WO2025122737A1 - Fluidic systems and apparatuses for mitigation of microfluidic bubbles and immunoassay methods using the same - Google Patents

Abstract

Methods for circumventing the formation of bubbles and/or mitigating the effects of bubble formation in a fluidic immunoassay are provided. Also described in a fluidic immunoassay method designed to circumvent or mitigate the effects of bubble formation. Additionally, instruments and apparatuses configured to remove bubbles from a region of interest for optical analysis are provided.

Description

FLUIDIC SYSTEMS AND APPARATUSES FOR MITIGATION OF MICROFLUIDIC

BUBBLES AND IMMUNOASSAY METHODS USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international (PCT) application claims priority to United States Provisional Patent Application No. 63/607,005, filed on December 6, 2023, the content of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present subject matter relates to methods for circumventing the formation of bubbles and/or mitigating the effects of bubble formation in a fluidic immunoassay. The present subject matter is also related to fluidic immunoassay methods designed to circumvent or mitigate the effects of bubble formation. Additionally, instruments and apparatuses configured to remove bubbles from a region of interest for optical analysis are described.

BACKGROUND

[0003] Healthcare providers depend on immunodiagnostic systems and methods to make informed decisions regarding patients’ disease diagnoses and subsequent prognoses. A point-of-care (POC) system for such testing, exhibiting an equal or better performance than sophisticated and expensive laboratory -based systems, would represent an indispensable tool for clinicians in providing prompt and accurate diagnoses and treatment plans.

[0004] For example, cardiovascular disease diagnosis is an area where POC immunodiagnostic platforms can be helpful, particularly, in early disease detection and for making a distinction between disease types. Cartridge-based, microfluidics-driven micro total analysis systems (pTAS) can deliver the POC immunodiagnostic needs thanks to their smaller footprint, their ability to actively perform a multi-step, integrated bioanalysis using a single platform, their improved assay performance due a better physio-chemical control at the micro scale, and their compatibility with portable analysis platforms. Results obtained using pTAS can be equal to or better than the laboratory-based, benchtop methods.

[0005] To do so, the bio-fluid needs to flow through a network of channels that are usually small in size with critical dimensions in the micrometer scale range. With this decreased channel size, the surface-to-volume ratio increases, thus making surface-based phenomena and their associated effects more prominent. Under such conditions, any deformities and/or particles that are stuck at the surface could become a nucleation point for air bubbles. Particularly, in a fluidics flowrate- driven experiment with active thermal actuation, dissolved air in the continuously moving fluids and thermal transfer consistently feeds the air bubbles. The bubbles thus increase in size and intensity over time. Additionally, fluidic bubbles can also reach inside the channels due to, e.g., imperfect fluidic connections, off-cartridge buffer reagents, and any other external effects or system components. Depending on their origin, type, size, intensity, and location inside the fluidic channels, the bubbles can disrupt a microfluidics bioanalysis. Accordingly, there is an ongoing need for methods and systems which prevent bubble formation and/or eliminate bubbles already generated during a bio-fluidics assay operation.

[0006] However, when running a bio-fluidics assay, microfluidics bubbles may be generated inside a fluidics channel and/or at an analysis region of interest (such as at or near an optical detector sensor). Presence of bubbles at a sensor region of interest can mask the immunoassay chemistry and interfere with subsequent optical analysis.

[0007] Some methods for removing bubbles from assays exist, such as pressurizing fluid, adding anti-foam agents to break the bubbles, including integrated bubble traps within a fluidics assay system, using external bubble traps to remove bubbles from the flow path, and/or decreasing the number of fluidics connections that can give rise to bubbles.

[0008] Despite these existing methods, bubbles still appear due to phenomena inherent to fluidics systems, such as natural thermal or electrochemical convection and micro- or nano-scale surface defects. These phenomena interfere with both reaction chemistry, surface binding and imaging analysis. In general, existing methods used for bubble removal are laborious, difficult to implement, and fall short of completely removing bubbles.

[0009] The methods, instruments and apparatus described herein provide a solution to the problem of bubble formation in a fluidic system for the detection of one or more analytes of interest in a fluid sample e.g., in an immunoassay).

BRIEF SUMMARY

[0010] In an aspect, a method of analyzing a fluid sample is provided. The method comprises providing an instrument configured to receive a fluidic cartridge and to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, and an optical platform, wherein the instrument and/or the optical platform is/are at a tilt angle or is/are movable to a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed, whereby the tilt angle dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region. [0011] In another aspect, a method for analyzing a fluid sample comprises (a) inserting a fluidic cartridge into an instrument configured to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, and (b) titling the instrument at a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed, whereby titling the instruments dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region.

[0012] In another aspect, a method for analyzing a fluid sample comprises inserting a fluidic cartridge into an instrument configured to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, whereby the inserting of the fluidic cartridge into the instrument results in all or a portion of the fluidic cartridge being at a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed and/or a horizontal surface plane, whereby the tilt of the fluidic cartridge dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region.

[0013] In another aspect, a method of analyzing a fluid sample is provided. The method comprises (a) inserting a fluidic cartridge into an instrument configured to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, and an optical platform, and (b) titling the optical platform at a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed, whereby titling the instruments dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region.

[0014] In another aspect, a method of analyzing a fluid sample is provided. The method comprises inserting a fluidic cartridge into an instrument configured to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, and an optical platform, whereby the inserting of the fluidic cartridge into the instrument results in all or a portion of the fluidic cartridge being at a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed and/or a horizontal surface plane, whereby the tilt of the fluidic cartridge dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region.

[0015] In another aspect, an apparatus for performing immunoassay of a fluid sample is provided. The apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a tilting mechanism configured to tilt the apparatus at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed. [0016] In another aspect, an apparatus for performing immunoassay of a fluid sample is provided. The apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, and a waste region, wherein the apparatus and/or the cartridge holder is/are at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed and/or a horizontal surface plane.

