Optical interrogation system having first and second pinhole plates and methods of use thereof

Abstract

Optical interrogation systems are provided. Systems of interest include: a first pinhole plate comprising one or more pinholes, each pinhole configured for optical alignment with one detection chamber of a plurality of detection chambers; a light source configured to illuminate each detection chamber of the plurality through one pinhole of the first pinhole plate that is optically aligned with the detection chamber; a second pinhole plate comprising one or more pinholes, each pinhole optically aligned with one pinhole of the first pinhole plate and configured for optical alignment with one detection chamber of the plurality; and an optical sensor configured to collect light from each detection chamber of the plurality through the pinhole of the second pinhole plate that is optically aligned with the detection chamber. Methods of using the optical interrogation systems of the invention to analyze sample fluids are also provided.

Description

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 563,780, filed March 11, 2024, and U.S. Provisional Patent Application No. 63 / 566,126, filed March 15, 2024, which are incorporated herein by reference in their entirety. Background Technology

[0002] introduction Point-of-care (POC) sample analysis systems are typically based on one or more reusable handheld analyzers (i.e., instruments or reading devices) that use single-use, disposable testing devices (e.g., cartridges or strips containing analytical elements (e.g., electrodes or optics for sensing analytes such as pH, oxygen, and glucose) and various types of proteins, enzymes, and blood cells) to perform sample testing. The disposable testing device may include fluid elements (e.g., catheters for receiving and delivering the sample to the sensing electrodes or optics), calibrator elements (e.g., an aqueous fluid for standardizing the electrodes and optics with known concentrations of analytes), and dyes with known extinction coefficients for standardizing the optics. The instrument or reading device may contain circuitry and other components for operating the electrodes or optics, performing measurements, and executing calculations. The instrument or reading device may also have the ability to display results and communicate those results to laboratory information systems and hospital information systems (LIS and HIS, respectively) (e.g., via computer workstations or other data management systems). Communication between the instrument or reading device and the workstation, and between the workstation and the LIS or HIS, may be conducted via, for example, an infrared link, a wired connection, wireless communication, or any other form of data communication capable of transmitting and receiving electrical information, or any combination thereof.

[0003] One benefit of point-of-care testing (POC) systems is the elimination of the time-consuming need to send samples to a central laboratory for testing. POC systems allow nurses or physicians (users or operators) to obtain reliable quantitative analytical results at the patient's bedside, with quality comparable to those obtained in a laboratory. In operation, the nurse selects a testing device with the desired panel for the test, draws a biological sample from the patient, dispenses the sample into the testing device, optionally seals the device, and inserts it into the instrument or reading device. While the specific order in which these steps occur may vary between different POC systems and providers, the intention to provide rapid sample testing results close to the patient remains constant. The instrument or reading device then performs the testing cycle, which involves performing all other analytical steps required for the test. This simplicity gives physicians a faster and deeper understanding of the patient's physiological condition and allows them to make more informed decisions about appropriate treatment by reducing turnaround time for diagnosis or monitoring, thus increasing the likelihood of successful patient outcomes.

[0004] As discussed herein, point-of-care sample testing systems typically include an instrument or analyzer configured to perform sample tests using single-use, disposable testing devices to determine analytes in biological samples. The type of sample test performed can vary and can be implemented using one or more disposable testing devices, including, for example, qualitative or semi-quantitative testing devices (e.g., lateral flow or microarray assays), quantitative testing devices (e.g., electrochemical assays), or combinations of qualitative or semi-quantitative and quantitative testing devices (e.g., a testing device having both lateral flow or microarray assays and electrochemical assays). To perform sample tests, the instrument or analyzer includes: an optical sensor configured to process signals from a qualitative or semi-quantitative testing device; and / or an electrical connector configured to process signals from a quantitative testing device (see, for example, U.S. Patent No. 9,194,859, which is incorporated herein by reference in its entirety). In particular, the optical sensor includes an optical imager configured to image the assay of the optical test cartridge. The measurement is a qualitative or semi-quantitative lateral flow test or microarray test (e.g., one or more lateral flow test strips or microarrays disposed in a conduit of an optical test chamber). The optical sensor further includes a processor configured to process the signal generated by the optical imager to display the qualitative or semi-quantitative test results. Summary of the Invention

[0005] Aspects of the present invention include an optical interrogation system, for example, for analyzing sample fluids (e.g., blood). The subject system includes: a first pinhole plate comprising one or more pinholes, each pinhole configured for optical alignment with one of a plurality of detection chambers; a light source configured to illuminate each of the plurality of detection chambers through a pinhole of the first pinhole plate, the pinhole being optically aligned with the detection chamber; a second pinhole plate comprising one or more pinholes, each pinhole optically aligned with a pinhole of the first pinhole plate and configured for optical alignment with one of the plurality of detection chambers; and an optical sensor configured to collect light from each of the plurality of detection chambers through a pinhole of the second pinhole plate optically aligned with the detection chamber. In some cases, the first and second pinhole plates each comprise an array of pinholes (e.g., 2 to 50 pinholes). In embodiments, the pinholes are spaced from 2 mm to 2.5 mm apart. In embodiments, the one or more pinholes of the first and second pinhole plates have a diameter ranging from 0.5 mm to 1.5 mm. In selected cases, the distance between the first and second pinhole plates ranges from 5 mm to 15 mm. In some implementations, the first and second pinhole plates have a thickness ranging from 20 mm to 50 mm. In selected types, the light source is composed of an array of optical fibers. In some such types, each fiber in the fiber array is optically aligned with a different pinhole of the first pinhole plate. In some cases, the light source also includes a fiber array plate having holes for mounting the fiber array. According to some embodiments, the light source may also include a microlens array positioned between the light source and the first pinhole plate, wherein each microlens in the array is optically aligned with a pinhole of the first pinhole plate. In some cases, the microlens array may be arranged within the microlens plate. In selected types, the light source includes an array of miniature light-emitting diodes (LEDs). In some such types, the light source includes miniature LEDs. In some embodiments, the light source includes a light guide plate (LGP). In some cases, the LGP may be made of cast-grade polymethyl methacrylate (PMMA). The optical sensor may vary. In some cases, the optical sensor is a CMOS sensor. In some cases, optical sensors are composed of photodiodes. In other cases, optical sensors are spectrometers.

[0006] The system of the present invention may additionally include optical elements positioned along an optical path between a light source and an optical sensor. For example, in some embodiments, the system includes a diffuser positioned along an optical path between the light source and a first pinhole plate. In some cases, the system includes a folded mirror or folded prism positioned along an optical path between the light source and the first pinhole plate. In embodiments, the system includes a bandpass filter positioned between a second pinhole plate and the optical sensor. According to some embodiments, the system includes a notch filter, for example, positioned between the first and second pinhole plates.

[0007] In some cases, the system may include a processor for use in conjunction with the aforementioned components. For example, embodiments of the system include a processor operatively connected to an optical sensor, a light source, and a memory storing instructions that, when executed by the processor, cause the processor to calculate the absorbance of each of the plurality of detection chambers. The processor of interest may be configured to: calculate the average intensity of incident light from the light source; calculate the average intensity of emitted light from each of the plurality of detection chambers; deactivate the light source and calculate the dark-image average intensity of the emitted light from each of the plurality of detection chambers; and calculate the absorbance of each of the plurality of detection chambers based on the average intensity of incident light from the light source, the average intensity of emitted light from each of the plurality of detection chambers, and the dark-image average intensity of the emitted light from each of the plurality of detection chambers. The processor may configure the intensity of the light source and the integration time of the optical sensor to adjust the dynamic range of the sample.

[0008] Various aspects of the invention also include methods for analyzing sample fluids. Methods of interest include: introducing sample fluid into a cartridge comprising a plurality of detection chambers; inserting the cartridge into an optical interrogation system of the invention (e.g., described above and herein); and illuminating the plurality of detection chambers with a light source to analyze the sample fluid. The sample fluid may vary and, in some embodiments, includes a blood sample (e.g., a whole blood sample). In some forms, analyzing the sample fluid includes performing a full metabolic assay (CMP). Attached Figure Description

[0009] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The following drawings are included in the drawings: Figure 1A-1B A light source including a light guide plate is depicted according to certain embodiments.

[0010] Figure 2A-2B A light source including a light guide plate is depicted according to certain embodiments.

[0011] Figures 3A-3E A light source optically coupled to an optical fiber array according to certain embodiments of the present invention is depicted.

[0012] Figures 4A-4B A first pinhole plate and a second pinhole plate according to certain embodiments of the present invention are presented.

[0013] Figures 5A-5E Aspects of an optical interrogation system according to certain embodiments are described.

[0014] Figures 6A-6C The filter arrangement of an optical interrogation system according to certain embodiments is described.

[0015] Figures 7A-7C A prototype of an optical interrogation system was depicted.

[0016] Figure 8 The image shows the light received from the pinhole relative to the mask used for intensity measurement.

[0017] Figure 9A-9G The experimental setup for performing stray light testing is shown. Figure 9A ) and results from stray light testing ( Figure 9B-9G ).

[0018] Figures 10A-10B The pinhole array is described. Figure 10A ) and the beam projection from the first pinhole plate having the pinhole array ( Figure 10B ).

[0019] Figure 11A-11B The absorption and optical density (OD) calculations using a pinhole array are described.

[0020] Figure 12 The absorbance spectrum of neutral density optical fiber on a UV1800 spectrometer was depicted.

[0021] Figures 13A-13D The test results for the neutral density filter are described.

[0022] Figures 14A-14B Stray light measurement was presented.

[0023] Figures 15A-15C A prototype of an optical interrogation system was depicted.

[0024] Figures 16A-16B The spectra of different light guide plates were presented.

[0025] Figure 17 The adjustments made to the light source, including the light guide plate, are described.

[0026] Figure 18 The experimental setup used to determine the power efficiency of the light guide plate is described.

[0027] Figure 19 The power efficiency results of the light guide plate are described.

[0028] Figure 20 An embodiment of the invention with a single pinhole plate and a reflector is depicted. Detailed Implementation

[0029] An optical interrogation system is provided. The system of interest includes: a first pinhole plate comprising one or more pinholes, each pinhole configured for optical alignment with one of a plurality of detection chambers; a light source configured to illuminate each of the plurality of detection chambers through a pinhole of the first pinhole plate, the pinhole being optically aligned with the detection chamber; a second pinhole plate comprising one or more pinholes, each pinhole optically aligned with a pinhole of the first pinhole plate and configured for optical alignment with one of the plurality of detection chambers; and an optical sensor configured to collect light from each of the plurality of detection chambers through a pinhole of the second pinhole plate optically aligned with the detection chamber. A method for analyzing sample fluids using the optical interrogation system of the present invention is also provided.

[0030] Before describing the invention in more detail, it will be understood that the invention is not limited to the specific embodiments described, and therefore variations are possible. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of the invention will be limited only by the appended claims.

[0031] Where a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, or any other stated value or intermediate value within the stated range, is covered within this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered within this invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both limits, the range excluding one or both of those included limits is also included in this invention.

[0032] This document presents certain ranges where numerical values ​​are preceded by the term "approximately". The term "approximately" is used in this document to provide textual support for the exact number that follows it, as well as numbers that are close to or approximate to the number following the term. In determining whether a number is close to or approximate to a specifically stated number, a close to or approximate unstated number may be a number that is substantially equivalent to the number that is given a specific statement in the context in which it is presented.

[0033] 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 to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

[0034] All publications and patents referenced in this specification are incorporated herein 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 methods and / or materials relating to the referenced publication. References to any publication are for its disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to prior rights to such publication due to prior invention. Furthermore, the publication dates provided may differ from the actual publication dates that may require independent verification.

[0035] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a / an” and “the” as used herein and in the appended claims include a plural of indicators. It should be further noted that claims may be drafted to exclude any optional elements. Therefore, the use of exclusive terms such as “uniquely,” “only,” or the use of a negative term in conjunction with the elements of the claim is intended to serve as a basis for reference.

[0036] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any of the described methods may be implemented in the order of the described events or in any other logically possible order.

[0037] Although the systems and methods have been or will be described with functional interpretations for the sake of grammatical fluency, it will be clearly understood that, unless expressly set forth in 35 USC §112, the claims will not be construed as necessary limitations on the construction limited in any way by the terms "means" or "steps," but will instead be given the full scope of meaning and equivalent meaning provided by the claims under the doctrine of judicial equivalence, and, where the claims are expressly set forth in 35 USC §112, will be given the full legal equivalent meaning in 35 USC §112.

[0038] Light interrogation system As discussed above, aspects of the present invention include optical interrogation systems. With respect to "optical interrogation" a system, it means a system configured to irradiate a substance (e.g., a sample) with light in a manner suitable for determining one or more properties of the substance. In some cases, the disclosed optical interrogation system improves the quality of data obtained during the irradiation of the substance compared to comparable prior systems. For example, embodiments of the system may improve the signal-to-noise ratio by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, and including 20% ​​or more. In some cases, the system is more compact and / or space-saving compared to comparable prior systems. For example, in some cases, the subject system constitutes a volume reduction of 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, and including 20% ​​or more, compared to comparable prior systems.

[0039] The optical interrogation system disclosed herein includes a first pinhole plate comprising one or more pinholes. The first pinhole plate is formed by a planar surface having pinholes located therein. The first pinhole plate can be constructed of any suitable material. In some cases, the plate comprises one or more metals, including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and combinations and alloys thereof. In additional embodiments, the first pinhole plate comprises a polymeric material, such as a plastic material. In some cases, the first pinhole plate comprises one or more rigid plastic materials, such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. In some cases, the pinhole plate is made of polyoxymethylene. In some cases, the inner surface of the pinhole may be coated with an anodized black material, for example, to minimize reflected light. The number of pinholes in the first pinhole plate can vary. In some cases, the first pinhole plate comprises a single pinhole. In other cases, the first pinhole plate comprises multiple pinholes. In some cases, the number of pinholes ranges from 2 to 1000, such as 2 to 100, 5 to 50, 10 to 25, and including 14 to 20. In some forms of a first pinhole plate comprising a plurality of pinholes, the pinholes in the plurality may be arranged in an array. The term "pinhole array" refers to a specific arrangement of pinholes organized according to a certain pattern or principle. In some cases, the pinholes in the array are arranged in rows and columns. In other embodiments, the pinholes in the array are arranged in a staggered pattern. In still other embodiments, the pinholes in the array are arranged in a concentric pattern. In still other cases, the pinholes are arranged in an irregular pattern, but remain aligned with the detection chamber to be interrogated.

