delivery edge any fluid sample that has not filled the imaging chamber.

[0088] An imaging chamber filled with a fluid sample can be observed and analyzed using an imaging member of an analyzer that operable connects to the analysis cartridge disclosed herein. An imaging member can be an optical imaging chamber or a digital imaging member. For example, an optical camera can image the imaging chamber and store the imaging information in a digital format, which can be analyzed by a computer processor. Similarly, a charge-coupled device (CCD) or complementary metal oxide semiconductor (CMOS) array sensor can image the imaging chamber and store the imaging information in a digital format, which can be analyzed by a computer processor. Certain details of storing and analyzing the images so obtained and stored are provided elsewhere in this disclosure.

[0089] In certain aspects, the analysis cartridges described herein comprise specific polymers for separating the top panel and the bottom panel of the analysis cartridge, for example, imaging chamber of the CBC module. In some cases, the top panel and the bottom panel comprise a double sided adhesive material holding the top and bottom panels together and also providing the gap between the top and bottom panels. In some cases, the top panel and the bottom panel comprise a polyethylene terephthalate (PET) film. The top panel and / or the bottom panel may also further comprise a coating of a hydrophobic material.

[0090] In some aspects, the results (e.g., CBC values) are communicated in about 4 minutes to about 40 minutes from the time the sample is collected. In other aspects, the results are communicated in about 4 minutes to about 30 minutes from the time the sample is collected. In yet other aspects, the results are communicated in about 4 minutes to about 20 minutes from the time the sample is collected. In some aspects, the results are communicated in about 40 minutes or less, about 39 minutes or less, about 38 minutes or less, about 37 minutes or less, about 36 minutes, or less about 35 minutes, less about 34 minutes or less, about 33 minutes or less, about 32 minutes or less, about 31 minutes or less, about 30 minutes or less, about 29 minutes or less, about 28 minutes or less, about 27 minutes or less, about 26 minutes or less, about 25 minutes or less, about 24 minutes or less, about 23 minutes or less, about 22 minutes or less, about 21 minutes or less, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, or about 4 minutes from the time the sample is collected.

[0091] In some aspects, the analysis cartridges are processed in an analyzer that contains software to execute one or more tasks, including the performance of the methods described herein. In some aspects, the analyzer contains software to automatically determine the next appropriate step in methods as described herein. For example, the instrument may contain software that determines amount or presence of an analyte of interest. The software may display this determination, such as on a graphical user interface.

[0092] In some aspects, the analyzer stores software that instructs a processor to execute a given task. In some aspects, the software stores machine readable instructions that instruct a processor to execute a given task. The machine-readable instructions may be one or more executable programs or portion(s) of an executable program for execution by a computer. The programs may be embodied in software stored on a non-transitory computer readable storage medium such as a CD-ROM, a floppy disk, a hard drive, a DVD, a Blu-ray disk, or a memory associated with the processors. Alternatively, the entire programs and / or parts thereof could alternatively be executed by a device other than the processors and / or embodied in firmware or dedicated hardware. Additionally or alternatively, processes may be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuitry, an FPGA, an ASIC, a comparator, an operational-amplifier (op-amp), a logic circuit, etc.) structured to perform the corresponding operation without executing software or firmware.

[0093] The machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. Machine readable instructions as described herein may be stored as data (e.g., portions of instructions, code, representations of code, etc.) that may be utilized to create, manufacture, and / or produce machine executable instructions. For example, the machine-readable instructions may be fragmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may require one or more of installation, modification, adaptation, updating, combining, supplementing, configuring, decryption, decompression, unpacking, distribution, reassignment, compilation, etc. in order to make them directly readable, interpretable, and / or executable by a computing device and / or other machine. For example, the machine-readable instructions may be stored in multiple parts, which are individually compressed, encrypted, and stored on separate computing devices, wherein the parts when decrypted, decompressed, and combined form a set of executable instructions that implement a program such as that described herein.