[0017] In another aspect, an apparatus for performing immunoassay of a fluid sample is described. The apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, an optical platform, and a tilting mechanism configured to tilt the optical platform at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed.

[0018] In another aspect, an apparatus for performing immunoassay of a fluid sample is described. The apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and an optical platform, wherein the apparatus and/or the cartridge holder is/are at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed and/or a horizontal surface plane.

[0019] In another aspect, an apparatus for performing immunoassay of a fluid sample is described. The apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a base or a plurality of legs, wherein the base or the plurality of legs orient the apparatus such that the cartridge holder is provided at a tilt angle measured relative to a planar surface upon which the apparatus is placed.

[0020] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0. 1 degrees to about 30 degrees. In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0. 1 degrees to about 30 degrees, measured relative to the planar surface upon which the apparatus is placed. In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0.1 degrees to about 30 degrees, measured relative to a horizontal surface plane.

[0021] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0.5 degrees to about 25 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed and/or a horizontal surface plane.

[0022] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0.75 degrees to about 20 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed and/or a horizontal surface plane.

[0023] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 1 degree, about 2 degrees, about 3 degrees, about 4 degrees, about 5 degrees, about 6 degrees, about 7 degrees, about 8 degrees, about 9 degrees, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed and/or a horizontal surface plane. In other embodiments, the cartridge holder is provided at a tilt angle of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 degrees to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed and/or a horizontal surface plane. [0024] In certain embodiments, the tilt angle is transient. In an embodiment, the instrument and/or cartridge holder is/are at the tilt angle for less than the time for performing the immunoassay. In certain embodiments, the tilt angle is achieved by mechanical movement of the instrument or a region or section of the instrument, such as the cartridge holder.

[0025] In an embodiment, the horizontal surface plane is with respect to ground.

[0026] In another aspect, an apparatus for performing immunoassay of a fluid sample is comprised of an optical detector, a light source, a cartridge holder, a region of interest, a waste region, an optical platform, wherein the optical platform is provided at or is movable to a tilt angle, measured relative to a planar surface upon which the apparatus is placed.

[0027] In certain embodiments, the optical platform is provided at a tilt angle of from about 0. 1 degrees to about 30 degrees, measured relative to the planar surface upon which the apparatus is placed.

[0028] In certain embodiments, the optical platform is provided at a tilt angle of from about 0.5 degrees to about 25 degrees, measured relative to the planar surface upon which the apparatus is placed.

[0029] In certain embodiments, the optical platform is provided at a tilt angle of from about 0.75 degrees to about 20 degrees, measured relative to the planar surface upon which the apparatus is placed.

[0030] In certain embodiments, the optical platform is provided at a tilt angle of from about 1 degree, about 2 degrees, about 3 degrees, about 4 degrees, about 5 degrees, about 6 degrees, about 7 degrees, about 8 degrees, about 9 degrees, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees, measured relative to the planar surface upon which the apparatus is placed. In other embodiments, the optical platform is provided at a tilt angle of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 degrees to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 degrees, measured relative to the planar surface upon which the apparatus is placed. [0031] In certain embodiments, the instrument or apparatus is provided at a tilt angle of from about 1 degree, about 2 degrees, about 3 degrees, about 4 degrees, about 5 degrees, about 6 degrees, about 7 degrees, about 8 degrees, about 9 degrees, about 10, degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees, measured relative to the planar surface upon which the instrument or apparatus is placed, such that the optical platform and/or cartridge holder is/are at the tilt angle. In other embodiments, the instrument or apparatus is provided at a tilt angle of from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 degrees to about 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44 or 45 degrees, measured relative to the planar surface upon which the apparatus is placed, such that the optical platform and/or cartridge holder is/are at the tilt angle.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a schematic view of an exemplary embodiment of an integrated fluidics system with flowrate control.

[0033] FIG. 2 shows an exemplary, non-limiting embodiment with tilt (right image) and without tilt (left image).

[0034] FIGS. 3A-3F show the improvement in optical imagining achieved using the method described herein as compared to a control method not involving any instrument tilt. Real time images were taken at the region of interest during experimentation at 37 °C with no instrument tilt at different times over a period of 150 minutes (top row), and at 37 °C with instrument tilt for the same duration (bottom row). As shown in FIG. 3A, analysis without any instrument tilt consistently showed the appearance of bubbles, which eventually masked the imaging, whereas, with instrument tilt, the bubbles dislodged from the region of interest, thus clearing the optical imaging path. See Example 1 below.

[0035] FIGS. 4A-4E similarly illustrate the improvement in optical imagining achieved using the method described herein, carried out at room temperature (e.g., about 25 °C). See Example 2 below.

DETAILED DESCRIPTION

[0036] Various aspects now will be described more fully hereinafter. Such aspects 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 disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art. [0037] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 pm to 8 pm is stated, it is intended that 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm are also explicitly disclosed, as well as the range of values greater than or equal to 1 pm and the range of values less than or equal to 8 pm. [0038] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “valve” includes a single valve as well as two or more of the same or different valves, reference to a “sensor” includes a single excipient as well as two or more of the same or different excipients, and the like.

[0039] The disjunctive “or” is inclusive, unless otherwise specified. For example, “X or Y” means “X, Y, or both X and Y” unless otherwise specified.

[0040] The word "about" when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, or about 55,” the term “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein.

[0041] The systems, devices, and methods of the present disclosure can comprise, consist essentially of, or consist of, the components or steps disclosed.

[0042] All ranges disclosed herein include all subranges contained therein, as well as all discreet values contained therein. Additionally, all ranges disclosed herein are inclusive of their endpoints, unless otherwise specified. For example, “X to Y” means “greater than or equal to X and less than or equal to Y” unless otherwise specified.