[0040] The size of the pinholes in the first pinhole plate can vary. In some cases, the diameter of the pinholes ranges from 0.1 mm to 5 mm, such as 0.2 mm to 4 mm, such as 0.3 mm to 3 mm, such as 0.4 mm to 2 mm, and includes 0.5 mm to 1 mm. In some cases, the pinholes have diameters of 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1.0 mm. When the first pinhole plate includes multiple pinholes, adjacent pinholes among these pinholes can be spaced at any suitable distance, wherein the distance is measured between the geometric centers of the pinholes. In some cases, the distance between adjacent pinholes ranges from 0.5 mm to 10 mm, such as 1 mm to 7 mm, such as 1.5 mm to 5 mm, and includes 2 mm to 2.5 mm. In some cases, the distance between adjacent pinholes is 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, or 2.5 mm. In embodiments, the pinholes may be spaced from 2 mm to 2.5 mm, 3 mm to 5 mm, 6 mm to 12 mm, or 13 mm to 25 mm for larger detection chambers or distributed chambers. The height / thickness of the first pinhole plate may also vary. In some cases, the pinhole plate has a height ranging from 1 mm to 50 mm, such as 2 mm to 40 mm, such as 3 mm to 30 mm, such as 4 mm to 25 mm, and including 20 mm to 25 mm. In some cases, the first pinhole plate has a thickness of 20 mm, 21 mm, 22 mm, 23 mm, or 24 mm. In some cases, the pinhole plate has a thickness ranging from 20 mm to 50 mm. In some cases, the pinhole diameter and plate thickness are sufficient to narrow the collimated light with a main ray angle of 1°–3° and minimize stray light and crosstalk signals to adjacent detection chambers.

[0041] Each pinhole in the first pinhole plate is configured for optical alignment with one of a plurality of detection chambers (e.g., micro-cubicles). In other words, the pinholes are sized and positioned within the plate such that the detection chambers used in conjunction with the subject system (e.g., in conjunction with a removable cartridge inserted into the system, as described further below) are optically aligned with the pinholes. "Optical alignment" means that one or more pinholes and detection chambers (when such detection chambers are present in the system) share an optical axis passing through the pinholes toward (or from) their respective detection chambers, as appropriate. For example, the pinholes may be sized relative to the detection chambers in such a way as to reduce the negative effects of stray light and associated optical density (OD) fluctuations. Good practice is to reduce the principal ray angle of the pinholes to avoid light reflection from the structural walls of the detection chambers. Suitable dimensions may include, but are not limited to, those presented above. Additionally, the pinholes may be matched with a specific array of detection chambers (e.g., in a cartridge) such that the first pinhole plate and the detection chambers are arranged according to the same pattern or principle. For example, both the detection chambers and pinholes can be arranged in rows and columns, in staggered patterns, or concentrically.

[0042] With respect to "detection chamber," it means any microfluidic component configured for the analysis of a sample (e.g., optical analysis). Exemplary detection chambers include, but are not limited to, microvolume cuvettes. The number of detection chambers in the plurality can vary. In some cases, the number of detection chambers in the plurality ranges from 2 to 100, such as 5 to 50, such as 10 to 25, and including 14 to 20. In some cases, the cartridge includes 10 or more detection chambers, such as 11 or more, such as 12 or more, such as 13 or more, such as 14 or more, such as 15 or more, such as 16 or more, such as 17 or more, such as 18 or more, such as 19 or more, and including 20 or more detection chambers. The shape and size of the detection chambers in the plurality can vary as needed. In some cases, the detection chambers in the plurality of chambers have an elongated structure (e.g., a length greater than the width). The elongated structure can have any convenient cross-sectional shape, wherein the cross-sectional shape of interest includes, but is not limited to, straight cross-sectional shapes (e.g., square, rectangle, trapezoid, triangle, hexagon, etc.), curved cross-sectional shapes (e.g., circular, elliptical), and irregular shapes (e.g., the bottom portion of a parabola coupled to the top portion of a plane). In some cases, the detection chambers in the plurality of chambers have a circular cross-section. In other embodiments, the detection chambers in the plurality of chambers have a square cross-section. In still other embodiments, the detection chambers in the plurality of chambers have a rectangular cross-section. In still other embodiments, the detection chambers in the plurality of chambers have an inverted conical cross-section. The volume of the detection chambers can also vary. In some cases, the detection chambers in the plurality of chambers have volumes ranging from 0.3 µl to 500 µl, such as 2 µl to 300 µl, such as 3 µl to 200 µl, such as 4 µl to 100 µl, and including 5 µl to 10 µl. In some configurations, the detection chambers in the plurality of chambers have a volume ranging from 0.3 µl to 50 µl. In some cases, the detection chambers in the plurality of chambers have a volume of 5 µl or greater, such as 6 µl or greater, such as 7 µl or greater, such as 8 µl or greater, such as 9 µl or greater, and including 10 µl or greater. In some cases, the detection chambers in the plurality of chambers have a diameter ranging from 0.1 mm to 20 mm, such as 0.5 mm to 15 mm, such as 1 mm to 10 mm, and including 1.5 mm to 2 mm. In some cases, the detection chambers in the plurality of chambers have a diameter of 1.5 mm or greater, such as 1.6 mm or greater, such as 1.7 mm or greater, such as 1.8 mm or greater, such as 1.9 mm or greater, and including 2 mm or greater.The adjacent detection chambers in the plurality may be spaced between 1 mm and 10 mm, such as 2 mm to 8 mm and including 4 mm to 5 mm. In some cases, the space between the detection chambers is sufficient to ensure that each detection chamber can be interrogated by the beam from the illuminator without interfering with its neighboring detection chambers. The detection chambers in the plurality may be arranged in any suitable pattern. In some cases, the array of detection chambers is arranged in an alternating pattern. In other cases, the array of detection chambers is arranged in a concentric pattern. The detection chambers in the subject box may be constructed of any suitable material. In some cases, the detection chambers are made of a polymer material, for example, transparent within the detection band. In some cases, the detection chambers in the plurality are made of polystyrene (PS), PMMA, CoC, or CoP. In some cases, the detection chambers are components of the subject optical interrogation system. In other embodiments, they are components of a removable box for use within the system.

[0043] The detection chamber may include: an inlet for receiving diluted sample fluid; and an outlet at which air and / or excess diluted sample fluid may escape when the detection chamber is filled. In some embodiments where the detection chambers of the plurality are formed of an elongated structure, the detection chamber includes an inlet located at a proximal end of the elongated structure and an outlet located at a distal end of the elongated structure. In some cases, the chamber is configured such that the detection chamber is arranged upright (i.e., vertically). In some such embodiments, the inlet may be arranged at the bottom such that the detection chamber is filled with diluted sample fluid from the bottom and rises via capillary action. In some cases, this arrangement is sufficient to minimize the formation of air bubbles when the sample fluid fills the detection chamber. In some alternative embodiments, the detection chamber is filled from top to bottom. In some cases, the detection chamber is light-accessible at certain windows. In some such cases, each of the plurality of detection chambers includes a first light-accessible window configured to allow light to enter and a second light-accessible window configured to allow light from a light source to exit. The rest of the detection chamber may or may not be light-accessible. In selected models, the detection chamber is opaque except for the first and second light-accessible windows.

[0044] The subject system additionally includes a light source configured to illuminate each of the plurality of detection chambers through a pinhole in a first pinhole plate, the pinhole being optically aligned with the detection chamber. In some cases, the system includes multiple light sources. In some such cases, the multiple light sources are positioned in an array. The spectra of one or more light sources may lie in any predetermined region of the electromagnetic spectrum detectable using a photosensitive array, whether or not they have been specially processed to extend the effective wavelength range detectable by such an array. In some embodiments, the predetermined wavelength or band is in the infrared spectrum. In some embodiments, the predetermined wavelength or band is in the ultraviolet spectrum. In some embodiments, the predetermined wavelength or band is in the visible spectrum.

[0045] In some cases, the light source consists of one or more light-emitting diodes (LEDs). In other cases, the light source consists of an LED array. The number of LEDs in the array can vary. The light source can consist of one or more LED emitters, including but not limited to, for example, two or more LED emitters, three or more LED emitters, four or more LED emitters, one LED emitter, two LED emitters, three LED emitters, four LED emitters, etc.

[0046] In some cases, the number of LEDs ranges from 4 to 20, such as 5 to 15, and includes 6 to 8. In some cases, a lighting component containing four LED emitters may contain two pairs of identical LEDs or a pair of LEDs with a first wavelength and a second pair of LEDs with a second wavelength. In cases where multiple LED emitters are used, any useful arrangement of the LED emitters can be found in the light source, including but not limited to, linear arrangements, staggered arrangements, array-like (e.g., "checkerboard") arrangements, etc. The useful LED emitters disclosed herein will vary, for example, based on the specific measurements to be performed by the system, the optical, electrical, or physical constraints of the system, etc.

[0047] LED emitters may include, but are not limited to, LED emitters having a peak minimum wavelength (λ) in nanometers (nm) between 340 and 750 nm, including but not limited to, those between 340 and 450, between 340 and 400, between 400 and 450, between 450 and 550, between 450 and 500, between 500 and 550, between 550 and 650, between 550 and 600, between 600 and 650, between 650 and 750, between 650 and 700, between 700 and 750, approximately 400 nm, approximately 580 nm, approximately 470 nm, approximately 628 nm, approximately 528 nm, approximately 674 nm, and so on.

[0048] In some cases, the light source contains two LED emitters of different wavelengths, wherein the distance between the different wavelengths will vary and can range from 5 nm to 300 nm or greater, including but not limited to, for example, distances of at least 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, and 160 nm. nm, separated by at least 165 nm, separated by at least 170 nm, separated by at least 175 nm, separated by at least 180 nm, separated by at least 185 nm, separated by at least 190 nm, separated by at least 195 nm, separated by at least 200 nm, separated by no more than 300 nm, separated by no more than 290 nm, separated by no more than 280 nm, separated by no more than 270 nm, separated by no more than 260 nm, separated by no more than 250 nm, separated by no more than 240 nm, separated by no more than 230 nm, separated by no more than 220 nm, separated by no more than 210 nm, separated by no more than 200 nm, separated by no more than 190 nm, separated by no more than 180 nm, separated by no more than 170 nm, separated by no more than 160 nm, separated by no more than 150 nm, separated by no more than 140 nm, separated by no more than 130 nm, separated by no more than 120 nm, separated by no more than 110 nm, separated by no more than 100 nm, etc.

[0049] In some cases, the disclosed LED emitters may be switchable (i.e., able to be turned on and off, including repeated on / off switching). In some cases, the wiring circuitry of a light source with two or more LED emitters is configured such that, or the programming controlling such a light source, is configured such that only one LED emitter can be switched on at a time. In this case, when the first LED emitter of the optic block is switched on, the second LED emitter of the optic block is switched off, and vice versa. In some cases, the switching of the LED emitters of the optic block includes a period of time in which neither of the two LED emitters of the optic block is switched on. In some cases, this period of time in which neither of the two LED emitters of the optic block is switched on occurs between switching the first emitter off and switching the second emitter on. In some cases, the LED's emission power can be adjusted from 0% to 100% via PWM or by adjusting the drive current.

[0050] In some cases, one or more LEDs in the array are configured to emit light at wavelengths ranging from 400 nm to 410 nm. In some cases, one or more LEDs in the array are configured to emit light at wavelengths ranging from 460 nm to 470 nm. In some cases, one or more LEDs in the array are configured to emit light at wavelengths ranging from 600 nm to 610 nm. In some cases, one or more LEDs in the array are configured to emit light at wavelengths ranging from 850 nm to 860 nm. In some cases, the LED array includes LEDs configured to emit light at 405 nm, 467 nm, 550 nm, 600 nm, and 850 nm. Each LED is turned on independently or simultaneously to illuminate the correct wavelength for each measurement. In some cases, the LEDs are side-lit micro light-emitting diodes (LEDs).

[0051] In some cases, the light source consists of a light guide plate and multiple multi-wavelength LEDs configured to emit light of different wavelengths (e.g., wavelengths discussed above). LGP illuminators allow for multiple spectra without the need for optical lenses, reducing the size and complexity of the light interrogation system. In some cases, the LEDs are side-lit micro-LEDs. For example, a system may include a side-lit LED illuminator consisting of a light guide plate (LGP) and multiple multi-wavelength LEDs. Commercially available light guide plates include, but are not limited to, those manufactured by Hexatron Technologies and Consun Technology Co. In some embodiments, the LGP includes a reflective film, for example, for incident light. Exemplary reflective films may be made of polyethylene terephthalate. In some cases, the LGP is engraved with reflective patterns (such as dotted or V-grooves) to reflect / refract the light beam toward a first pinhole plate. The LGP may be made of any suitable material, including but not limited to cyclic olefin polymers (CoP), cyclic olefin copolymers (CoC), poly(methyl methacrylate) (PMMA), and polystyrene (PS). In some cases, the LGP is made of cast-grade PMMA. In other cases, the LGP is composed of low melt flow rate (MFR) PMMA. In some cases, the light source includes a collimator (e.g., to provide uniform illumination). In embodiments, the main collimator is arranged in a honeycomb pattern. In some cases, the collimator also includes a film for use as a diffuser. Exemplary films include, but are not limited to, Hexatron Technologies' Opto90 PET225 film. Additional diffusers that may be used include, for example, Hexatron Technologies' Opto90 Frost diffuser, etc. Another type of low autofluorescence reflector film that may be used includes, for example, Consun Technology's 0.3 mm PET reflector film.