[0094] In another example, the machine-readable instructions may be stored in a state in which they may be read by a computer, but require addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc. in order to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may need to be configured (e.g., settings stored, data input, network addresses recorded, etc.) before the machine-readable instructions and / or the corresponding program(s) can be executed in whole or in part. Thus, the disclosed machine-readable instructions and / or corresponding program(s) are intended to encompass such machine-readable instructions and / or program(s) regardless of the particular format or state of the machine-readable instructions and / or program(s) when stored or otherwise at rest or in transit.

[0095] The machine-readable instructions described herein can be represented by any past, present, or future instruction language, scripting language, programming language, etc. For example, the machine-readable instructions may be represented using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0096] The machine readable instructions may be stored on a non-transitory computer and / or machine readable medium such as a hard disk drive, a flash memory, a read-only memory, a compact disk, a digital versatile disk, a cache, a random-access memory and / or any other storage device or storage disk in which information is stored for any duration (e.g., for extended time periods, permanently, for brief instances, for temporarily buffering, and / or for caching of the information). As used herein, the term non-transitory computer readable medium is expressly defined to include any type of computer readable storage device and / or storage disk and to exclude propagating signals and to exclude transmission media.

[0097] METHODS

[0098] The analysis cartridges disclosed herein can be used for analyzing a fluid sample, for example, a blood sample for its cellular components.

[0099] A suitable fluid sample analyzed in the methods disclosed herein can be blood, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, or the like. Additional fluid samples that could be analyzed in the methods disclosed herein can be readily identified by a person of ordinary skill in the art and such embodiments are within the purview of the disclosure. Any structural elements of the analysis cartridges described elsewhere in this disclosure, for example, those described under “Devices” above, are applicable to the methods disclosed herein. For example, the structure of the imaging chamber as discussed above is also applicable to the methods disclosed herein.

[0100] Certain aspects of the disclosure provide a method of analyzing a fluid sample, for example, a blood sample, in the analysis cartridges disclosed herein. In some cases, the methods comprise delivering to an imaging chamber a fluid sample in a small volume, for example, a fluid sample between 0.1 pl and 2 pl, such as 0.1 pl, 0.2 pl, 0.3 pl, 0.4 pl, 0.5 pl, 0.6 pl, 0.7 pl, 0.8 pl, 0.9 pl, 1 .0 pl, 1 .1 pl, 1 .2 pl, 1 .3 pl, 1 .4 pl, 1 .5 pl, 1 .6 pl, 1 .7 pl, 1 .8 pl, 1 .9 pl, or 2.0 pl.

[0101] The fluid sample, such as a blood sample loaded into the imaging chamber can be analyzed to obtain, store, and process images of the fluid sample. Such images can be used to determine cellular composition of the fluid sample. For example, depending on the detection of certain types and number of blood cells in the images obtained from the imaging chamber, the methods comprise providing OBC values for the blood sample.

[0102] In some cases, the methods comprise using a DMF chip to load a fluid sample into the imaging chamber of an analysis cartridge.

[0103] In some cases, the methods comprising using a sample chamber and a sample deposition member to load a fluid sample into the imaging chamber of an analysis cartridge. In some cases, using a sample chamber and a sample deposition member to load a fluid sample into the imaging chamber of an analysis cartridge further comprise using a sample drain to receive excess fluid sample beyond the fluid sample filled into the imaging chamber.

[0104] Thus, in certain cases, the disclosure provides a method of analyzing a fluid sample in an analysis cartridge that comprises a sample delivery module comprising a sample chamber, a sample deposition member that delivers the fluid sample into the sample chamber, and a sample drain fluidically connected to the sample chamber, wherein excess fluid sample beyond the fluid sample filled into the imaging chamber is received in a sample drain fluidically connected to the sample chamber. In some cases, the excess fluid sample beyond the fluid sample filled into the imaging chamber is received in two or more channels of the sample drain fluidically connected to the sample chamber. Similarly, in some cases, the disclosure provides a method of analyzing a fluid sample in an analysis cartridge that comprises a sample delivery module comprising a DMF chip that delivers the fluid sample to the sample delivery edge, wherein the DMF chip comprises: a first substrate; a second substrate; a gap separating the first substrate from the second substrate; a plurality of electrodes that generate electrical actuation forces on droplets of fluid sample between the first and the second substrates to transport the liquid droplet.