[0043] By reserving the right to proviso out or exclude any individual members of any such group, including any sub-ranges or combinations of sub-ranges within the group, that can be claimed according to a range or in any similar manner, less than the full measure of this disclosure can be claimed for any reason. Further, by reserving the right to proviso out or exclude any individual components or groups thereof, or any members of a claimed group, less than the full measure of this disclosure can be claimed for any reason.

[0044] The disclosures of any patent, patent application, or publication referenced in this disclosure are incorporated herein by reference in their entireties in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.

[0045] For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0046] In an aspect, the apparatus takes advantage of height-driven density mismatch effects - i.e., between microfluidics bubbles and surrounding fluid - to dislodge bubbles from a region of interest.

[0047] The region of interest may be, for example, a portion of an immunoassay system positioned or configured to be analyzed by an optical instrument.

[0048] In certain embodiments, the region of interest is provided in a fluidics cartridge.

[0049] In certain embodiments, dislodged bubbles are directed away from a region of interest. In certain embodiments, dislodged bubbles are directed away from a region of interest and toward an output.

[0050] In certain embodiments, one or more absorbent pads are included in a fluidics cartridge for waste removal.

[0051] In certain embodiments, the output is a waste port or waste container.

[0052] In certain embodiments, the waste port or container is provided as part of a fluidics cartridge.

[0053] In other embodiments, the waste port or container is removably attachable to a fluidics cartridge or is downstream of the cartridge in a fluidics arrangement or system.

[0054] In certain embodiments, the method involves inserting a fluidic cartridge into an instrument configured to optically analyze a fluid sample suspected of containing one or more analytes of interest. In an embodiment, the instrument is tilted at a tilt angle measured relative to a planar surface upon which the instrument is placed. In an embodiment, the fluidic cartridge when inserted into the instrument is a tilt angle measured relative to a planar surface upon which the instrument is placed or with respect to a horizontal surface plane. The titling dislodges bubbles present at a region of interest and/or causes the bubbles to move such that they do not interfere with measurement of the region of interest. In an embodiment, in response to the tilt angle, the bubbles move toward an output on the cartridge, such as a waste region. In certain embodiments, the instrument comprises a cartridge holder, a region of interest, and a waste region. [0055] In certain embodiments, the method involves inserting a fluidic cartridge into an instrument having an optical platform and configured to optically analyze a fluid sample suspected of containing one or more analytes of interest. The optical platform is tilted at a tilt angle measured relative to a planar surface upon which the instrument is placed. Said titling dislodges bubbles from a region of interest and/or causes the bubbles to move such that they do not interfere with measurement of the region of interest. In an embodiment, in response to the tilt angle, the bubbles move toward an output on the cartridge, such as a waste region. In certain embodiments, the instrument comprises a cartridge holder, a region of interest, and a waste region, and an optical platform.

[0056] In certain embodiments, a waste region described herein comprises a waste outlet or waste port.

[0057] In certain embodiments, a waste region described herein comprises a waste container.

[0058] In certain embodiments, a waste container described herein may be removably attachable to the cartridge.

[0059] In certain embodiments, a waste region described herein is in fluidic connection with a waste container external to the cartridge.

[0060] In certain embodiments, a waste region described herein comprises one or more absorbent pads.

[0061] In certain embodiments, an apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a tilting mechanism configured to tilt the apparatus at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed.

[0062] In another aspect, an apparatus for performing immunoassay of a fluid sample is provided. The apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and an optical platform. In an embodiment, the apparatus comprise a tilting mechanism configured to tilt the optical platform at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed. In an embodiment, the cartridge holder is at a tilt angle, such that insertion of a cartridge into the cartridge holder results in the cartridge being at a tilt angle. In an embodiment, the tilt angle is with respect to a horizontal surface plane. In embodiments, the tilt angle is with respect to a planar surface upon which the apparatus is placed.

[0063] In another aspect, an apparatus for performing immunoassay of a fluid sample comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a base or a plurality of legs, wherein the base or the plurality of legs orient the apparatus such that the cartridge holder is provided at a tilt angle. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured with respect to a horizontal surface plane.

[0064] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0. 1 degrees to about 30 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured with respect to a horizontal surface plane.

[0065] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0.5 degrees to about 25 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured with respect to a horizontal surface plane.

[0066] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 0.75 degrees to about 20 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured with respect to a horizontal surface plane.

[0067] In certain embodiments, the cartridge holder is provided at a tilt angle of from about 1 degree, about 2 degrees, about 3 degrees, about 4 degrees, about 5 degrees, about 6 degrees, about 7 degrees, about 8 degrees, about 9 degrees, about 10, degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees. In embodiments, the tilt angle is measured relative to the planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured with respect to a horizontal surface plane.

[0068] In another aspect, an apparatus for performing immunoassay of a fluid sample is provided. The apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and an optical platform.

[0069] In certain embodiments, the optical platform is provided at a fixed tilt angle, measured relative to a planar surface upon which the apparatus is placed or relative to a horizontal surface plane.

[0070] In embodiments, the tilt angle is transitory. For example, the tilt angle is present for a portion of time required to perform an immunoassay on the instrument. For example, if the immunoassay time is 15 minutes, the tilt angle is present for 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 100% of the immunoassay time.

[0071] In certain embodiments, the optical platform is adjustable such that it may be provided at one or more different tilt angles, measured relative to a planar surface upon which the apparatus is placed or relative to a horizontal surface plane. [0072] In certain embodiments, the apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a tilting mechanism. The tilting mechanism is configured to tilt the apparatus at a selected tilt angle. In embodiments, the tilt angle is measured relative to a planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured relative to a horizontal surface plane.