[0052] In embodiments where the light source includes an LGP, the light source may include one or more prisms (e.g., two prisms). Possible prisms include, but are not limited to, K9 prisms. Prisms may have widths ranging from 5 mm to 20 mm in some cases, such as 7 mm to 15 mm, or 8 mm to 12 mm. In some cases, the prism has a width of 10 mm. In some embodiments, the prism(s) may be positioned at a distance ranging from 10 mm to 50 mm from the LGP, such as 20 mm to 40 mm, 25 mm to 35 mm, and including 29 mm to 31 mm. Where employed, the prism can be used to direct light to the LGP. In some embodiments (e.g., where it is desirable to generate more uniform illumination power from the LGP), the light source includes a reflective cavity defined by internal reflectors (e.g., a top internal reflector and a bottom internal reflector), which may be made of any suitable reflective material. In some cases, the internal reflectors may be spaced from 1 mm to 20 mm apart, such as 2 mm to 10 mm, 3 mm to 7 mm, and including 4 mm to 6 mm. In some embodiments, the internal reflectors have a thickness ranging from 1 mm to 10 mm, such as 2 mm to 6 mm, and including 3 mm to 5 mm. Light is emitted (e.g., by an LED) into the reflective cavity, and the reflector confines the light within the cavity, after which the light is reflected to the LGP (e.g., by prism reflection). In some cases, the light source comprises multiple sets of LEDs, such as LEDs on both the top and bottom surfaces of the internal reflector, such that light is emitted from these LEDs into the reflective cavity and guided to the LGP by the prism.

[0053] The size of the light source can vary. In some cases, the light source containing the LGP is characterized by an LED angle ranging from 20° to 80°, such as 40° to 70°, such as 41° to 50°, such as 42° to 47°, and including 44° to 46°. In embodiments, the LGP has a rectangular or square surface area with one or more sides having a length ranging from 20 mm to 80 mm, such as 30 mm to 70 mm, such as 40 mm to 60 mm, such as 45 mm to 55 mm, and including 49 mm to 51 mm. In selected cases, the LGP has a size of 50 mm x 50 mm. In some cases, the LGP includes a non-functional boundary surrounding the functional area of ​​the LGP. The width of the boundary can range from 1 mm to 10 mm, such as 2 mm to 8 mm, such as 3 mm to 5 mm. In some embodiments, the light source containing the LGP has a range from 0.01 mw / mm². 2 Up to 10 mw / mm 2The radiant power is measured and dimming control is provided for each LED. In some cases, assuming an ambient temperature change of <5 °C, a light source with an LGP exhibits a power change of less than 0.01% over 10 minutes.

[0054] Figure 1A-1B A light source including an LGP is depicted according to certain embodiments of the present invention. Figure 1A A side view of a light source 100 is shown, which includes a reflective cavity 101 defined by an internal reflector 102. It also includes a top LED 103a and a bottom LED 103b, mounted using mounting plates 104a and 104b, respectively. Light emitted by LEDs 103a and 103b is guided to an LGP 106 via prisms 105a and 105b. The LGP 106 then projects light in a direction perpendicular to its surface. Figure 1B A top view of light source 100 and its dimensions are presented. (See attached image.) Figure 1B As shown, prisms 105a and 105b guide light to LGP 106 at an LED angle θ. The boundary of LGP 106 is defined by distance d1. The effective width of LGP 106 is defined by distance d2. The distance between LGP 106 and prisms 105a and 105b is defined by distance d3. The width of prisms 105a and 105b is defined by distance d4.

[0055] Figure 2A-2B Alternative depictions of light sources including LGPs according to certain embodiments of the invention are presented. For example... Figure 2A-2B As shown, the light source 200 includes a reflecting cavity 201, mounting plates 204a and 204b, prisms 205a and 205b, and an LGP 206. These components are as described above regarding... Figure 1A-1B The arrangement is as described. Screws 207 for mounting the light source 200 (e.g., to the first pinhole plate) are also shown.

[0056] In some cases, the light source is a laser source. In embodiments, the laser can be any convenient laser, such as a continuous-wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the flow cytometer includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In other cases, lasers of interest include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof. In still other cases, the subject flow cytometer includes solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, and combinations thereof. Alternatively, the light source is or includes an arc lamp. In some cases, the light source is a white light lamp, such as a xenon lamp.

[0057] According to some embodiments, the light source may also include one or more optical adjustment components. In some embodiments, the optical adjustment components are located between the light source and the detection chamber and may include any means capable of changing the spatial width of illumination from the light source or some other characteristic of the illumination (such as, for example, illumination direction, wavelength, beam width, beam intensity, and focus). The optical adjustment protocol may include any convenient means of adjusting one or more characteristics of the light source, including but not limited to lenses, mirrors, filters, fiber optics, wavelength splitters, pinholes, slits, collimation protocols, and combinations thereof. In some embodiments, the system of interest includes one or more focusing lenses. In one example, the focusing lens may be a reducing lens. In other embodiments, the system of interest includes fiber optics. In selected cases, the system includes a tapered mixing bar configured to integrate incident light from a light source (e.g., an LED array) and generate a uniform output (total internal reflection) in a smaller area. In some cases, the system of the present invention includes an optional diffuser configured to improve light uniformity. In some embodiments, the system includes a folded mirror. In other embodiments, the system includes a folded prism.

[0058] In some embodiments, a light source is optically coupled to an array of optical fibers. The term "optical fiber array" refers to a plurality of optical fibers arranged according to a certain principle or pattern such that the fibers of the array can be optically coupled to pinholes in a first pinhole plate. In embodiments, each fiber in the optical fiber array is optically aligned with a different pinhole in the first pinhole plate. The optical fibers may be constructed of an elongated structure having a near end and a far end, wherein the elongated structure is made of a light-transparent material configured to transmit light from the near end to a pinhole in the first pinhole plate. In some forms, the optical fibers of the optical fiber array are bundled together at their near ends. This bundle of optical fibers can then be optically coupled (e.g., via a suitable connector, such as an SMA connector) to a light source, such as a suitable light source described above. In some embodiments, the transparent material includes glass materials, such as, but not limited to, silica (e.g., fused silica). In other embodiments, the transparent material includes polymer materials. In such embodiments, the transparent material may include one or more materials, such as, but not limited to, poly(methyl methacrylate) (PMMA), polystyrene, and poly(perfluorobutylene vinyl ether) (CYTOP). The number of optical fibers in the fiber array can vary and, in some cases, matches the number of pinholes in the first pinhole plate. For example, the number of optical fibers can range from 2 to 1000, such as 2 to 100, 5 to 50, 10 to 25, and 14 to 20. Each optical fiber may include a coating / cladding and an optically transparent core. In embodiments, the diameter of the core ranges from 0.1 mm to 2 mm, such as 0.2 mm to 1 mm, and includes 0.3 mm to 0.5 mm. In embodiments, the diameter of the coating ranges from 0.1 mm to 2 mm, such as 0.2 mm to 1.5 mm, such as 0.4 mm to 1 mm, and includes 0.5 mm to 0.7 mm.

[0059] In some embodiments, the optical fibers of the array comprise or are constituted by optical tubes. The optical tube may include an inner cavity and a coating to increase the amount of reflection of light waves traveling within it. In other embodiments, no coating is included. The optical tube may be made of any suitable material. In some embodiments, the optical tube is made of a thermoplastic polymer. In some embodiments, the thermoplastic polymer is polycarbonate. In some cases, optically transparent materials such as PMMA, CoC, Cop, or glass may be used to mold the optical tube. In this embodiment, the number of optical fibers in the fiber array may vary and, in some cases, match the number of pinholes in the first pinhole plate. For example, the number of optical fibers may range from 2 to 1000, such as 2 to 100, such as 5 to 50, such as 10 to 25, and including 14 to 20. In embodiments, each optical tube is constituted by an unclad, optically transparent core. In embodiments, the core may have various diameters to match the pinholes, ranging from, for example, 0.1 mm to 5 mm, such as 0.2 mm to 1 mm, and including 0.3 mm to 0.5 mm. In some embodiments, the cross-section of the light tube is circular or square.

[0060] In some embodiments where the light source is optically coupled to the fiber optic array, the system of the present invention may additionally include a fiber optic array plate having holes for mounting the fiber optic array. The fiber optic array plate may be constructed of any suitable material. In some cases, the fiber optic array plate comprises one or more metals, such as aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and combinations and alloys thereof. In additional embodiments, the fiber optic array plate comprises a polymeric material, such as a plastic material. In some cases, the fiber optic array plate comprises one or more rigid plastic materials, such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. In some cases, the holes of the fiber optic array plate match the pinholes of a first pinhole plate. In other words, the holes of the fiber optic array plate are arranged according to the same principle or pattern as the first pinhole plate. In some cases, the distance between adjacent holes of the fiber optic array plate ranges from 0.5 mm to 10 mm, such as 1 mm to 7 mm, such as 1.5 mm to 5 mm, and includes 2 mm to 3 mm. In some cases, adjacent holes are spaced 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, or 2.5 mm apart. In some cases, the fiber optic array panel has a thickness ranging from 1 mm to 100 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 20 mm, such as 5 mm to 15 mm, and including 9 mm to 11 mm. In some cases, the holes in the fiber optic array panel have diameters ranging from 0.1 mm to 5 mm, such as 0.2 mm to 2 mm, such as 0.3 mm to 1 mm, such as 0.4 mm to 0.8 mm, and including 0.5 mm to 0.7 mm.

[0061] In some embodiments, the system further includes a microlens array. The microlens array comprises a plurality of microlenses (i.e., microlenses) arranged in an array, the microlenses being adapted to focus light emitted from the optical fiber onto a detection chamber. In some cases, the microlenses in the array are configured to be optically aligned with the aperture of the fiber array plate. In other words, the microlenses in the array are arranged according to the same principles or patterns as the fiber array plate and the derived first pinhole plate. The microlenses in the array have any suitable diameter. Diameters of interest range from 0.5 mm to 5 mm, such as 1 mm to 2 mm, such as 1.2 mm to 1.8 mm, and including 1.3 mm to 1.5 mm. In some embodiments, the microlenses in the array are arranged within a microlens plate, for example configured to hold the microlenses in a specific configuration. The microlens plate can be constructed from any suitable material. In some cases, the microlens plate comprises one or more metals, such as aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and combinations and alloys thereof. In additional embodiments, the microlens plate comprises a polymeric material, such as a plastic material. In some cases, the fiber optic array panel comprises one or more rigid plastic materials, such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. In some cases, the thickness of the microlens plate ranges from 0.5 mm to 10 mm, such as 0.9 mm to 1.1 mm. In some embodiments, the microlens plate has a thickness of 1 mm.

[0062] Figures 3A-3B A light source optically coupled to a fiber optic array according to certain embodiments is depicted. For example... Figure 3A As shown, optical fiber 301 consists of fibers with a diameter ΦD i The optical fiber core 301a is composed of optical fiber cores with a diameter ΦD O The coating 301b is used to characterize the area. Each fiber optic is aligned to a hole within a fiber array plate 302 having a width W and a length L. Each hole in the fiber array plate is characterized by a diameter ΦD. Adjacent holes in the fiber array plate 302 are spaced apart by a distance PE_x along the x-direction and by a distance PE_y along the y-direction; these distances may be the same or different. A small lens array 303 is also shown. Figure 3B The array of small lenses is shown in more detail below. Figure 3B As shown, the microlens array 303 consists of microlens plates with diameters ΦD. L It is composed of a small lens (i.e., a microlens) 304. Figure 3A It also demonstrates how the near end of fiber 301 is bundled into fiber bundle 305, which itself is optically coupled to a suitable light source 306.

[0063] The optical interrogation system of the present invention also includes a second pinhole plate. The second pinhole plate of interest includes one or more pinholes, each pinhole optically aligned with a pinhole of a first pinhole plate and configured for optical alignment with one of the plurality of detection chambers. The second pinhole plate is formed by a planar surface having pinholes located therein. The second pinhole plate can be constructed of any suitable material. In some cases, the plate includes one or more metals, including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (e.g., stainless steel), copper, tin, and combinations and alloys thereof. In additional embodiments, the second pinhole plate includes a polymeric material, such as a plastic material. In some cases, the second pinhole plate includes one or more rigid plastic materials, such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. In some cases, the pinhole plate is made of polyoxymethylene. In some cases, the inner surface of the pinhole may be coated with an anodized black material, for example, to minimize reflected light. The number of pinholes in the second pinhole plate can vary. In some cases, the second pinhole plate includes a single pinhole. In other cases, the second pinhole plate includes a plurality of pinholes. In some cases, the number of pinholes ranges from 2 to 1000, such as 2 to 100, 2 to 50, 10 to 25, and including 14 to 20. In some cases, the number of pinholes in both the first and second pinhole plates ranges from 2 to 50. In some forms of the second pinhole plate including a plurality of pinholes, the pinholes may be arranged in an array according to the same principle or pattern as the first pinhole plate. In some cases, the pinholes in the array are arranged in rows and columns. In other embodiments, the pinholes in the array are arranged in a staggered pattern. In still other embodiments, the pinholes in the array are arranged in a concentric pattern. In still other cases, the pinholes are arranged in an irregular pattern, but remain aligned with the detection chamber to be interrogated.