[0105] Moreover, in some cases, the disclosure provides a method of analyzing a fluid sample in an analysis cartridge that comprises a sample delivery module comprising a sample chamber and a sample deposition member that deposits the fluid sample into the sample chamber.

[0106] When the analysis cartridge comprises a monitor, the methods disclosed herein comprise monitoring via a monitor the front edge of the fluid sample as the fluid sample fills the imaging chamber. The monitor can comprise a camera that monitors the front edge of the fluid sample. Alternatively, the monitor can comprise one or more electrodes that monitor the front edge of the fluid sample.

[0107] Accordingly, in certain embodiments, the disclosure provides a method of analyzing a fluid sample, wherein when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample delivery module to stop delivering any additional fluid sample to the sample delivery edge of the imaging chamber and, optionally, move any excess fluid sample away from the sample delivery edge.

[0108] In certain cases, the disclosure provides a method of analyzing a fluid sample, wherein when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the digital microfluidics chip to move away from the sample delivery edge any fluid sample that has not filled into the imaging chamber.

[0109] In some cases, the disclosure provides a method of analyzing a fluid sample, wherein the sample delivery module comprises the sample deposition member for depositing the fluid sample into the sample chamber, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample deposition member to move away from the sample delivery edge any fluid sample that has not filled the imaging chamber.

[0110] It may be desirable to analyze a control sample. The control sample may be analyzed concurrently with the sample from the subject as described above. The results obtained from the subject sample can be compared to the results obtained from the control sample. Standard curves may be provided, with which assay results for the sample may be compared. Using samples taken from multiple donors, standard curves can be provided for reference levels in normal healthy subjects.

[0111] Thus, in view of the above, a method for determining the presence, amount, or amount of analyte in a test sample is provided. The method comprises assaying the test sample for a target analyte and comparing it to a control. The calibrator is optionally, and is preferably, part of a series of calibrators in which each of the calibrators differs from the other calibrators in the series by the concentration of the analyte.

[0112] As noted above, in certain embodiments, the analysis cartridges described herein comprise specific polymers for separating the top panel and the bottom panel of the analysis cartridge, for example, imaging chamber of the CBC module. In some cases, the top panel and the bottom panel comprise a double sided adhesive material holding the top and bottom panels together and also providing the gap between the top and bottom panels. In some cases, the top panel and the bottom panel comprise a polyethylene terephthalate (PET) film. The top panel and / or the bottom panel may also further comprise a coating of a hydrophobic material.

[0113] Accordingly, certain methods of the disclosure provide fabricating an analysis cartridge, particularly, an imaging chamber. Certain such methods comprise: producing a top panel having certain features, producing a bottom panel having certain features, and gluing the two panels together using an adhesive of a specific thickness.

[0114] In some cases, the top and / or the bottom panels are PET films.

[0115] Certain such methods produce an analysis cartridge comprising a top panel and a bottom panel separated by a desirable distance. In some cases, such desirable distance is such that a fluid sample containing cells, for example, a blood sample produces a monolayer of cells.

[0116] In certain embodiments, parts of the analysis cartridge are provided and a user can connect them together to produce an analysis cartridge or a portion of an analysis cartridge. For example, parts made from hard plastic can be provided such that the parts mechanically engage with each and a user can click together the different parts of the analysis cartridge.

[0117] In some cases, the methods comprise adding a layer of a hydrophobic agent to the top and / or the bottom panels. An example of such hydrophobic agent is fluoropel.

[0118] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0119] Clause 1. An analysis cartridge, comprising: an imaging chamber comprising a top panel and a bottom panel separated by a suitable gap to produce a monolayer of cells between the top and the bottom panels, the imaging chamber having a sample delivery edge, wherein the gap between the top panel and the bottom panel is accessible to a fluid sample delivered to the sample delivery edge, and a sample delivery module configured to deliver the fluid sample to the sample delivery edge thereby allowing the fluid sample to fill the imaging chamber.