[0073] In certain embodiments, the apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, an optical platform, and a tilting mechanism. The tilting mechanism is configured to tilt the optical platform at a selected tilt angle. In embodiments, the tilt angle is measured relative to a planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured relative to a horizontal surface plane.

[0074] In certain embodiments, the apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a base, wherein the base orients the apparatus such that the cartridge holder is provided at a tilt angle. In embodiments, the tilt angle is measured relative to a planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured relative to a horizontal surface plane.

[0075] In certain embodiments, the apparatus comprises an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a plurality of legs, wherein the plurality of legs orient the apparatus such that the cartridge holder is provided at a tilt angle. In embodiments, the tilt angle is measured relative to a planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured relative to a horizontal surface plane.

[0076] In certain embodiments, an instrument or apparatus described herein may have one or more components that may be provided at or adjusted to a selected tilt angle. In certain embodiments, the tilt angle is from about 0.1 degrees to about 30 degrees, measured relative to the planar surface upon which the instrument or apparatus is placed. In certain embodiments, the tilt angle is from about 0.5 degrees to about 25 degrees. In embodiments, the tilt angle is measured relative to a planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured relative to a horizontal surface plane.

[0077] In certain embodiments, the tilt angle is from about 0.75 degrees to about 20 degrees, measured relative to the planar surface upon which the instrument or apparatus is placed. In certain embodiments, the tilt angle is about 1 degree, about 2 degrees, about 3 degrees, about 4 degrees, about 5 degrees, about 6 degrees, about 7 degrees, about 8 degrees, about 9 degrees, about 10, degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees, 16 degrees, 17 degrees, 18 degrees, 19 degrees, or 20 degrees. In embodiments, the tilt angle is measured relative to a planar surface upon which the apparatus is placed. In embodiments, the tilt angle is measured relative to a horizontal surface plane.

[0078] In certain embodiments, the instrument or apparatus has an adjustable cartridge holder configured to be adjusted to a selected tilt angle as measured relative to a planar surface upon which the instrument or apparatus is placed. In certain such embodiments, the cartridge holder can be adjusted to any selected tilt angle. In other embodiments, the cartridge holder can be adjusted to one of a plurality of defined, discrete tilt angles. In certain embodiments, the tilt angle of the cartridge holder is manually adjustable. In certain embodiments, the tilt angle of the cartridge holder is mechanically adjustable. In certain embodiments, the tilt angle of the cartridge holder may be adjusted in response to a command or instruction provided by computer software code, optionally implemented by an electronic board in electrical communication with the instrument or apparatus.

[0079] In certain embodiments, the instrument or apparatus has an adjustable optical platform configured to be adjusted to a selected tilt angle as measured relative to a planar surface upon which the instrument or apparatus is placed. In certain such embodiments, the optical platform can be adjusted to any selected tilt angle. In other embodiments, the optical platform can be adjusted to one of a plurality of defined, discrete tilt angles. In certain embodiments, the tilt angle of the optical platform is manually adjustable. In certain embodiments, the tilt angle of the optical platform is mechanically adjustable. In certain embodiments, the tilt angle of the optical platform may be adjusted in response to a command or instruction provided by computer software code, optionally implemented by an electronic board in electrical communication with the instrument or apparatus.

[0080] In certain embodiments, the instrument or apparatus has an adjustable base. In such embodiments, the base is adjustable to provide a tilt angle for the instrument or apparatus. In certain such embodiments, the base can be adjusted to any selected tilt angle. In other embodiments, the base can be adjusted to one of a plurality of defined, discrete tilt angles. In certain embodiments, the tilt angle of the base is manually adjustable. In certain embodiments, the tilt angle of the base is mechanically adjustable. In certain embodiments, the tilt angle is provided by computer software code, optionally implemented by an electronic board in electrical communication with the instrument or apparatus.

[0081] In certain embodiments, the instrument or apparatus has a plurality of legs or feet. In such embodiments, at least one leg or foot is of an adjustable height. In certain embodiments, the plurality of legs or feet are all of adjustable heights. In certain embodiments having a plurality of legs or feet, each of the legs or feet having an adjustable height are adjustable. In certain embodiments, the legs or feet may be adjusted manually to different height positions. In other embodiments, the legs or feet nay be adjusted manually to discrete, defined height positions. In certain embodiments, the legs or feet may be adjusted pneumatically to different height positions. In other embodiments, the legs or feet nay be adjusted pneumatically to discrete, defined height positions. In certain embodiments, the legs or feet may be adjusted in response to a command or instruction provided by computer software code, optionally implemented by an electronic board in electrical communication with the instrument or apparatus.

[0082] In certain embodiments, the optical imaging system is an instrument such as a dark-field optical microscope or dark-field spectrophotometer. In certain embodiments, the optical imaging system is a fluorescent analyzer, such as a fluorospectrometer.

[0083] In an aspect, a tilt angle provides a height difference between the region where bubbles are located (e.g., a region of interest) and another region (e.g., a waste outlet) of the instrument, apparatus, and/or cartridge.

[0084] Due to the provided height difference, gravity-driven density mismatch effects are generated that push or otherwise direct the bubbles to another region (e.g., a waste region) of the instrument, apparatus and/or cartridge, thus clearing the region of interest (e.g., an optical region of interest).

[0085] As described herein, certain embodiments involve providing the entire instrument or apparatus (as opposed to only an optical platform, cartridge, or cartridge holder) at a tilt angle. In such embodiments, since the whole instrument e.g., with a cartridge installed in or on an optical platform or cartridge holder) is tilted, the tilt angle does not affect instrumental conditions such as optical focus. In this way, analysis conditions remain undisturbed and the method of removing bubbles does not interfere with optical detection or imagining processes.