[0064] The size of the pinholes in the second pinhole plate can vary. In some cases, the diameter of the pinholes ranges from 0.1 mm to 5 mm, such as 0.2 mm to 4 mm, such as 0.3 mm to 3 mm, such as 0.4 mm to 2 mm, and includes 0.5 mm to 1 mm. In some cases, the pinholes have diameters of 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1.0 mm. When the second pinhole plate includes multiple pinholes, adjacent pinholes can be spaced at any suitable distance, wherein the distance is measured between the geometric centers of the pinholes. In some cases, the distance between adjacent pinholes ranges from 0.5 mm to 10 mm, such as 1 mm to 7 mm, such as 1.5 mm to 5 mm, and includes 2 mm to 2.5 mm. In some cases, the distance between adjacent pinholes is 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, or 2.5 mm. In embodiments, the pinholes may be spaced from 2 mm to 2.5 mm, 3 mm to 5 mm, 6 mm to 12 mm, and, for example, 13 mm to 25 mm for larger detection chambers or distributed chambers. In selected cases, adjacent pinholes of the first and second pinhole plates are spaced from 2 mm to 2.5 mm apart. The height / thickness of the second pinhole plate can also vary. In some cases, the pinhole plate has a height ranging from 1 mm to 50 mm, such as 2 mm to 40 mm, such as 3 mm to 30 mm, such as 4 mm to 25 mm, and including 20 mm to 25 mm. In some cases, the second pinhole plate has a thickness of 20 mm, 21 mm, 22 mm, 23 mm, or 24 mm. In some cases, the pinhole plate has a thickness ranging from 20 mm to 50 mm. In some cases, the second pinhole plate of the present invention includes a light trap pocket configured to prevent stray light emitted from a light source from being collected by the optical sensor. In this configuration, the light-trapping recess may be formed by a void within the interior of a second pinhole plate, which is configured to absorb stray light. In some cases, the light-trapping recess is coated with an anodized black material to avoid internal reflections. In some cases, the pinhole diameter and plate thickness are sufficient to narrow the collimated light with a principal ray angle of 1°–3° and minimize stray light and crosstalk signals to adjacent detection chambers. The first and second pinhole plates may be the same or different in size. In some cases, the pinholes in the first pinhole plate have a larger diameter than those in the second pinhole plate. In other cases, the pinholes in the second pinhole plate have a larger diameter than those in the first pinhole plate. In some embodiments, the pinhole image forms a “super photodiode” to measure the light intensity of all cuvettes.The sum of the pixels in the pinhole image represents the transmitted light power of each cuvette and is used in absorbance calculations.

[0065] The distance separating the first and second pinhole plates can vary, for example, depending on the size of the cartridge / detection chamber used. In some embodiments, the distance between the first and second pinhole plates ranges from 2 mm to 100 mm, such as 3 mm to 75 mm, such as 4 mm to 50 mm, and includes 5 mm to 15 mm. The pinholes of the subject second pinhole plate are each configured for optical alignment with one of a plurality of detection chambers (e.g., a micro-cube). In other words, the pinholes are sized and positioned such that the detection chamber used in conjunction with the subject system (e.g., in conjunction with a removable cartridge inserted into the system, as described further below) is optically aligned with the pinhole. For example, the pinholes can be sized relative to the detection chamber in such a way as to reduce the negative effects of stray light and associated optical density (OD) fluctuations. Suitable dimensions may include, but are not limited to, those presented above. Following common optical engineering practices and using light-tracing simulations, the diameters of the first and second pinholes, as well as the principal ray angle, can be tuned to avoid stray light reflections from the structured walls of the detection chamber. In some cases, the first and second pinhole plates are components of the box received by the system of the present invention rather than the system itself.

[0066] As discussed above, embodiments of the present invention include a first pinhole plate and a second pinhole plate, each pinhole plate having a single pinhole. In some such embodiments, the system includes a mechanical positioning device configured to adjust the position of the cartridge relative to the first and second pinhole plates such that each detection chamber of the cartridge can be illuminated at different times. Any convenient device (e.g., a motor) can be used in the mechanical positioning device to facilitate this movement of the cartridge.

[0067] The disclosed aspects of the optical interrogation system also include optical sensors configured to collect light from each of the plurality of detection chambers through pinholes in a second pinhole plate optically aligned with the detection chamber. The optical sensors of the optical interrogation system can vary. Sensors of interest may include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-mode charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors, or combinations thereof, and other detectors. In some embodiments, the collected light is measured using a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor.

[0068] In some cases, optical sensors are multidimensional arrays of pixels that form part of a high-resolution photosensitive array. As used herein, the term "high-resolution" refers to a resolution equal to or greater than that of a standard lens-based optical microscope. The resolution of a standard lens-based optical microscope is defined as the shortest distance between two points on a specimen that an observer or camera system can still distinguish as separate entities. Pixels per inch (PPI) or pixels per centimeter (PPCM) are measurements of the pixel density of an optical sensor. The resolution of an optical sensor is the number of pixels that contribute to the final image and is typically measured in megapixels (meaning millions of pixels). For example, a photosensitive array comprising 1280 x 720 pixels has 921,600 pixels, or less than 1 million pixels, and a photosensitive array comprising 1920 x 1080 pixels has 2,073,600 pixels, or approximately 2.1 million pixels, with a resolution of [missing information].

[0069] Microfabrication techniques (e.g., photolithography and plasma deposition) can be used to construct multilayer sensor structures in confined spaces. CCDs offer advantages for contact optical microscopy applications, including the ability to detect light on exposed surfaces. Various CCD configurations can be used: a full-frame architecture can be used to maximize the image-available portion of the chip, but requires an external shutter to prevent image ghosting during readout; while a frame-transfer architecture avoids image ghosting, but requires a masked, non-photosensitive area of ​​the parallel register, approximately the same size as the photosensitive area of ​​the parallel register, resulting in an imaging integrated circuit having approximately half the photosensitive area of ​​the full-frame architecture. Due to the small area of ​​individual pixels in the array used according to the various aspects discussed herein, the amount of charge collected in each pixel will be small under many imaging conditions; however, because the specimen is in contact or nearly in contact with the pixel, the effective acceptance angle of the pixel for photons emitted from the specimen is greater than the effective acceptance angle achieved by lenses in conventional microscopy.

[0070] CMOS devices offer alternative advantages for these applications, including lower manufacturing costs, signal processing via electronics embedded in individual pixels, and the ability to read out independently addressed pixel values ​​without sequential transfer. In some CMOS embodiments, thinned back-illuminated arrays are used; while previously requiring expensive and complex fabrication methods, these arrays can be fabricated inexpensively using bonding wafer processes, such as those using a silicon-on-insulator substrate and a buried oxide layer as etch stops to produce a uniformly thinned, optimally thinned light-absorbing back layer (see U.S. Patent No. 7,425,460, which is incorporated herein by reference as an example). Light entering conventional (front-illuminated) imaging integrated circuits typically passes through capping layers that scatter the light and whose metal circuitry blocks the underlying photosensitive layer; in back-illuminated imaging integrated circuits, the photosensitive layer is closer to the surface, above the metal circuitry carrier layer, resulting in generally less light blocking (a larger "fill factor") and thus higher effective quantum efficiency.

[0071] Multi-dimensional pixel arrays can be optical sensors or photodetectors formed from semiconductor materials used in very large-scale or larger integrated circuits. A defining property of semiconductor materials is that they can be doped with impurities that controllably alter their electronic properties; in some embodiments, the array is formed substantially of a crystalline inorganic solid (such as silicon); and in other embodiments, the array is formed substantially of a compound semiconductor composed of at least two different kinds of elements. Compound semiconductors can be composed of elements from Groups 13-15 (conventional Groups III-V), such as elements from Group 13 (conventional Group III, boron, aluminum, gallium, indium) and Group 15 (conventional Group V, nitrogen, phosphorus, arsenic, antimony, bismuth). Possible chemical formulas for compound semiconductors can range from binary (two elements, such as gallium (III) arsenide (GaAs)), ternary (three elements, such as indium gallium arsenide (InGaAs)), and quaternary (four elements, such as aluminum gallium indium phosphide (AllnGaP)) alloys. In some embodiments, the pixel array is a light sensor or photodetector, such as one or more PDs, for example, a silicon photodiode (PD) having one or more undoped intrinsic semiconductor regions sandwiched between p-type and n-type semiconductor regions. Alternatively, other light sensors or detectors, with or without filters for controlling wavelength, may be used without departing from the spirit and scope of the invention. The spectral response of the multidimensional pixel array can range from 300 nm to 1000 nm. This provides the ability to cover a wide spectrum of LED wavelengths.

[0072] Signals from optical sensors provide information for each pixel of an image, including or deducible from intensity, wavelength, and optical density. Intensity values ​​can be assigned on any scale, for example, from 0 units to 4095 units (“IVlJ”). Optical density (“OD”) is a measure of the amount of light absorbed relative to the amount of light transmitted through a medium; for example, a higher “OD” value indicates a greater amount of light absorbed during transmission. OD can be quantitatively described using optical density units (“OD”) or fractions thereof; for example, MilliOD is one-thousandth of OD. One “OD” unit represents a 90% reduction in light intensity. “OD” or “MilliOD” as quantitative values ​​can be used for images acquired or derived from transmitted light (e.g., transmitted blue light).

[0073] In some embodiments, information from the optical sensor is split into multiple channels, such as three channels, which provides particular utility for determining a four-part LDC. However, the invention is not limited to the three-channel embodiment. The first of the three channels can be directed toward information relating to light emitted from the sample at a first wavelength (e.g., 540 nm, which appears green). The second channel can be directed toward information relating to light emitted from the sample at a second wavelength (e.g., 660 nm, which appears red). The third channel can be directed toward information relating to light passing through the sample at a third wavelength (e.g., 413 nm, which is used to determine the blue optical density "OD"). These wavelength values ​​and the number of channels have particular utility when performing LDC on whole blood samples. However, the invention is not limited to these specific wavelengths or number of channels. Additional channels can be implemented to collect information and / or transmittance values ​​at different wavelengths. This information can then be used to assess other components within the sample and / or improve the accuracy of the analysis. For example, in applications where it is desirable to further distinguish basophils within the sample, a fourth and fifth channel can be added. The fourth channel can be directed toward information relating to light passing through the sample at a fourth wavelength (e.g., 540 nm), used to determine the green OD, and the fifth channel can be directed toward information relating to light passing through the sample at a fifth wavelength (e.g., 660 nm), used to determine the red OD. These OD values ​​can further be used to identify basophils.

[0074] In some cases, the optical sensor is an optical spectrometer configured to measure properties on one or more portions of the electromagnetic spectrum. In other cases, the optical spectrometer is a miniaturized optical spectrometer. Such miniaturized optical spectrometers are described, for example, in U.S. Patent Application Publication No. 2017 / 0010154.

[0075] In some cases, the system includes one or more optical filters configured to allow the collection of light at certain wavelengths. Various optical filters may be employed depending on the requirements for analyzing a particular analyte. In some cases, one or more bandpass filters are included. In some cases, one or more low-pass filters are included. In still other cases, one or more high-pass filters are included. In still other cases, the one or more optical filters constitute some combination of bandpass, low-pass, and high-pass filters. In some cases, the one or more spectral filters are configured to remove autofluorescence from the sample and consumable (i.e., cartridge) plastic.

[0076] The optical filters disclosed herein include, but are not limited to, illumination filters having a center wavelength (CWL) between 350 nm and 750 nm, including, but not limited to, those between 350 and 450, between 350 and 400, between 400 and 450, between 450 and 550, between 450 and 500, between 500 and 550, between 550 and 650, between 550 and 600, between 600 and 650, between 650 and 750, between 650 and 700, between 700 and 750, approximately 409 nm, approximately 583 nm, approximately 475 nm, approximately 638 nm, approximately 535 nm, approximately 690 nm, and so on.

[0077] The illumination filters disclosed herein also include, but are not limited to, illumination filters having a full width at half maximum (FWHM) ranging from 5 nm to 100 nm, for example, approximately 5 nm to 10 nm, approximately 10 nm to 15 nm, approximately 15 nm to 20 nm, approximately 20 nm to 25 nm, approximately 25 nm to 30 nm, approximately 30 nm to 35 nm, approximately 35 nm to 40 nm, approximately 40 nm to 45 nm, approximately 45 nm to 50 nm, approximately 50 nm to 55 nm, approximately 55 nm to 60 nm, approximately 60 nm to 65 nm, approximately 65 nm to 70 nm, approximately 70 nm to 75 nm, approximately 75 nm to 80 nm, approximately 80 nm to 85 nm, approximately 85 nm to 90 nm, approximately 90 nm to 95 nm, approximately 95 nm to 100 nm, approximately 10 nm to 90 nm, approximately 10 nm to 80 nm, approximately 10 nm to 70 nm, approximately 10 nm to 70 nm, approximately 10 nm to 100 nm, approximately 10 nm to 90 nm, approximately 10 nm to 8 ... nm to 60 nm, approximately 10 nm to 50 nm, approximately 10 nm to 40 nm, approximately 10 nm to 30 nm, approximately 10 nm to 20 nm, approximately 20 nm to 90 nm, approximately 30 nm to 90 nm, approximately 40 nm to 90 nm, approximately 50 nm to 90 nm, approximately 60 nm to 90 nm, approximately 70 nm to 90 nm, approximately 80 nm to 90 nm, 65 nm, 22 nm, 36 nm, 24 nm, 18 nm, 25 nm, etc.

[0078] In some cases, illumination filters as described herein may be characterized by having a specific combination of CWL and FWHM, including, for example, the combinations of CWL and FWHM described above. For example, in some cases, the illumination filters disclosed herein may be characterized as having a 409 nm CWL and 65 nm FWHM, a 583 nm CWL and 22 nm FWHM, a 475 nm CWL and 36 nm FWHM, a 638 nm CWL and 24 nm FWHM, a 535 nm CWL and 18 nm FWHM, a 690 nm CWL and 25 nm FWHM, and so on.

[0079] In some cases, multi-band filters are positioned in front of the second pinhole plate to narrow the wavelength of the incident light and reject any fluorescence signal from the fluid sample or spontaneous fluorescence emission from the detection chamber, thereby minimizing polychromatic radiation from the incident light source (e.g., an LED). In some embodiments, this may be sufficient to improve the linearity of high absorbance to, for example, 3OD. Alternatively, single-band filters are positioned in front of each pinhole, which gives each detection chamber a signal band that matches its characteristic absorption peak. For example, a 340 nm bandpass filter with a 10 nm FWHM can effectively measure NADH absorbance from the response to 3OD and reject fluorescence emission radiation at 470 nm.

[0080] In some cases, the system includes a notch filter configured to filter out stray light from the detection chamber. As understood in the art, a notch filter weakens signals within a short frequency range while allowing signals of all other frequencies to pass through unaffected. In some cases, the frequency range corresponds to the frequency of light emitted by a light source, for example, to prevent such light from becoming incident on the optical sensor. Where employed, the notch filter may be located between a first pinhole plate and a second pinhole plate, such as between the detection chamber and the second pinhole plate. Alternatively, a notch filter may be used between the second pinhole plate and the optical sensor.