[0120] Clause 2. The analysis cartridge of clause 1 , wherein the sample delivery module delivers to the imaging chamber between 0.1 pl and 2 pl of the fluid sample.

[0121] Clause 3. The analysis cartridge of clause 1 or 2, wherein the sample delivery module comprises a sample chamber, a sample deposition member that delivers the fluid sample into the sample chamber, and a sample drain fluidically connected to the sample chamber, wherein the sample drain receives excess fluid sample beyond the fluid sample filled into the imaging chamber.

[0122] Clause 4. The analysis cartridge of clause 3, wherein the sample drain comprises two or more channels fluidically connected to the sample chamber.

[0123] Clause 5. The analysis cartridge of clause 1 or 2, wherein, the sample delivery module comprises a digital microfluidics (DMF) chip that delivers the fluid sample to the sample delivery edge, wherein the DMF chip comprises: a first substrate; a second substrate; a gap separating the first substrate from the second substrate; a plurality of electrodes that generate electrical actuation forces on droplets between the first and the second substrates to transport the droplets.

[0124] Clause 6. The analysis cartridge of clause 1 or 2, wherein the sample delivery module comprises a sample chamber and a sample deposition member that deposits the fluid sample into the sample chamber.

[0125] Clause 7. The analysis cartridge of any one of the preceding clauses, wherein the sample delivery module further comprises a monitor that monitors the front edge of the fluid sample as the fluid sample fills the imaging chamber.

[0126] Clause 8. The analysis cartridge of clause 7, wherein the monitor comprises a camera that monitors the front edge of the fluid sample.

[0127] Clause 9. The analysis cartridge of clause 7, wherein the monitor comprises one or more electrodes that monitor the front edge of the fluid sample.

[0128] Clause 10. The analysis cartridge of any one of clauses 6 to 9, wherein when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample delivery module to stop delivering any additional fluid sample to the sample delivery edge of the imaging chamber and, optionally, move any excess fluid sample away from the sample delivery edge.

[0129] Clause 11. The analysis cartridge of clause 10, wherein the sample delivery module comprises the DMF chip, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the DMF chip to move away from the sample delivery edge any fluid sample that has not filled into the imaging chamber.

[0130] Clause 12. The analysis cartridge of clause 10, wherein the sample delivery module comprises the sample deposition member for depositing the fluid sample into the sample chamber, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample deposition member to move away from the sample delivery edge any fluid sample that has not filled the imaging chamber.

[0131] Clause 13. The analysis cartridge of any one of the preceding clauses, comprising: the top panel and the bottom panel comprising a deposition of a hydrophobic agent, and a double sided adhesive material holding the top and bottom panels together and also providing the gap between the top and bottom panels.

[0132] Clause 14. The cartridge of clause 13, wherein, the top panel and the bottom panel comprise a PET film.

[0133] Clause 15. A method of analyzing a fluid sample, comprising loading the fluid sample in any one of the analysis cartridges of clauses 1 to 14 and analyzing the fluid sample.

[0134] Clause 16. The method of clause 15, comprising analyzing between 0.1 pl to 2 pl of the fluid sample.

[0135] Clause 17. The method of clause 15 or 16, wherein the analysis cartridge comprises a sample delivery module comprising a sample chamber, a sample deposition member that delivers the fluid sample into the sample chamber, and a sample drain fluidically connected to the sample chamber, wherein excess fluid sample beyond the fluid sample filled into the imaging chamber is received in a sample drain fluidically connected to the sample chamber.

[0136] Clause 18. The method of clause 17, wherein the excess fluid sample beyond the fluid sample filled into the imaging chamber is received in two or more channels of the sample drain fluidically connected to the sample chamber. Clause 19. The method of clause 15 or 16, wherein, the analysis cartridge comprises a sample delivery module comprising a DMF chip that delivers the fluid sample to the sample delivery edge, wherein the DMF chip comprises: a first substrate; a second substrate; a gap separating the first substrate from the second substrate; a plurality of electrodes that generate electrical actuation forces on droplets of fluid sample between the first and the second substrates to transport the liquid droplet.