[0086] In an aspect of certain embodiments of the present invention, tilting the region of interest and an observation apparatus (e.g., an optical imaging system) at the same angle relative to a common planar surface allows one to maintain all characteristics of the imaging approach relative to the region of interest - i.e., focus/autofocus, incident light angle, angle of observation, and the like.

[0087] In certain embodiments, the tilt angle is adjustable.

[0088] In other embodiments, the tilt angle is fixed i.e., essentially permanent).

[0089] In certain embodiments, tilting may be motorized and optionally automated.

[0090] In embodiments, the tilt angle is transitory. In embodiments, the tilt angle exists for a period of time that is less than the time for an immunoassay run time. That is, the tilt angle is present temporarily, such as during a wash cycle or another part of the assay cycle. In an embodiment, the apparatus is capable of detection of whether bubbles have moved away from a region of interest and/or dissipated and can adjust the tilt angle based on the detection.

[0091] Where the tilting is automated using a motor and/or a pneumatic mechanism, tilting may be done temporarily, such as during a wash cycle or another part of the diagnostic cycle through the use of a motor that automatically tilts the instrument, apparatus, optical platform, cartridge, or cartridge holder, and then resets after bubbles have dissipated. In some embodiments, the motor may reset the instrument, apparatus, optical platform, cartridge, or cartridge holder after a defined period of time.

[0092] In certain embodiments, an immunoassay method described herein may comprise a flowrate-controlled process involving an external flowrate-controlled fluidics system in fluidic communication with the cartridge and/or cartridge holder.

[0093] FIG. 1 depicts an exemplary, non-limiting embodiment of a flowrate-controlled system in which a pump, valves, and sensors external to an optical detection assembly are provided. As shown in FIG. 1, an electronic board 1 is provided in electrical communication with a device (e.g., a computer, server, or mobile device) that contains software code (e.g., automation code), a pneumatic pump 2 having a first pump port 2a and a second pump port 2b, and one or more sensors (e.g., a pressure sensor 4 and/or a fluid flow sensor 6). The software code defines a fluidics operation, which is implemented by the electronic board 1 to cause the pneumatic pump 2 to generate a positive pressure via the second pump port 2b. The first pump port 2a is in direct pneumatic communication with a first solenoid valve 3a, which is normally in a closed position. The second pump port 2b is in direct pneumatic communication with a second solenoid valve 3b, which is normally in an open position. The pair of solenoid valves 3 is in direct pneumatic communication with a fluid reservoir 5 via a fluid reservoir input 5a. A net positive pressure applied upon the fluid reservoir 5 via the fluid reservoir input 5a causes flow of fluid out of the fluid reservoir 5 via a fluid reservoir output 5b. The fluid reservoir output 5b is in direct fluid communication with a fluid flow sensor 6, which is configured to measure a parameter of the fluid flow (e.g., a volume of fluid or a rate of fluid flow).

[0094] In certain embodiments, the fluid flows past the fluid flow sensor 6 to reach an isolation valve 7, which is also in electrical communication with the electronic board 1.

[0095] Downstream of the fluid flow sensor 6, and downstream of the isolation valve 7 if present, is a fluidics cartridge holder 8 configured to receive a fluidics cartridge 9. When a fluidics cartridge 9 is inserted into or onto the cartridge holder 8, fluid held undisturbed by the isolation valve 7 is allowed to incubate over a region of interest 10. In certain embodiments, the region of interest 10 is an optical sensor region which can be analyzed by an optical instrument (e.g., a spectrophotometer) (collectively, 15a, 15b, 16a, 16b, 17). Fluid flows to the region of interest 10 via an input Ila that is in fluid communication with the isolation valve 7. The input Ila may be provided on the cartridge holder 8 or on the fluidics cartridge 9. Additionally, an output 11b is provided either on the cartridge holder 8 or on the fluidics cartridge 9. An optional waste container 12 may be provided in fluid communication with the output 11b. An optional thermal controller 13 may be provided, for example a thermal controller 13 with one or more heating rods 14, which heat the cartridge 9 and/or cartridge holder 8. The optical instrument (collectively, 15a, 15b, 16a, 16b, 17) for analysis of the sample at the region of interest 10 includes a camera 15a with a lens 15b and a light source 16a with a lens 16b to provide incident light. The camera 15a detects, via lens 15b, light reflected at the region of interest 10. The camera 15a and light source 16a are electronically connected to a device or assembly of devices 17 (e.g., a computer) configured for light generation and image processing.

[0096] In certain embodiments and applications, based on an interval of time or amount of fluid defined by the software code, the isolation valve 7 may be toggled by the electronic board to a closed position to isolate fluid in a substantially undisturbed (e.g., substantially static) state to prevent backflow in a direction back toward the pneumatic pump 2.

[0097] In other embodiments and applications, such as in a continuous flow assay, the isolation valve 7 may remain in an open position to permit continuous flow to the fluidics cartridge 9. In certain embodiments, the isolation valve 7 may omitted.

[0098] In such embodiments, the fluid reservoir provides fluid sample, including any optional buffers and/or detectable particles that may bind to analyte(s) of interest.

[0099] In certain embodiments, the fluid reservoir provides detectable particles having binding members bound thereto.

[0100] In certain embodiments, the detectable particles are functionalized nanoparticles that are capable of binding to the analyte of interest such that the bound analyte (i.e., analyte plus nanoparticle) can subsequently bind to a functionalized region of interest. In certain embodiments, the flowing fluid sample containing analyte and bound nanoparticles is permitted to incubate at the region of interest to allow for binding at the region of interest. In an aspect, incubating at the region of interest allows for accurate analyte detection. In an aspect, incubation can be achieved effectively and efficiently by including the isolation valve, which maintains the fluid sample at the region of interest in a substantially undisturbed (i.e., static or substantially static) state.