[0081] In some embodiments, the optical interrogation system includes an array of optical fibers optically coupled to one or more optical sensors. In embodiments, each fiber in the fiber array is optically aligned with a different pinhole in a second pinhole plate (or a single pinhole plate in the embodiments described below). The optical fibers may be constructed of an elongated structure having a near end and a far end, wherein the elongated structure is made of an optically transparent material configured to transmit light from the near end to the one or more optical sensors. In some forms, the optical fibers of the fiber array are bundled together at their far ends. The fiber bundle can then be optically coupled (e.g., via a suitable connector, such as an SMA connector) to an optical sensor, such as those described above. In some embodiments, the transparent material includes a glass material, such as, but not limited to, silica (e.g., fused silica). In other embodiments, the transparent material includes a polymer material. In such embodiments, the transparent material may include one or more materials, such as, but not limited to, poly(methyl methacrylate) (PMMA), polystyrene, and poly(perfluorobutylene vinyl ether) (CYTOP). The number of optical fibers in the fiber array may vary and, in some cases, match the number of pinholes in the second pinhole plate. For example, the number of optical fibers can range from 2 to 1000, such as 2 to 100, 5 to 50, 10 to 25, and 14 to 20. Each optical fiber may include a coating / cladding and an optically transparent core. In embodiments, the diameter of the core ranges from 0.1 mm to 2 mm, such as 0.2 mm to 1 mm, and includes 0.3 mm to 0.5 mm. In embodiments, the diameter of the coating ranges from 0.1 mm to 2 mm, such as 0.2 mm to 1.5 mm, such as 0.4 mm to 1 mm, and includes 0.5 mm to 0.7 mm.

[0082] The fiber optic array optically coupled to one or more optical sensors may also be composed of optical tubes. The optical tube may include an inner cavity and a coating to increase the amount of light waves reflected traveling within it. In other embodiments, no coating is included. The optical tube may be made of any suitable material. In some embodiments, the optical tube is made of a thermoplastic polymer. In some embodiments, the thermoplastic polymer is polycarbonate. In some cases, optically transparent materials such as PMMA, CoC, Cop, or glass may be used to mold the optical tube. In this embodiment, the number of fibers in the fiber optic array may vary and, in some cases, match the number of pinholes in the first pinhole plate. For example, the number of fibers may range from 2 to 1000, such as 2 to 100, such as 5 to 50, such as 10 to 25, and including 14 to 20. In embodiments, each optical tube consists of an unclad, optically transparent core. In embodiments, the core may have various diameters to match the pinhole, ranging from, for example, 0.1 mm to 5 mm, such as 0.2 mm to 1 mm, and including 0.3 mm to 0.5 mm. In some embodiments, the cross-section of the light tube is circular or square. In embodiments, the system also includes a small lens array for use with an optical fiber array optically coupled to one or more optical sensors. In additional embodiments, the system includes an optical fiber array plate. These elements are as described above and can be adapted for use on the collection side, in addition to or instead of being used on the illumination side.

[0083] Figure 3C-3E An embodiment of an optical fiber array including optical tubes is described. Figure 3C An array of optical fibers including light tubes 312, which are optically coupled to a light source 311, is shown. The light tubes 312 are configured to illuminate a detection chamber through individual pinholes (not shown) in a first pinhole plate 313. An array of optical fibers including light tubes 315, configured to collect light from individual pinholes (not shown) in a second pinhole plate 314, is also shown. The fiber array including light tubes 315 is optically coupled to an optical sensor 316. Figure 3D An alternative view of the top of the same light interrogation system is presented. Figure 3E The diagram illustrates a light tube that can be used in or adapted for use in this system.

[0084] Figures 4A-4B An optical interrogation system according to certain embodiments of the present invention is described. Figure 4A A second pinhole plate 403 with pinholes is depicted, through which the detection chamber 401 can be illuminated. Figure 4B A silhouette diagram of the same system is presented. For example... Figure 4BAs shown, the system includes: a first pinhole plate 402 positioned adjacent to a first light-accessible window of the detection chamber 401 near an entrance 405; and a second pinhole plate 403 positioned adjacent to a second light-accessible window of the detection chamber 401 near an exit 406. An optical sensor 404 is also shown.

[0085] Figure 5A Components of an optical interrogation system according to certain embodiments of the present invention are depicted. For example... Figure 5A As shown, a housing with a detection chamber 501 is located between pinhole plates 503 and 502 (described in more detail above), the detection chamber having an inlet 505 and an outlet 506. An optical sensor 504 is also shown. Figure 5A In this embodiment, the optical sensor is a CMOS image sensor that overlays the array of detection chambers behind the pinhole plate 503 to simultaneously measure the transmitted light power of all detection chambers 501. The collected beam projects a holographic image onto the CMOS image sensor. The pinhole image 508 forms a "super photodiode" to measure the light intensity of all cuvettes. The sum of the pixel electrons of the pinhole image is the transmitted light power of each cuvette and is used in absorbance calculations.

[0086] Figure 5B Components of an optical interrogation system according to certain embodiments of the present invention are described. Figure 5B Including the above about Figure 5A The same components are described. Additionally, a micro-LED array 511, a tapered optical mixing rod 512, a diffuser 513, a folded mirror 514, and a focusing lens 515 are included. Spectral filters 510 are also depicted; in this embodiment, these spectral filters are bandpass or lowpass optical filters placed behind the second pinhole plate 503 to remove the sample fluid (509) and the autofluorescence of the box plastic. Figure 5C Components of an optical interrogation system according to certain embodiments of the present invention are described. Figure 5C Including the above about Figure 5A The components described are the same as those described, with the addition of a light guide plate (LGP) 516 and a side-lit micro LED 517. Figure 5D A miniature spectrometer 518 configured to receive light from a detection chamber 501 is depicted. Figure 5E A pinhole image 550 is depicted. Region 551 is used for intensity measurement (e.g., mask).

[0087] Figures 6A-6C Different optical interrogation system arrangements configured to reduce stray light are presented. These include a first pinhole plate 602, a second pinhole plate 603, a detection chamber 601, an optical sensor 604, and a notch filter 610. Figure 6AIn one embodiment, the notch filter 610 is positioned between the second pinhole plate 603 and the optical sensor 604. Figure 6B and Figure 6C In this embodiment, the notch filter 610 is positioned between the first pinhole plate 602 and the second pinhole plate 603, that is, between the detection chamber 601 and the second pinhole plate 603. Figure 6C In one embodiment, the second pinhole plate 603 includes a light trap recess 615 configured to prevent stray light from being collected by the optical sensor 604. Figures 6A-6C In this design, the first pinhole plate 602 has pinholes with a diameter of Φ1, and the second pinhole plate 603 has pinholes with a diameter of Φ2. Additionally, the detection chamber 601 has a diameter of d'. For example... Figure 6C As shown, the first pinhole plate 602 and the second pinhole plate 603 have thicknesses d1 and d2, respectively. The distance separating the first pinhole plate 602 and the second pinhole plate 603 is characterized by a distance d3, which approximately corresponds to the thickness of the box.

[0088] In some alternative embodiments, the optical interrogation system includes a single pinhole plate (i.e., instead of a first and a second pinhole plate) and a reflector (e.g., a mirror) configured to reflect light from each detection chamber back to the pinhole plate. The reflector may be made of any suitable reflective material. In some embodiments, the detection chamber at one end is metallized, treated, or coated to have a mirror finish. In other cases, the system includes a mirror plate, for example, located above the top of the detection chamber. The single pinhole plate may have any of the dimensions described above with respect to the first pinhole plate. In some cases, the pinhole plate has a d / Φ ratio >10 (where d is the thickness of the pinhole plate and Φ is the pinhole diameter), such as >25. In some cases, this ratio may be sufficient to ensure that collimated light passes through the pinhole. According to this embodiment, the system also includes an optical sensor configured to collect light from each of the plurality of detection chambers through a pinhole in the second pinhole plate that is optically aligned with that detection chamber. An incident optical fiber or tube collimates the light so that it passes through a pinhole to the bottom layer of the cuvette and then through the liquid sample. The light is reflected by a reflector, and a sensor (or an optical fiber or tube optically coupled to it) captures the reflected light through the same pinhole. The light intensity is then measured by an optical sensor (e.g., a PD or CMOS sensor array), and the absorbance of the liquid sample is calculated. The reflected light doubles the light path and lowers the height of the cuvette, thereby reducing the liquid volume. The two light guides (i.e., the optical fibers used for illumination and collection) can be coaxial with a covering between them, or they can be separated into groups.

[0089] Figure 20 An embodiment of an optical interrogation system comprising a single pinhole plate and a reflector is presented. For example... Figure 20As shown, optical fiber 2004 (e.g., as part of an optical fiber array optically coupled to a light source; not shown) illuminates detection chamber 2001 through pinholes in pinhole plate 2002. Light passing through detection chamber 2001 is reflected back by reflector 2003 along its original path (i.e., through detection chamber 2001 and the pinholes in pinhole plate 2002) and received by optical fiber 2005 optically coupled to an optical detector (not shown). Pinhole plate 2002 has a thickness d, and the pinholes have a diameter Φ. Figure 20 In the embodiments described above, the d / Φ ratio is >10. Although not explicitly shown, the arrangement described above can be materialized for each pinhole in the pinhole plate 2002 as needed.

[0090] In some cases, a processor is operatively connected to an optical sensor, a light source, and a memory storing instructions thereon that, when executed by the processor, cause the processor to calculate the absorbance from the cartridge. As described in further detail below, the cartridge of the present invention includes a plurality of detection chambers. Therefore, the processor can be configured to calculate the absorbance of each of the plurality of detection chambers.

[0091] In some cases, calculating absorbance involves calculating the average intensity of incident light from the light source. The system may be configured to, for example, measure the average intensity of the first pinhole plate for each detection chamber before loading the cartridge into the system. The processor may be additionally configured to calculate the average intensity of emitted light from each of the plurality of detection chambers. This may include measuring the light intensity again after the cartridge containing the sample fluid has been inserted into the system. The processor may be additionally configured to deactivate the light source and calculate the dark-image average intensity of the emitted light from each of the plurality of detection chambers. Subsequently, the processor may calculate the absorbance of each of the plurality of detection chambers based on the average intensity of the incident light from the light source, the average intensity of the emitted light from each of the plurality of detection chambers, and the dark-image average intensity of the emitted light from each of the plurality of detection chambers. In some cases, the processor is configured to calculate the absorbance of each detection chamber as follows: .

[0092] Where A is absorbance, and I... 入射 It is the average intensity of the incident light from the light source, I abs_i It is the average intensity of the emitted light from each of the plurality of detection chambers, and I 暗 It is the dark image average intensity of the emitted light from each of the multiple detection chambers.

[0093] In some cases, the processor is configured to repeatedly calculate the absorbance for each detection chamber until a change in absorbance is measured. In other words, the absorbance measured above is considered the initial absorbance at the first time point. The processor can repeat the absorbance calculation until a significant change in absorbance is measured at a later time point. In some cases, the processor is configured to calculate the analyte concentration based on the rate of change in absorbance. For example, in some models, the processor is configured to calculate the rate of change as follows: .

[0094] Where ROC is the rate of change, and A 结束 The absorbance is calculated at time point T2 when a significant change in absorbance is measured, and A 开始 The first absorbance is calculated at time point T1. In some cases, a series of absorbances Ai are measured at time points Ti during the reaction, and the ROC is calculated using linear regression curve fitting.

[0095] In some cases, absorbance is read directly from a sensor (e.g., a spectrometer) at one or more wavelengths for absorbance calculations. An alternative is to capture the entire absorbance spectrum, for example, from 340 nm to 850 nm. In some cases, this provides more data than a single wavelength for calculating the concentration of the reaction product.

[0096] The systems and methods disclosed herein can employ analysis using imaging or signal analysis, algorithms for assisting image or signal analysis, and cutoff values. Analysis performed, for example, to extract one or more density distribution feature values, will vary in fluorescence intensity and may include cases where the density distribution features are based on or not based on color features of the image. Thus, density distribution feature values ​​can be color feature values ​​or non-color feature values. Color feature values ​​will typically depend on image information extracted from one or more color channels of the image, which is affected by staining of the specimen. Non-color feature values ​​can be derived from image information (extracted from the entire image or a portion thereof) and are independent of the image's color mode (e.g., color, grayscale, binary, etc.) or one or more color channels, but are generally unaffected by any staining of the specimen.

[0097] Density distribution feature values ​​extracted from density distribution features will individually or in combination indicate the density distribution of cells in the specimen and / or whether the analyzed image or region of interest (ROI) contains a morphological assessment region or a sample detection region. Therefore, one or more morphological assessment regions of a specimen can be identified based on one or more extracted density distribution feature values. Thus, the density distribution features analyzed in the subject method will include those that can be automatically extracted from digital images and analyzed to identify one or more morphological assessment regions of a specimen, which can be used in assessments performed by an automated digital cell morphology analyzer. When employing a combination of multiple density distribution feature values, the useful number of individual density distribution feature values ​​in such a combination will vary and can range from 2 to 20 or more, including but not limited to 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 The combinations of multiple density distribution feature values ​​can include those derived entirely from non-color features, entirely from color features, or from a combination of color and non-color features.

[0098] The useful density distribution characteristics of a specimen will vary and may include, but are not limited to, cell density within an image or region of interest (ROI), variations in cell density within an image or ROI, variations in cell density between images or ROIs, cell size within an image or ROI, variations in cell size within an image or ROI, variations in cell size between images or ROIs, cell shape within an image or ROI, variations in cell shape within an image or ROI, variations in cell shape between images or ROIs, cell pallor within an image or ROI, variations in cell pallor within an image or ROI, variations in cell pallor between images or ROIs, cell color within an image or ROI, variations in cell color within an image or ROI, variations in cell color between images or ROIs, and the number and combination of overlapping cells within an image or ROI. Therefore, useful density distribution characteristics include, but are not limited to, cell count, coefficient of variation (CV) index of cell count, cell size, CV index of cell size, index defining cell shape, CV of cell shape index, index defining central pallor of cells, index defining cell color, and the count and combination of overlapping cells.