[0137] Clause 20. The method of clause 15 or 16, wherein the analysis cartridge comprises a sample delivery module comprising a sample chamber and a sample deposition member that deposits the fluid sample into the sample chamber.

[0138] Clause 21. The method of any one of clauses 15 to 20, comprises monitoring via a monitor the front edge of the fluid sample as the fluid sample fills the imaging chamber.

[0139] Clause 22. The method of clause 21 , wherein the monitor comprises a camera that monitors the front edge of the fluid sample.

[0140] Clause 23. The method of clause 21 , wherein the monitor comprises one or more electrodes that monitor the front edge of the fluid sample.

[0141] Clause 24. The method of any one of clauses 21 to 23, wherein when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample delivery module to stop delivering any additional fluid sample to the sample delivery edge of the imaging chamber and, optionally, move any excess fluid sample away from the sample delivery edge.

[0142] Clause 25. The method of any one of clauses 21 to 23, wherein the sample delivery module comprises the DMF chip, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the digital microfluidics chip to move away from the sample delivery edge any fluid sample that has not filled into the imaging chamber.

[0143] Clause 26. The method of any one of clauses 21 to 23, wherein the sample delivery module comprises the sample deposition member for depositing the fluid sample into the sample chamber, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample deposition member to move away from the sample delivery edge any fluid sample that has not filled the imaging chamber.

[0144] Clause 27. A method of fabricating an analysis cartridge of any one of clauses 1 to 14, comprising: producing a top panel having certain features, producing a bottom panel having certain features, and gluing the two panels together using an adhesive of a specific thickness.

[0145] Clause 28. The method of clause 27, comprising depositing a hydrophobic agent to the top panel and / or the bottom panel.

[0146] Clause 29. The method of clause 27 or 28, wherein the adhesive comprises a double sided adhesive holding the top and bottom panels together and providing the gap between the top and bottom panels.

[0147] Clause 30. The method of any one of clauses 27 to 29, wherein, the top panel and the bottom panel comprise a PET film.

[0148] The preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMSWHAT IS CLAIMED IS:1 . An analysis cartridge, comprising: an imaging chamber comprising a top panel and a bottom panel separated by a suitable gap to produce a monolayer of cells between the top and the bottom panels, the imaging chamber having a sample delivery edge, wherein the gap between the top panel and the bottom panel is accessible to a fluid sample delivered to the sample delivery edge, and a sample delivery module configured to deliver the fluid sample to the sample delivery edge thereby allowing the fluid sample to fill the imaging chamber.

2. The analysis cartridge of claim 1 , wherein the sample delivery module delivers to the imaging chamber between 0.1 pl and 2 pl of the fluid sample.

3. The analysis cartridge of claim 1 or 2, wherein the sample delivery module comprises a sample chamber, a sample deposition member that delivers the fluid sample into the sample chamber, and a sample drain fluidically connected to the sample chamber, wherein the sample drain receives excess fluid sample beyond the fluid sample filled into the imaging chamber.

4. The analysis cartridge of claim 3, wherein the sample drain comprises two or more channels fluidically connected to the sample chamber.

5. The analysis cartridge of claim 1 or 2, wherein, the sample delivery module comprises a digital microfluidics (DMF) chip that delivers the fluid sample to the sample delivery edge, wherein the DMF chip comprises: a first substrate; a second substrate; a gap separating the first substrate from the second substrate; a plurality of electrodes that generate electrical actuation forces on droplets between the first and the second substrates to transport the droplets.

6. The analysis cartridge of claim 1 or 2, wherein the sample delivery module comprises a sample chamber and a sample deposition member that deposits the fluid sample into the sample chamber.

7. The analysis cartridge of any one of the preceding claims, wherein the sample delivery module further comprises a monitor that monitors the front edge of the fluid sample as the fluid sample fills the imaging chamber.

8. The analysis cartridge of claim 7, wherein the monitor comprises a camera that monitors the front edge of the fluid sample.

9. The analysis cartridge of claim 7, wherein the monitor comprises one or more electrodes that monitor the front edge of the fluid sample.