[0101] In certain embodiments, the detectable particles comprise a binding member having binding affinity for the analyte of interest. [0102] In certain embodiments, the detectable particles comprise a binding member that specifically binds the analyte of interest.

[0103] In certain embodiments, the region of interest comprises a binding member having binding affinity for the analyte of interest. In certain embodiments, the region of interest comprises a binding member that specifically binds the analyte of interest. In certain embodiments, the region of interest comprises a binding member that binds the conjugate formed between the analyte of interest and the detectable particle.

[0104] The detectable particles comprise or consist essentially of a metal, preferably a transition metal or a noble metal. In certain embodiments, the detectable particles comprise or consist essentially of one or more metals selected from gold, silver, platinum, palladium, iridium, osmium, rhodium, ruthenium and alloys thereof. In certain embodiments, the detectable particles are nanoparticles having at least a plasmonic material embedded therein (e.g., gold, aluminum, silver or a metamaterial).

[0105] In certain embodiments, the detectable particles consist of a metal selected from gold, silver, platinum, palladium, iridium, osmium, rhodium, and ruthenium. For example, in certain embodiments, the detectable particles comprise or consist essentially of gold. In embodiments, the detectable particles consist of gold.

[0106] The detectable particles have an average diameter ranging from about 1 nm to about 1500 nm, or from about 25 nm to about 500 nm, or from about 50 nm to about 250 nm or from 100 to 200 nm.

[0107] In certain embodiments, the detectable particles resonate at a wavelength ranging from about 250 nm to about 1000 nm, or about 300 nm to about 950 nm, or about 350 nm to about 900 nm, or about 400 nm to about 850 nm, or about 450 nm to about 800 nm.

[0108] In certain embodiments, the detectable particles have a shell-core structure, wherein the core is magnetic and the shell is a transition metal. In embodiments, the core is iron, an oxide of iron, or an iron alloy. In embodiments, the core is iron or iron (II, III) oxide (i.e., FesC ). In embodiments, the shell is gold.

[0109] In certain embodiments, the diameter of the magnetic core i.e., the average magnetic core diameter of a plurality of detectable particles) may be in the range of from about 1 nm to about 300 nm, or from about 25 nm to about 250 nm, or from about 50 nm to about 200 nm, or from about 75 nm to about 150 nm and the thickness of the shell may be in the range of from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm. [0110] In certain core-shell embodiments, the diameter of the magnetic core may be in the range of from 0.5 nm to about 60 nm, or from about 1 nm to about 40 nm, or from about 3 nm to about 30 nm, or from about 5 nm to about 25 nm, and the shell may have a thickness in the range of from about 1 nm to about 100 nm, or from about 5 nm to about 80 nm, or from about 5 nm to about 60 nm, or from about 10 nm to about 45 nm.

[0111] Optionally, an intermediate layer may be provided between the core and shell of the detectable particles (i.e., the intermediate layer may be provided as a first shell between the core and the outer shell). The intermediate layer may be comprised of silica. The diameter of the magnetic core (<?.#., the average magnetic core diameter of a plurality of detectable particles) may be in the range of from about 1 nm to about 300 nm, or from about 25 nm to about 250 nm, or from about 50 nm to about 200 nm, or from about 75 nm to about 150 nm. The thickness of the intermediate layer may be in the range of from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm. The thickness of the shell may be in the range of from about 0.5 nm to about 50 nm, or from about 1 nm to about 40 nm, or from about 5 nm to about 30 nm, or from about 10 nm to about 25 nm. The detectable particle may have a diameter (i.e., an average diameter) in the range of from about 25 nm to about 500 nm, or from about 50 nm to about 450 nm, or from about 75 nm to about 350 nm, or from about 100 nm to about 300 nm.

[0112] In other embodiments, the detectable particles do not have a core-shell structure. That is, in such embodiments, the detectable particles consist essentially of a transition metal or alloy thereof. For example, the detectable particles may consist essentially of gold.

[0113] In other embodiments, other optically detectable markers or labels may be used. For example, fluorescent or chemiluminescent particles may be used. In certain non- limiting embodiments, europium beads may be used.

[0114] In certain embodiments, a pump - preferably a bidirectional pump configured to generate positive and negative pressures - is provided. The pump is in electrical communication with a computer or an electronic board in further electrical communication with a computer. The computer stores software code defining a fluidic operation, wherein the software code defines one or more of a minimum and/or a maximum fluid volume, fluid flowrate, incubation time, and optionally one or more cycles such as push-pull (i.e., wash) cycles.

[0115] In certain embodiments, the flowrate-controlled fluidics system comprises one or more valves, such as solenoid valves or one or more pairs of solenoid valves and an isolation valve to prevent backflow from the fluidics cartridge. The flowrate-controlled fluidics system may also comprise one or more sensors, such as a flowrate or fluid volume flow sensor, and one or more pressure sensors or pressure regulators.

[0116] In certain embodiments, a feedback loop is formed between the one or more sensors, pump, and solenoid valve, all in electrical communication with the electronic board and/or computer. The feedback loop serves to monitor (via the one or more sensors) the amount of fluid pumped and/or the rate of fluid being pumped and/or the amount of time elapsed during the fluidics operation. The measuremen t(s) by the one or more sensors serve as bases for the software code algorithm (i.e., the defined fluidic operation) to adjust flow of fluid by generating a positive pressure or a negative pressure using the pump. In certain embodiments, a fluid reservoir is provided, which may be present in an external fluidic system connected to the cartridge or cartridge holder, or which may be provided within (i.e., integrated into) a fluidics cartridge).

Utilizing such a flowrate-controlled system is one effective means to account for increased fluidics resistance that may arise as a result of raising or tilting the instrument, apparatus, cartridge, cartridge holder, or optical platform.