[0099] According to the subject-matter approach, extracting density distribution features from an image or ROI may include one or more image processing steps, which may employ one or more image processing algorithms. Useful image processing steps may include, but are not limited to, splitting channels of a multi-channel image, generating one or more image masks (e.g., foreground mask, color mask, threshold mask, combined mask (e.g., combined color mask and foreground mask)), space filling or aperture closure (e.g., aperture closure in the generated mask), noise filtering, segmentation (e.g., cell segmentation), and so on.

[0100] Image processing steps typically involve processing digital images that can vary and may be in binary (e.g., black and white), grayscale, or color formats. Images in various formats can be further converted between formats as needed using appropriate image processing algorithms. For example, a color image can be "split" into individual color channels to produce a separate grayscale image for each color channel. For instance, a red, green, and blue (RGB) image can be split into separate red, green, and blue channels to produce a grayscale image of the red channel, a grayscale image of the green channel, and a grayscale image of the blue channel. Color images can be converted between different color spaces and split into any convenient and suitable color channels for a specific color space, including but not limited to RGB, CMYK, HSV, CIE, Lab, CIELUV, YCbCr, and so on. Binary and grayscale images can be applied to the channels of a color image; for example, in cases where multiple binary or grayscale images are applied to multiple channels of a color image, the color image can be constructed or "merged" from binary and / or grayscale images. When a color image is split into individual color channels to produce a grayscale image, the individual grayscale images can be referenced by their previous channel names. For example, a grayscale image generated from the red channel can be referred to as "red" in subsequent steps, and / or any value generated from the "red" channel can be referenced by its previous channel name. For example, the average "red" intensity refers to the average intensity value derived from the grayscale image generated from the red channel. Corresponding nomenclature can be used to refer to images and values ​​derived from other color spaces.

[0101] Therefore, a digital color image can be processed as a color image (i.e., a multi-channel image), or it can be converted or split into two or more individual color channels prior to processing. When split into two or more individual color channels, any number of the resulting split images can be used in additional processing steps, including but not limited to all split images (i.e., all individual channels of the image) or only one of the split images (i.e., only one of the individual channels of the image) or one or more of the split images (i.e., individual channels of the image), including but not limited to two or more, three or more, two, three, etc.

[0102] Digital color or monochrome images can be segmented prior to processing. As used herein, when the terms “segmented” and “segmentation” relate to image processing, they generally refer to dividing or dividing an image into meaningful structures or segments. Various methods for image segmentation can be found in the methods described herein or in preparing an image for processing according to the methods described herein. The choice of a particular segmentation method or combination of segmentation methods will depend on a variety of factors, including the type of image captured, the nature of the image’s subject matter, the desired outcome of the image processing, the color or monochrome features used, one or more of the desired density distribution features to be extracted, etc.

[0103] In some cases, image segmentation can be achieved using one or more of threshold-based segmentation, edge-based segmentation, and region-based segmentation. Specific image segmentation methods include, but are not limited to, thresholding methods, clustering methods, compression-based methods, histogram-based methods, edge detection methods, bi-clustering methods, region growing methods, partial differential equation-based methods (e.g., parametric methods, level set methods, fast marching methods, etc.), variational methods, graph partitioning methods (e.g., Markov random field methods), watershed transform methods, model-based segmentation methods, multi-scale segmentation methods, semi-automatic segmentation methods, trainable segmentation methods, and so on.

[0104] Other digital image processing image transformations that can be found useful in the described methods include, but are not limited to, point processing transformations (e.g., negative transformations), logarithmic transformations, inverse logarithmic transformations, root n transformations, power n transformations, gamma correction, contrast transformations (e.g., contrast stretching), window centering correction, histogram equalization, etc.), filtering (i.e., neighborhood) transformations (e.g., mean filters, Gaussian filters, median filters, image gradient filters, Laplacian filters, normalized cross-correlation (NCC) filters, etc.), and so on.

[0105] Embodiments of the system of the present invention may include any convenient power source for supplying operating power to system components, and this may vary depending on the desired usage environment or use case. The nature of the power source may vary and may or may not include a power management circuitry system. Typically, the system receives power, i.e., power distributed throughout the system (i.e., via a wired connection). In some cases, the system is configured to receive power via a wire and plug from an external source (such as, for example, a wall socket, etc.). In embodiments, the system includes a power supply unit configured to modulate the power received from the external source into a configuration that can be utilized by components of the system (e.g., one or more rectifier circuits, transformers, etc.). In other cases, the system includes a battery unit such that the system does not need to be connected to an external power source, such as a wall socket. When present, the battery unit may include a battery as a disposable battery or a rechargeable battery. For rechargeable batteries, any convenient protocol may be used to recharge the battery, including but not limited to wireless charging protocols (such as inductive charging). In some applications, the system may have a battery life ranging from 0.1 hours to 120 days, from 14 days to 30 days, from 8 hours to 30 days, from 8 hours to 12 days, from 12 hours to 24 hours, and from 0.5 hours to 10 hours. Some embodiments are configured to receive power from both an external power source and the battery cell. In some embodiments, one or more cartridges may include a dedicated power source; however, more typically, the cartridge receives power via a sample fluid analysis system, for example, via a wired electrical connection to the sample fluid analysis system.

[0106] As discussed above, embodiments of the present invention include one or more processors or controllers and associated memory operatively coupled thereto. The processor of the present invention can be used to implement various functions of the system, such as as described in more detail above. In other words, the memory operatively coupled to the processor may include instructions stored thereon that, when executed by the one or more processors or controllers, cause the one or more processors or controllers of the sample fluid analysis system to control one or more aspects of sample analysis performed by the system. In some embodiments, the one or more memories are located within the system. In other cases, the one or more memories are present on or within a cartridge. In this case, when the cartridge is received within the housing of the system, the instructions on these memories can be executed by the processor. The control unit / processor (and / or one or more cartridges) of an embodiment of the sample fluid analysis system may be configured, for example, to control internal timing, execute various algorithms, perform result calculations, and the hardware components (e.g., mechanical components) of the operating system, including, for example, controlling the interface between the housing and one or more cartridges removably coupled to the housing.

[0107] Any convenient processor and memory may be used in embodiments of the subject system, including embodiments of a sample fluid analysis system or cartridge. For example, any readily available commercially available processor or memory may be used. In particular, in embodiments, the processor may include a general-purpose processor or controller or microcontroller or other processor or combination thereof configured to control aspects of the system. In each case, the processor and memory are operatively connected to each other. This operative connection may take any convenient form, such that instructions and data can be obtained by the processor via any convenient input technology, such as via wired or wireless network connection, Bluetooth® connection, shared memory, bus, or any other functionally similar communication protocol.

[0108] Additionally, according to some embodiments, the system may include a display and an operator input device. The operator input device may be, for example, a keyboard, mouse, touchscreen, keypad, etc. As described above, embodiments of the sample fluid analysis system include, and in some cases, embodiments of the box may also include, a processing module comprising one or more processors that have access to one or more memories storing instructions for controlling aspects of the system (i.e., the sample fluid analysis system and (one or more) boxes) to perform sample analysis. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other aspects. The processor may be a commercially available processor or one or more other processors that are available or will become available. The processor executes the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor's coordination and execution of the functions of various computer programs written in various programming languages, such as Java, Perl, C++, other high- or low-level languages, and combinations thereof, as known in the art. The operating system coordinates and executes the functions of other components of the processing module, typically in cooperation with the processor. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all based on known technologies. The processor can be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0109] The memory of the processing module can be any of a variety of known or future memory storage devices. Examples include any universally available random access memory (RAM), magnetic media (such as resident hard disks or magnetic tapes), optical media (such as compact optical discs for reading and writing), flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including compact optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. This type of memory storage device typically reads from and / or writes to program storage media (such as compact optical discs, magnetic tapes, removable hard disks, or floppy disks, respectively). Any of these program storage media, or other program storage media now in use or that may be developed later, can be considered a computer program product. As will be understood, these program storage media typically store computer software programs and / or data. Computer software programs (also known as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0110] In some embodiments, a computer program product is described having a computer-usable medium in which control logic (computer software program, including program code) is stored. When executed by a processor, the control logic causes the processor to perform the functions described herein, including, for example, controlling aspects of a sample fluid diluent. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. It will be apparent to those skilled in the art that implementing a hardware state machine to perform the functions described herein will be obvious. Further details regarding computer-controlled systems are provided below. The system may also include, for example, a communication connector unit (e.g., a Universal Serial Bus (USB) connector and associated circuitry) to transmit any relevant data (e.g., results of sample analysis) to a remote device (such as a personal computer, laptop computer, PDA, mobile phone, smartphone, set-top box, etc.). The term remote device is used herein to refer to any device outside the system. Various technologies may be employed. For example, the communication connector may employ any of the following technologies or families of technologies (but not limited to): USB, FireWire, SPI, SDIO, RS-232 port, or any other suitable electrical connector to allow data communication between the sample fluid analysis system and the remote device. The communication connector unit provides the ability to communicate with a remote device having a suitable interface for operative coupling with the communication connector. In some aspects, the communication connector is configured to communicate with smartphones, such as Apple iPhones or Samsung Galaxy phones. It should also be understood that more than one communication connector unit can be implemented on a system, such as a sample fluid analysis system and / or multiple communication units on one or more cartridges.

[0111] It should be understood that the term "communication connector" is used in this disclosure to refer to any various connection interfaces, such as male or female connectors. Taking USB as an example, the communication connector can be any of various USB plugs or USB sockets / ports. Since USB sockets are typically found on computers and other devices, using a corresponding USB plug as a communication connector will allow the sample fluid analysis system, cartridge, sample acquisition device, or other aspects of the system of this disclosure to be directly plugged into the USB socket where applicable, thereby avoiding the use of cables. In other aspects, a suitable USB socket can be used on one aspect of the system to enable communication using USB cables (similar to many other devices such as digital cameras, smartphones, smartwatches, etc.). It should be understood that in some cases, the communication connector unit can implement wireless technology, in which case the connection interface will be a corresponding transmitter, receiver, and / or transceiver.

[0112] Various functional features can be performed using a communication connector unit. For example, a communication connector can be used to transfer data from the system to a remote device. This remote device can store the data and / or further process the data and / or combine the data with other additional information. The data may include not only analyte measurements, but also information such as user settings / preferences, recorded data, rates of change in analyte levels, and / or analyte levels exceeding thresholds. In other cases, the sample fluid analysis system may be configured to store and / or further process this data and / or combine this data with other additional information.

[0113] Depending on the system's application environment, the remote device can also transmit data (e.g., raw data or any additional data (e.g., data for further processing)) (wired or wireless) to a second remote device (e.g., in a doctor's office, hospital, or third-party location) via a separate communication channel. The second remote device can be, for example, a personal computer, laptop computer, PDA, smartphone, set-top box, etc. For instance, data can be transferred from the sample fluid analysis system to the user's personal computer, stored therein, and then transmitted via an internet connection on the personal computer to a remote server at the hospital. The doctor at the hospital can then access and review the data on the server. In some aspects, the sample fluid analysis system can be configured to receive program updates from the remote device via a communication connector unit.

[0114] In some aspects, the communication connector unit is coupled to the housing of the sample fluid analysis system. While the communication connector is not required to be located on the sample fluid analysis system, in some cases, it may be included as part of the system, eliminating the additional cost of a separate communication connector for each additional housing. If the sample fluid analysis system includes a communication connector of the first technology (e.g., a USB plug), the additional housing may have the option to include additional capabilities, such as, for example, new wireless communication protocols.

[0115] In embodiments, the remote device (such as the remote device described above) includes a network interface for connecting it to a network (e.g., the Internet). A user interface application operated by the remote device provides the user with options such as viewing data on a monitor, storing data on a storage medium (e.g., CD-ROM, memory card, etc.), further analyzing and / or manipulating the data, transferring data to other devices, and / or printing the data (e.g., charts, reports) on a printer. The remote device may also include a network interface (e.g., a network interface card (NIC), modem, router, RF front end, etc.) for connecting the remote device to a network. For example, in some aspects, the sample fluid analysis system may be coupled to the remote device via a USB connection, which may be a personal computer or laptop computer connected to the Internet using a wireless interface. In some aspects, the sample fluid analysis system may be coupled to the remote device via a micro-USB connection, which is a smartphone with an RF front end for accessing a mobile network. The user interface application provides a user interface for using the network connection of the remote device, for example, to forward data to a doctor, hospital, healthcare provider, and / or other third party located at a second remote device on the network. The recipient can then take appropriate action at the second remote device.

[0116] In some cases, the system includes a display unit coupled to its housing. The display unit may be configured to include a display and / or a display port for coupling a monitor to the system. The display unit may display aspects of the sample analysis results determined using various aspects of the system, which may include any desired analytical results determined by the system, such as, for example, analyte concentration, rate of change of analyte concentration, and / or analyte concentration exceeding a threshold.

[0117] The display unit may be configured to include a dot-matrix display. Alternatively, other display types may be used, such as liquid crystal displays (LCDs), plasma displays, light-emitting diode (LED) displays, or seven-segment displays. The display may be monochrome (e.g., black and white) or multicolor (i.e., having a range of colors). The display unit may be configured to provide alphanumeric displays, graphical displays, video displays, audio displays, auditory or vibratory outputs, or combinations thereof. The display unit may also be configured to provide information, for example, related to sample analysis, such as current analyte concentrations and predictive aspects (e.g., predictive analyte concentrations, such as trend information).

[0118] In some aspects, for example, the display unit may be configured to include a touchscreen display, in which a user may input information or commands via the display area using, for example, a stylus, a finger, or any other suitable input device, such as where the touchscreen is configured as a user interface in an icon-driven or motion-driven environment.

[0119] The systems including touchscreens disclosed herein may include the same functionality and basic design as the systems without touchscreens disclosed herein. In some cases, the touchscreen system will include a larger display unit compared to the system's display unit without a touchscreen, in order to accommodate the additional area required to accommodate any touchscreen buttons that may be used. In some cases, the system does not have a display (i.e., no display).

[0120] In some cases, the system may include input elements coupled to its housing, which enable the user to input, select, etc. (In some cases, the box may also include input elements coupled to its housing.) In some cases, a touchscreen may be used with or without input elements.