10. The analysis cartridge of any one of claims 6 to 9, wherein when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample delivery module to stop delivering any additional fluid sample to the sample delivery edge of the imaging chamber and, optionally, move any excess fluid sample away from the sample delivery edge.1 1. The analysis cartridge of claim 10, wherein the sample delivery module comprises the DMF chip, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the DMF chip to move away from the sample delivery edge any fluid sample that has not filled into the imaging chamber.

12. The analysis cartridge of claim 10, wherein the sample delivery module comprises the sample deposition member for depositing the fluid sample into the sample chamber, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample deposition member to move away from the sample delivery edge any fluid sample that has not filled the imaging chamber.

13. The analysis cartridge of any one of the preceding claims, comprising:the top panel and the bottom panel comprising a deposition of a hydrophobic agent, and a double sided adhesive material holding the top and bottom panels together and also providing the gap between the top and bottom panels.

14. The cartridge of claim 13, wherein, the top panel and the bottom panel comprise a PET film.

15. A method of analyzing a fluid sample, comprising loading the fluid sample in any one of the analysis cartridges of claims 1 to 14 and analyzing the fluid sample.

16. The method of claim 15, comprising analyzing between 0.1 pl to 2 pl of the fluid sample.

17. The method of claim 15 or 16, wherein the analysis cartridge comprises a sample delivery module comprising a sample chamber, a sample deposition member that delivers the fluid sample into the sample chamber, and a sample drain fluidically connected to the sample chamber, wherein excess fluid sample beyond the fluid sample filled into the imaging chamber is received in a sample drain fluidically connected to the sample chamber.

18. The method of claim 17, wherein the excess fluid sample beyond the fluid sample filled into the imaging chamber is received in two or more channels of the sample drain fluidically connected to the sample chamber.

19. The method of claim 15 or 16, wherein, the analysis cartridge comprises a sample delivery module comprising a DMF chip that delivers the fluid sample to the sample delivery edge, wherein the DMF chip comprises: a first substrate; a second substrate; a gap separating the first substrate from the second substrate; a plurality of electrodes that generate electrical actuation forces on droplets of fluid sample between the first and the second substrates to transport the liquid droplet.

20. The method of claim 15 or 16, wherein the analysis cartridge comprises a sample delivery module comprising a sample chamber and a sample deposition member that deposits the fluid sample into the sample chamber.

21. The method of any one of claims 15 to 20, comprises monitoring via a monitor the front edge of the fluid sample as the fluid sample fills the imaging chamber.

22. The method of claim 21 , wherein the monitor comprises a camera that monitors the front edge of the fluid sample.

23. The method of claim 21 , wherein the monitor comprises one or more electrodes that monitor the front edge of the fluid sample.

24. The method of any one of claims 21 to 23, wherein when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample delivery module to stop delivering any additional fluid sample to the sample delivery edge of the imaging chamber and, optionally, move any excess fluid sample away from the sample delivery edge.

25. The method of any one of claims 21 to 23, wherein the sample delivery module comprises the DMF chip, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the digital microfluidics chip to move away from the sample delivery edge any fluid sample that has not filled into the imaging chamber.

26. The method of any one of claims 21 to 23, wherein the sample delivery module comprises the sample deposition member for depositing the fluid sample into the sample chamber, and when the monitor detects that the front edge of the fluid sample crosses a predetermined location in the imaging chamber, the monitor activates a mechanism that causes the sample deposition member to move away from the sample delivery edge any fluid sample that has not filled the imaging chamber.

27. A method of fabricating an analysis cartridge of any one of claims 1 to 14, comprising: producing a top panel having certain features, producing a bottom panel having certain features, and gluing the two panels together using an adhesive of a specific thickness.

28. The method of claim 27, comprising depositing a hydrophobic agent to the top panel and / or the bottom panel.

29. The method of claim 27 or 28, wherein the adhesive comprises a double sided adhesive holding the top and bottom panels together and providing the gap between the top and bottom panels.

30. The method of any one of claims 27 to 29, wherein, the top panel and the bottom panel comprise a PET film.

Images