[0117] FIG. 2 shows an exemplary, non-limiting embodiment wherein the instrument is titled at a tilt angle, resulting in bubbles being dislodged from the region of interest 10 and directed toward an output lib connected to a waste container 12. The instrument shown in FIG. 2 is fed by a pneumatic, flowrate-controlled external fluidics control. One non-limiting embodiment of the external fluidics control is shown in FIG. 1. Due to the height difference provided by the instrument tilt, gravity -driven density mismatch effects are generated that push or otherwise direct the bubbles to another region (e.g., the output 11b and waste container 12), thereby clearing the region of interest 10 of bubbles. In an aspect, the channel in which the region of interest 10 is located is filled with fluid, thereby allowing the bubbles to be expelled.

[0118] In other words, raising the instrument height could add resistance to the fluid flow, but since the external fluidics system is driven by a flowrate-controlled feedback loop designed to maintain a constant flowrate over a defined period of time, the system compensates for any increased fluidics resistance. Thus, the apparatus, instrument and method described herein maintains essentially uniform fluidics conditions throughout a fluidics operation despite a change in height or tilt angle (and thus the introduction of a density mismatch).

[0119] In certain embodiments, a thermal controller may be included, optionally a thermal controller in combination with one or more heating elements (e.g., heating rods). In certain embodiments, a thermal controller is provided to heat the cartridge, cartridge holder, and/or optical platform. [0120] In certain embodiments, heat provided by a thermal controller contributes further to the inventive method. In such embodiments, applied heat helps to grow bubbles, thereby causing them to move toward the waste outlet or container due to gravity-driven effects.

[0121] In certain embodiments of the inventive methods and apparatuses described herein, a tilt angle may be provided for a chosen period of time during an immunoassay operation. In other embodiments, the tilt angle may be permanent. In other embodiments, the tilt angle may be provided transiently.

EXAMPLES

[0122] Further aspects of the present subject matter will be apparent to persons of ordinary skill in the art based on the following non-limiting Examples.

EXAMPLE 1

[0123] A real-time IL-6 assay with continuous flow-based binding of functionalized magnetic gold nanoparticles (mGNPs) was performed to assess the efficacy of the apparatus and method. The nanoparticle concentration was 0.25 pg/mL mGNPs (at a volume of 1500pL).

[0124] The mGNPs were pre-functionalized with IL-6 markers.

[0125] A piezoelectric pump system similar to the exemplary system shown in FIG. 1 was used to provide fluid flow at a constant rate. Standard silicone tubing was used in said flow system.

[0126] Fluidic lanes were primed with filtered PBST buffer (0.2 micron filter.) The mGNPs were introduced at a flow rate of 10 L/min at 37C.

[0127] Pictures were taken at time stamps of T=10 min until sample ran out.

[0128] Settings for the optical detector instrument: Exposure of 400 ms; Gain of 26.1 dB; Gamma = 1; LED power = 12 W. Focus settings were never changed.

[0129] The entire instrument body was titled at an angle of ~8.6 degrees. It was concluded that tilting almost completely resolved the bubble issue for region of interest exposure. Focus remained the same.

[0130] Sample loading and incubation: The back of the imaging instrument was raised by 8.5 degrees and the 37C heater was powered on. A diagnostic cartridge was placed on the optical platform to equilibrate before running experiment (for ~ 15 minutes). Sample tubing was replaced at the beginning of every experiment to ensure no contamination. A fiberglass insulation cover was placed over the cartridge to reduce infinite temperature field. 550pL buffer (or less) was loaded first into tubing using Ventus/Kipling software and control. Once loaded, any air gap was pushed out and 150pL buffer was loaded at approximately 100 pL/min flowrate into the desired cartridge channel for baseline imaging. After filling the cartridge with liquid, tubing was disconnected, and any excess buffer was expelled. The reservoir was replaced with 20p L particle sample. Ventus/Kipling software and control was used to push sample to the end of the tubing to ensure no air gap between inlet and end of tubing. The tubing was placed on the desired cartridge inlet. Using Lab VIEW software-based fluidics control, 60 p L of sample was pushed across the region of interest at a 100 pL/min flow rate. A sample image TO was taken. Then, using Lab VIEW software-based fluidics control, 10 pL/min sample was pushed for 150 minutes.

Images were acquired automatically (using an image capture software) every 10 minutes.

[0131] Washing (after 150 minutes passed): Sample tubing was disconnected from the inlet and any remaining sample was expelled from the tubing. The sample reservoir was replaced with a wash buffer Eppendorf reservoir containing 1 mL PBST buffer. The buffer was pushed to the end of the tubing (connected to the same lane). Using Lab VIEW software-based fluidics control, a wash cycle was run at a flowrate of 200 pL/min for 5 minutes. An image was taken every minute into the wash cycle.

[0132] Results are shown in FIGs. 3A-3D. As evidenced by the images and data shown in FIGS. 3A-3D, it can be concluded that there was a clear increase in binding (signal) over time without the observation of any bubbles interfering with imaging and analysis. For example, FIG. 3C and FIG. 3D show observable mGNP peak growth over time, either in the form of mGNP density, histogram peak growth or increase number of particles.

EXAMPLE 2

[0133] To ascertain the detrimental effect of bubbles on the ability to accurately identify or detect magnetic gold nanoparticles, an assay was run at a flow rate of 50 pL/min at 37C, with no instrument tilt and with a tilt of 6°.

[0134] FIG. 3F shows results of the assay both without and with instrument tilt.

[0135] In the absence of tilt, bubbles accumulated during the course of a normal fluidic experiment. The bubbles hid magnetic gold nanoparticles from detection, with only 24,695 counted.