[0121] Some aspects of the subject system are also described in U.S. Patent Application Publications 2018 / 0126381; 2019 / 0056304; 2019 / 0056384; 2019 / 0054466; and 2022 / 0274109, and U.S. Patent Applications 5,096,669; 7,177,767; 7,765,069; and 7,998,411, the disclosures of which are incorporated herein by reference in their entirety. Additional information regarding the sample analysis system (including aspects of the system that can be removably coupled to determine analyte levels for the purpose of performing sample analysis) is described in U.S. Patent Application Publication 2011 / 0256024A1 and U.S. Patent Application Publication 2012 / 0149245A1, each of which is incorporated herein by reference in its entirety. Further details regarding the subject device can be found in U.S. Provisional Patent Application No. 63 / 525,611 (Agent No. ADDV-129PRV), filed July 7, 2023.

[0122] As mentioned above, embodiments of the subject system can be configured as a receiving box, which may include, for example, one or more detection chambers. A housing may house the functional elements of the system. Embodiments of the device, and particularly embodiments of the housing, can have any convenient shape and size, and such embodiments can vary, for example, based on the intended application environment or usage environment of the system, or, for example, the desired throughput of the system. That is, it should be understood that the housing can have various shapes depending on specific design considerations. As described herein, embodiments of the system can be utilized in a variety of different environments, including, but not limited to, home environments, bedside testing environments, ambulance environments, emergency room environments, doctor's office environments, pharmacy environments, small clinics or pop-up clinics, hospital laboratory environments, or core laboratory environments. Therefore, the size and shape of embodiments of the system can be determined to suit the desired environment. One aspect of the desired environment for using embodiments of the systems disclosed herein is the number of sample analyses (e.g., determining one or more analyte levels across several samples) that can be performed within a fixed amount of time and / or the number of sample analyses that can be performed simultaneously. For example, in some home environments, it is expected that the device will be configured to run a sample analysis only once, while in some core laboratory environments, it is expected that the system will be configured to run one, two, three, four, five, six, seven, eight, nine, ten, dozens, hundreds, thousands or more sample analyses simultaneously.

[0123] In some cases, the size and / or shape of an embodiment of the system may be defined by the number of sample analyses that can be performed simultaneously by an embodiment of the device. In this case, an embodiment capable of performing multiple sample analyses simultaneously may be configured to receive and simultaneously accommodate multiple cartridges and / or multiple sample collection devices. Embodiments of the housing may be configured to simultaneously accommodate one or more cartridges, such as 1 cartridge, 2 cartridges, 3 cartridges, 4 cartridges, 5 cartridges, 6 cartridges, 7 cartridges, 8 cartridges, 9 cartridges, 10 cartridges, 20 cartridges, 30 cartridges, 40 cartridges, 50 cartridges, 100 cartridges, or 500 or more cartridges. Embodiments of the system may be configured to simultaneously receive one or more samples, such as 1 sample, 2 samples, 3 samples, 4 samples, 5 samples, 6 samples, 7 samples, 8 samples, 9 samples, 10 samples, 20 samples, 30 samples, 40 samples, 50 samples, 100 samples, or 500 or more samples. Similarly, embodiments of the sample fluid analysis system and cartridge can be configured to perform one or more sample analyses simultaneously (i.e., determine the levels of one or more analytes in one or more samples), such as performing one sample analysis at a time, performing two sample analyses simultaneously, performing three sample analyses simultaneously, performing four sample analyses simultaneously, performing five sample analyses simultaneously, performing six sample analyses simultaneously, performing seven sample analyses simultaneously, performing eight sample analyses simultaneously, performing nine sample analyses simultaneously, performing ten sample analyses simultaneously, performing twenty sample analyses simultaneously, performing thirty sample analyses simultaneously, performing forty sample analyses simultaneously, performing fifty sample analyses simultaneously, performing one hundred sample analyses simultaneously, or performing five hundred or more sample analyses simultaneously. That is, embodiments of the device and cartridge can be configured to determine one analyte level for a sample at a time, simultaneously determine two analyte levels, simultaneously determine three analyte levels, simultaneously determine four analyte levels, simultaneously determine five analyte levels, simultaneously determine six analyte levels, simultaneously determine seven analyte levels, simultaneously determine eight analyte levels, simultaneously determine nine analyte levels, simultaneously determine ten analyte levels, simultaneously determine twenty analyte levels, simultaneously determine thirty analyte levels, simultaneously determine forty analyte levels, simultaneously determine fifty analyte levels, simultaneously determine one hundred analyte levels, or simultaneously determine five hundred or more analyte levels. Similarly, embodiments of the system and cartridge can be configured to determine the level of a single analyte for one sample at a time, or simultaneously determine two samples, three samples, four samples, five samples, six samples, seven samples, eight samples, nine samples, ten samples, twenty samples, thirty samples, forty samples, fifty samples, one hundred samples, or five hundred or more samples.

[0124] In selected cases, the housing has a shape close to a cuboid. In other cases, the housing includes features configured for user bedside detection of grip (e.g., in a manner similar to the i-STAT described above). ® This is part of the device. The dimensions of the housing can vary. In some cases, the housing has a length ranging from 10 cm to 75 cm, such as 15 cm to 50 cm. In some cases, the housing has a width ranging from 5 cm to 50 cm, such as 10 cm to 20 cm. In some cases, the housing has a height ranging from 3 cm to 20 cm, such as 5 cm to 15 cm. The housing of the present invention can be made of any suitable material. For example, in some cases, the housing comprises one or more rigid plastic materials, such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, and other polymeric plastic materials. Examples of polymeric materials include acrylonitrile-butadiene-styrene (ABS), polylactic acid (PLA), acrylic-styrene-acrylonitrile (ASA), polyethylene terephthalate (PET), ethylene glycol-modified polyethylene terephthalate (PETG), polyarylether ketone (PAEK), polyetherimide (PEI), polycarbonate (PC), polypropylene (PP), aliphatic polyamide (PPA), polyoxymethylene (POM), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), polyphenylene sulfone (PPSU), polyether ether ketone (PEEK), and nylon, as well as their composites and mixtures.

[0125] Embodiments of the system may be configured to be substantially sealed, such that the contents of the sample and / or the contents of the cartridge are disposed of at one or more waste disposal locations and are not otherwise discharged from the system (i.e., leaked out of the system). In embodiments, for example, the system is substantially sealed, such that substances cannot enter the main device and / or cartridge and / or other aspects of the system except through dedicated inlet points, such as substances entering the sample fluid analysis system from the cartridge via the cartridge interface. In embodiments, the system may be configured for self-cleaning of certain aspects of the system, and in some cases, one or more cartridge types may be configured to facilitate this self-cleaning function.

[0126] The housing of interest is configured to receive a box. The housing can be configured to receive the box internally in any suitable manner. For example, in some cases, the housing includes sockets, such as slots (i.e., ports), with dimensions suitable for receiving the box. In some cases, the housing may include bays for one or more box sockets, and in some cases, one or more covers over these sockets. The covers may be made of a transparent material, allowing the user to easily determine whether and / or which sockets in the box bays are occupied by a box or a particular type of box. Alternatively, the housing may include a retractable platform configured to be actuated between an extended position and a retracted position. When the platform is in the extended position, the user can place a box on the platform. When the platform moves to the retracted position, the box is received inside the housing. In some cases, the housing is configured such that the box can only be inserted in a single orientation (i.e., to prevent mis-design). In other words, the housing is configured such that if the box is inserted in an orientation different from the specified orientation, the user will be unable to insert the box (e.g., without damaging the box or the housing). For example, the associated sockets of different types of boxes and box interfaces can be shaped or sized such that only specific box types can be loaded into the dedicated sockets of the box interface. In other examples, the associated sockets of different types of boxes and box interfaces include other physical or mechanical features, such as pins or other keying techniques, to prevent boxes other than those of a specific type from being loaded into the dedicated box sockets. For example, in some cases, a specific type of box may be shaped with a chamfered cross-section so that the socket of the box interface cannot receive that box unless it also includes a similar chamfered cross-section.

[0127] The housing can be configured to dock with the cartridge when it is received therein. "Dock" in this context means a connection in a functional and signal-communication relationship with the cartridge. The cartridge interface may include at least one socket and is configured to functionally interconnect a sample fluid analysis system with the cartridge, i.e., one or more contents within the cartridge become functionally usable by the sample fluid analysis system for performing sample analysis. Therefore, the cartridge interface may include, for example, one or more mechanical interconnections with the cartridge (e.g., gears, push rods, other mechanical engagement features, etc.), electrical interconnections with the cartridge (e.g., wired or wireless connections), fluid interconnections with the cartridge (e.g., piping systems or other fluid paths), or any other interconnections necessary for the contents of the cartridge to be functionally usable by the sample fluid analysis system. In other words, the apparatus of the present invention can be configured to cause various functions (e.g., sample fluid metering, sample fluid separation, mixing, etc., which will be described in more detail below) to be performed within the cartridge. Additionally, the apparatus of the present invention can be configured to transmit signals (e.g., electrical signals, optical signals / light) to and from the cartridge for interrogating the sample fluid within the cartridge.

[0128] In some cases, the system includes a mechanical positioning device, which can be used to adjust the position of the box within the system. For example, in embodiments of the invention (where fewer pinholes exist in the first and second pinhole plates than in the detection chambers present in the box), the mechanical positioning device can be configured to adjust the position of the box such that each detection chamber is optically aligned with at least one pinhole at a certain point in time, thus enabling optical interrogation of the contents of the detection chambers. In embodiments, the housing is configured to mechanically hold or secure the box in place. In some cases, the box and housing are configured to hold the box in place using spring loading. For example, the housing may include a box interface with a socket having flexible members (e.g., tabs) configured to grip or press against the sides of the box when it is loaded into the housing. In some cases, the housing and box are configured to hold the box in place using gravity or magnetic interaction or other mechanical (e.g., spring) interaction, etc. In some cases, the box may be latched or otherwise secured or locked in place, for example by user or robot features. In embodiments, any convenient number of latches or locks may be provided. In some cases, the housing and the box are configured to engage using a press-fit between the box and the housing. The physical interfaces (i.e., the physical interface of the housing and the physical interface of the box) can be removably coupled to each other by incorporating any of a variety of releasable engagement mechanisms (e.g., snaps, sliders, magnetic elements, Velcro, clasps, hooks, hinges, locks, latches, etc.). The physical interfaces, as well as the entire housing of various aspects of the system, can be form-fitted to provide a tight fit for robust coupling and additional functional features (e.g., portability) when coupled as a single unit.

[0129] In some embodiments, the housing may be configured to enable additional capabilities. That is, the housing may be configured to allow the addition of one or more additional cartridge interfaces, enabling the sample fluid analysis system to simultaneously interface with different types of cartridges and / or more than one cartridge. In other words, embodiments of the systems disclosed herein are modular, at least in terms of the housing being configured to add the ability to receive different and / or additional cartridges, for example, to add one or more additional cartridge interfaces to the sample fluid analysis system. In other embodiments, the cartridge interfaces of the housing may be reconfigurable, enabling it to receive different types of cartridges upon reconfiguration. For example, the cartridge interface may be configured to receive an adapter that, when used, allows different or additional types of cartridges to interface with the housing.

[0130] As stated above, embodiments of the housing are used in conjunction with one or more boxes, i.e., the housing can be configured to dock with one or more boxes. It should be understood that the housing and the one or more boxes are removably coupled to each other. Therefore, in this disclosure, the following terms are used interchangeably: references to boxes(s) removably coupled to the housing; references to a sample fluid analysis system removably coupled to boxes(s); references to a removably coupled housing and boxes(s); and references to one or more boxes loaded into or received by the housing, or similar terms. Furthermore, when these aspects of embodiments of the system of the invention are said to be “coupled,” “inserted,” “loaded,” or “received,” it means that at least two aspects are currently coupled (but remain removably coupled).

[0131] In some cases, a single cartridge may be loaded into the housing for the desired sample analysis, and in others, more than one cartridge may be loaded into the housing. Loading a cartridge into the housing may include inserting the cartridge into the housing, such that one side of the cartridge is fully or substantially exposed to the housing. In other cases, causing the sample fluid analysis system to dock with the cartridge includes applying a connector or interface between one side of the cartridge and one side of the housing. Such a connector or interface may include any convenient mechanical and / or electrical and / or fluid interconnection or any other functional interconnection that makes the contents of the cartridge usable by the housing for performing sample analysis.

[0132] In embodiments, the system may be configured to indicate the type of cartridge required to perform a specific sample analysis, such as determining the level of a specific analyte in a specific type of sample. For example, the system may include a display unit for displaying the type of cartridge that needs to be loaded into the sample fluid analysis system for performing a specific type of sample analysis. Similarly, in embodiments, the system may be configured to provide an indication that a cartridge is loaded into a housing or, in some cases, a specific type of cartridge is loaded into the housing. In embodiments, the device may include an indicator element; for example, the housing or its cartridge interface may include an indicator element configured to indicate whether a cartridge is loaded into the housing and / or the type of cartridge loaded into the sample fluid analysis system and / or the status of the cartridge loaded into the housing (e.g., the level or some resource present in the cartridge) and / or any other information related to using the device for sample analysis. For example, the indicator element may include one or more indicator lights (such as LEDs) or a display interface. Any convenient technology may be used to identify the presence and / or type of cartridge loaded into the sample fluid analysis system, including, for example, mechanical technology (e.g., keying or unique pin structures associated with different types of cartridges), or electronic technology (e.g., digital codes stored in non-volatile memory, RFID technology or other wireless identifiers, magnetic codes, etc.), or optical technology (such as barcodes, 2D barcodes or other optical identifiers, or combinations thereof, that can be read by a camera on the sample fluid analysis system).

[0133] Embodiments of the system of the present invention may utilize any convenient technique to identify whether a cartridge is loaded into the sample fluid analysis system and / or the type of cartridge loaded into the sample fluid analysis system, such as, for example, one or more mechanical / physical indicators (e.g., specific patterns or pins, tabs, etc. may be present on the cartridge, and the relative patterns of such elements may be present on the socket of the cartridge interface of the sample fluid analysis system), or one or more electrical indicators (e.g., indicator electrodes, magnetic codes, software identifiers used in conjunction with the system's processor and memory, wireless communication, RFID tags, etc.), or one or more optical indicators (e.g., barcodes or 2D barcodes and cameras configured to be used in conjunction with the system's processor and memory to identify the presence and / or type of cartridge loaded into the sample fluid analysis system).