[0136] With a 6° tilt of the instrument, bubbles were substantially reduced over the same course of a normal fluidic experiment. Elimination of bubbles allowed magnetic gold nanoparticles to be identified and quantified. With the tilt, 76,432 particles were counted - more than three times the number of particles detected without tilt. EXAMPLE 3 [0137] The assay of Example 1 was run again, but at room temperature. As in Example 1, it can be concluded that there was a clear increase in signal over time without the observation of any bubbles interfering with imaging and analysis. Results are shown in FIGS. 4A-4E.

[0138] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

Claims

CLAIMS IT IS CLAIMED:

1. A method of analyzing a fluid sample, comprising:

(a) inserting a fluidic cartridge into an instrument configured to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, and

(b) tilting the instrument at a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed, whereby tilting the instruments dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region.

2. A method of analyzing a fluid sample, comprising:

(a) inserting a fluidic cartridge into an instrument configured to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, and an optical platform, and

(b) tilting the optical platform at a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed, whereby tilting the instruments dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region.

3. A method of analyzing a fluid sample, comprising: providing an instrument configured to receive a fluidic cartridge and to optically analyze a fluid sample suspected of containing an analyte of interest, the instrument comprising a cartridge holder, a region of interest, and a waste region, and an optical platform, wherein the instrument and/or the optical platform is/are at a tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the instrument is placed, whereby the tilt angle dislodges bubbles from a region of interest and/or causes the bubbles to move toward the waste region.

4. The method of any one of claims 1-3, wherein the tilt angle is from about 0.1 degrees to about 30 degrees, measured relative to the planar surface upon which the apparatus is placed.

5. The method of any one of claims 1-3, wherein the tilt angle is from about 0.5 degrees to about 25 degrees, measured relative to the planar surface upon which the apparatus is placed.

6. The method of any one of claims 1-3, wherein the tilt angle is from about 0.75 degrees to about 20 degrees, measured relative to the planar surface upon which the apparatus is placed.

7. The method of any one of claims 1-3, wherein the tilt angle is about 1 degree, about 2 degrees, about 3 degrees, about 4 degrees, about 5 degrees, about 6 degrees, about 7 degrees, about 8 degrees, about 9 degrees, about 10, degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, about 15 degrees, about 16 degrees, about 17 degrees, about 18 degrees, about 19 degrees, or about 20 degrees, measured relative to the planar surface upon which the apparatus is placed.

8. The method of any one of claims 1-3, further comprising an external fluidics control configured to provide a fluid sample to the region of interest at an essentially constant flowrate.

9. The method of claim 8, wherein the external fluidics control comprises a bidirectional pneumatic pump, a plurality of solenoid valves, an isolation valve, a flowrate sensor, and a pressure sensor.

10. The method of claim 9, wherein the external fluidics control further comprises a programmable electronic board in electrical communication with the bidirectional pneumatic pump, the plurality of solenoid valves, the isolation valve, and the flowrate sensor, and wherein the electronic board is in electrical communication with a device storing software code defining a fluidics operation.

11. The method of claim 9, wherein the fluidic cartridge or optical platform is heated by one or more heating elements heated via a thermal controller.

12. An apparatus for performing immunoassay of a fluid sample, comprising: an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a tilting mechanism configured to tilt the apparatus at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed.

13. An apparatus for performing immunoassay of a fluid sample, comprising: an optical detector, a light source, a cartridge holder, a region of interest, a waste region, an optical platform, and a tilting mechanism configured to tilt the optical platform at a selected tilt angle, wherein the tilt angle is measured relative to a planar surface upon which the apparatus is placed.

14. An apparatus for performing immunoassay of a fluid sample, comprising: an optical detector, a light source, a cartridge holder, a region of interest, a waste region, and a base or a plurality of legs, wherein the base or the plurality of legs orient the apparatus such that the cartridge holder is provided at a tilt angle measured relative to a planar surface upon which the apparatus is placed.

15. An apparatus for performing immunoassay of a fluid sample, comprising: an optical detector, a light source, a cartridge holder, a region of interest, a waste region, an optical platform, wherein the optical platform is provided at a tilt angle, measured relative to a planar surface upon which the apparatus is placed.

16. The apparatus of any one of claims 12-15, wherein the tilt angle is from about 0.1 degrees to about 30 degrees, measured relative to the planar surface upon which the apparatus is placed.

17. The apparatus of any one of claims 12-15, wherein the tilt angle is from about 0.5 degrees to about 25 degrees, measured relative to the planar surface upon which the apparatus is placed.

18. The apparatus of any one of claims 12-15, wherein the tilt angle is from about 0.75 degrees to about 20 degrees, measured relative to the planar surface upon which the apparatus is placed.

19. The apparatus of any one of claims 12-15, wherein the tilt angle is about 1 degree, about 2 degrees, about 3 degrees, about 4 degrees, about 5 degrees, about 6 degrees, about 7 degrees, about 8 degrees, about 9 degrees, about 10, degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, about 15 degrees, about 16 degrees, about 17 degrees, about 18 degrees, about 19 degrees, or about 20 degrees, measured relative to the planar surface upon which the apparatus is placed.

20. The apparatus of claim 12, further comprising a fluidics arrangement configured to provide a fluid sample to the region of interest at an essentially constant flowrate.

21. The apparatus of claim 20, wherein the external fluidics arrangement comprises a bidirectional pneumatic pump, a plurality of solenoid valves, an isolation valve, a flowrate sensor, and a pressure sensor.

22. The apparatus of claim 21, wherein the external fluidics arrangement further comprises a programmable electronic board in electrical communication with the bidirectional pneumatic pump, the plurality of solenoid valves, the isolation valve, and the flowrate sensor, and wherein the electronic board is in electrical communication with a device storing software code defining a fluidics operation.

23. The apparatus of any one of claims 12-15, further comprising a thermal controller and one or more heating elements.