[0134] Methods for analyzing sample fluids As discussed above, aspects of the present invention also include methods for analyzing sample fluids. Aspects of the present invention include: introducing a sample fluid into a cartridge comprising a plurality of detection chambers; inserting the cartridge into an optical interrogation system (e.g., as described above); and illuminating the plurality of detection chambers with a light source to analyze the sample fluid.

[0135] In some embodiments, the method includes introducing a sample fluid into a cartridge comprising a plurality of detection chambers, each detection chamber including a first light-reachable window configured to allow light to enter and a second light-reachable window configured to allow light to exit. The method may additionally include inserting the cartridge into the sample fluid analysis system of the present invention (e.g., as described above). The sample fluid (e.g., blood) may be added to the cartridge before or after insertion into the system. In some cases, the method includes introducing the sample fluid into the cartridge before insertion into the system. In other cases, the method includes introducing the sample fluid into the cartridge after insertion into the system. Additionally, the method includes illuminating the plurality of detection chambers with a light source; and calculating the absorbance of each of the plurality of detection chambers to analyze the sample fluid (e.g., using one or more of the equations / algorithms described above).

[0136] When using embodiments of the systems disclosed herein, one or more cartridges are typically used and consumed for the purpose of performing a single sample analysis (e.g., determining the analyte level of the sample). That is, typically one or more cartridges are loaded into the sample fluid analysis system before the system initiates a sample analysis and remain in place (e.g., latched or locked or otherwise loaded into position) in the sample fluid analysis system until the system has completed the desired sample analysis, after which their contents may be depleted or otherwise consumed, and the cartridges need to be removed. However, in some cases, a single cartridge may be utilized for more than one sample analysis. That is, in some cases, a single cartridge may be utilized across multiple sample analyses for a particular desired sample analysis performed by the system.

[0137] The detection chambers for use in the subject box can be any microfluidic component configured for analyzing (e.g., optical analysis) samples. Exemplary detection chambers include, but are not limited to, microvolume cuvettes. In embodiments, at least a subset of the plurality of detection chambers includes one or more reagent beads / beads comprising a dried reagent. The dried reagent may be, for example, a lyophilized reagent or a printed reagent. In some cases, the reagent is configured for rapid dissolution, as described in U.S. Patent 5,413,732, the disclosure of which is incorporated herein by reference. With regard to “at least a subset,” it means that a plurality (though not necessarily all) of the detection chambers includes reagent beads. In some cases, some of the detection chambers (e.g., one, two, three) do not include reagents or reagent beads, such as when those detection chambers are intended to be used as controls in an assay. In some alternative embodiments, some of the detection chambers (e.g., one, two, three) include reagent beads that do not include any dried reagent, such as when those detection chambers are intended to be used as controls in an assay. In various embodiments, reagent beads are microparticles having a general shape (such as spherical, cylindrical, cubic, dodecahedral, elliptical, or other regular or irregular shapes). In some embodiments, reagent beads are formed from polymers (such as latex, glass, silica, or polystyrene). In other embodiments, reagent beads are formed from magnetic materials such that they exhibit magnetic properties when placed in a magnetic field and have no residual magnetism once removed from the magnetic field. Reagent beads may have a diameter, width, and / or length from about 0.1 µm to about 35 µm, from about 0.1 µm to about 20 µm, or from about 0.1 µm to about 10 µm. Reagent beads may be coated with a reagent capable of binding to a target antigen in a sample. The reagent may include antibodies, antibody fragments, ion carriers, enzymes, a group of enzymes, peptides having a cleavable detectable moiety, optical marker dyes that identify the type of assay beads, and / or combinations thereof.

[0138] In some cases, the cartridge is pre-filled with reagents and ready for immediate use. In some embodiments, all or part of the reagents may be present. The device may need to be stored under appropriate conditions to maintain the reactivity of the reagents. For example, depending on the reagents present, the device may need to be stored in a refrigerator or freezer before use. When the reagents are not sensitive to room temperature, the device may be stored at room temperature.

[0139] In some embodiments, the one or more dry reagents may include one or more non-fluorescent or fluorescent dyes, such as eosin, methylene blue, acridine orange (also known as “Basic Orange 15” or “ACO”) or Astrazon orange (also known as “AO” or Basic Orange 21), components that bind to nucleic acid DNA in cells (e.g., blood cells, such as WBCs), anticoagulants, antibodies, antibody fragments, ion carriers, enzymes, a group of enzymes, peptides having cleavable detectable moieties, substrates, optical marker dyes that identify the type of assay beads, and / or combinations thereof.

[0140] In some embodiments, the kit is designed for measuring clinical chemistry tests in a blood sample. Clinical chemistry tests refer to a set of tests routinely ordered to determine a subject's overall health status. In some cases, clinical chemistry tests include metabolic tests. Clinical chemistry tests help assess, for example, the body's electrolyte balance and / or the status of several major organs. In some cases, the measurements are performed on blood samples typically drawn from a vein. Examples of clinical chemistry tests detectable by the assays disclosed herein include, but are not limited to, the Basal Metabolic Mechanism (BMP), Comprehensive Metabolic Mechanism (CMP), electrolyte tests, lipid tests, liver tests, kidney function tests, and thyroid function tests. The Basal Metabolic Mechanism (BMP) includes eight tests, all of which are found in the CMP. The BMP provides information on the current health status of the kidneys and respiratory system, as well as electrolyte and acid / base balance and blood glucose levels. The CMP measures liver and kidney health, blood glucose levels, blood acid / base balance, fluid and electrolyte balance, and important blood proteins. In some cases, CMP measures glucose, calcium, total albumin and globulin, bilirubin, BUN (blood urea nitrogen), creatinine, albumin, sodium, potassium, bicarbonate, chloride, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Electrolyte tests are used to detect problems with fluid and electrolyte balance. For example, electrolyte tests measure blood levels of carbon dioxide, chloride, potassium, and sodium. Lipid tests are used to assess a subject's risk of cardiovascular disease. For example, lipid tests measure the amount of cholesterol and other fats in the blood, such as total cholesterol, LDL (low-density lipoprotein), HDL (high-density lipoprotein), and triglycerides. Liver tests (liver function packages) are used to screen for, detect, assess, and monitor acute and chronic liver inflammation (hepatitis), liver disease, and / or damage. Liver tests measure various enzymes, proteins, and other substances produced by the liver. For example, liver function tests include albumin, total protein, ALP, ALT, AST, gamma-glutamyl transferase (GGT), bilirubin, lactate dehydrogenase (LD), and prothrombin time (PT). Kidney function tests (renal function tests) include tests such as albumin, creatinine, BUN, and eGFR to assess kidney function. Thyroid function tests are used to assess thyroid function and help diagnose thyroid diseases. Thyroid function tests measure thyroid hormones such as thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH). In some cases, high TSH levels indicate that the thyroid gland is not producing enough thyroid hormone (primary hypothyroidism). Conversely, low TSH levels usually indicate that the thyroid gland is producing too much thyroid hormone (hyperthyroidism).In other cases, the presence of elevated TSH and low free T4 (FT4) or free T4 index (FTI) indicates primary hypothyroidism due to disease in the thyroid gland. Low TSH and low FT4 or FTI indicate hypothyroidism due to problems involving the pituitary gland. Low TSH and elevated FT4 or FTI are also found in individuals with hyperthyroidism. These clinical chemistry tests are well known in the art and are further described in the assays section of this disclosure.

[0141] In some cases, such as when the kit is used in a complete metabolic assay (CMP) program, the reagents include one or more of the following: 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA), 2-chloro-4-nitrophenyl-α-maltotriose (CNPG3), 2-methyl-4-isothiazolin-3-one hydrochloride (MIT), 4,7,13,16,21-pentaoxa-1,10-diazabicyclo[8.8.5]tezorisane (Kryptofix) 221), 4-aminoantipyrine hydrochloride, adenosine 5'-bisphosphate, adenosine 5'-triphosphate, α-ketoglutarate, amylase, azoarsenic III, sodium salt, ascorbic acid oxidase (cucumber genus), bilirubin oxidase, bromocresol purple, calcium acetate, creatine amidohydrolase (actinobacillus genus), creatine acylaminohydrolase (Pseudomonas genus), copper sulfate, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), β-galactosidase, glucose-6-phosphate dehydrogenase (yeast), glutamate dehydrogenase (bovine liver), glutamine synthetase, hexokinase (yeast), imidazole, lactate Acid dehydrogenase, L-alanine, L-aspartic acid, L-glutamic acid, magnesium chloride, magnesium sulfate, malate dehydrogenase (pig heart), manganese chloride, N-acetylcysteine, β-nicotinamide adenine dinucleotide (NAD), reduced β-nicotinamide adenine dinucleotide (NADH), o-nitrophenyl-β-D-galactopyranoside (ONPG), peroxidase (horseradish), phosphoenolpyruvate, phosphoenolpyruvate carboxylase, p-NPP, potassium ferrocyanide, potassium iodide, pyruvate kinase, sarcosine oxidase (microorganisms), sodium potassium tartrate, urease (canavon bean), zinc sulfate, and other buffers, surfactants, excipients, and preservatives.

[0142] Some aspects of the subject box are also described in U.S. Patent Application Publications 2018 / 0126381; 2019 / 0056304; 2019 / 0056384; 2019 / 0054466; and 2022 / 0274109, and U.S. Patent Applications 5,096,669; 7,177,767; 7,765,069; and 7,998,411, the disclosures of which are incorporated herein by reference in their entirety. In some instances, certain aspects of this box are provided in U.S. Provisional Application No. 63 / 612,715 (Attorney’s Case No. ADDV-151PRV), filed December 20, 2023, the disclosure of which is incorporated herein by reference. In some cases, certain aspects of this box are provided in U.S. Provisional Application No. 63 / 563,861 (Agent No. ADDV-143PRV), filed March 11, 2024.

[0143] As used herein, “sample,” “test sample,” or “biological sample” refers to a sample that contains or is suspected of containing the analyte of interest. Samples may originate from any suitable source. In some cases, samples may include liquids, flowing particulate solids, or fluid suspensions of solid particles. In some cases, samples may be treated prior to the analysis described herein. For example, a sample may be isolated or purified from its source prior to analysis; however, in some embodiments, untreated samples containing the analyte may be directly measured. The source of the analyte molecules may be synthetic (e.g., produced in a laboratory), environmental (e.g., air, soil, fluid samples such as water supply systems, etc.), animal (e.g., mammals), plant, or any combination thereof. In a particular example, the source of the analyte is human matter (e.g., bodily fluids, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, feces, tissues, organs, etc.). Tissues may include, but are not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervical tissue, skin, etc. The sample can be a liquid sample or a liquid extract of a solid sample. In some cases, the sample may be derived from an organ or tissue, such as a biopsy sample, which can be dissolved through tissue disintegration / cell lysis.

[0144] Fluid samples of a wide range of volumes can be analyzed. In a few exemplary embodiments, the sample volume may be approximately 0.5 nL, approximately 1 nL, approximately 3 nL, approximately 0.01 μL, approximately 0.1 μL, approximately 1 μL, approximately 5 μL, approximately 10 μL, approximately 50 μL, approximately 100 μL, approximately 1 mL, approximately 5 mL, approximately 10 mL, and so on. In some cases, the volume of the fluid sample is between approximately 0.01 μL and approximately 10 mL, between approximately 0.01 μL and approximately 1 mL, between approximately 0.01 μL and approximately 100 μL, between approximately 0.1 μL and approximately 10 μL, between approximately 1 μL and approximately 100 μL, between approximately 10 μL and approximately 100 μL, or between approximately 10 μL and approximately 75 μL.

[0145] In some cases, samples may undergo pre-analytical processing. Pre-analytical processing can provide additional functionality, such as non-specific protein removal and / or mixing functionality that can be implemented efficiently but inexpensively. General methods for pre-analytical processing may include the use of electrodynamic trapping, AC electrodynamics, surface acoustic waves, isovelocity electrophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art.

[0146] In some cases, fluid samples may be concentrated before use for assay. For example, in embodiments where the source of the analyte molecules is human bodily fluids (e.g., blood, serum), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. Fluid samples may be concentrated by approximately 1, 2, 3, 4, 5, 6, 10, 100 times, or more before use.

[0147] In some embodiments, the sample disclosed herein is whole blood. Samples used for hematology are typically whole blood. A whole blood sample consists of red blood cells, white blood cells, and platelets suspended in a protective yellow liquid called plasma. In some embodiments, samples used for immunoassays and clinical chemistry assays are typically serum or plasma. In some embodiments, whole blood samples are obtained from a subject. In some embodiments, the subject is a living subject, including animals and humans. After aspirating the whole blood sample from a sample tube, a portion of the whole blood sample must be removed from the whole blood sample so that serum or plasma can be separated from that portion for subsequent use in immunoassays or clinical chemistry assays.

[0148] In some embodiments, the sample of this disclosure is venous blood. As used herein, the term "venous blood" refers to a blood sample taken from a vein and examined for a specific substance released by nearby organs and tissues. An elevated level of the substance can be a sign of disease in an organ or tissue. In some embodiments, venous blood is collected via a venous blood collection procedure. For example, in venous blood collection, a needle is inserted into a vein to collect a blood sample for testing.

[0149] In some embodiments, the sample disclosed herein is capillary blood. As used herein, “capillary blood” or “capillary sample” refers to a blood sample obtained by pricking the skin. Capillary blood is typically obtained by pricking the fingers of adults and the heels of infants and young children. Capillaries are tiny blood vessels located near the surface of the skin. Compared to venous plasma, capillary plasma typically contains higher concentrations of protein, calcium, and chloride, and lower levels of potassium, sodium, and urea nitrogen.

[0150] In some embodiments, the sample disclosed herein is plasma. As used herein, the term "plasma" refers to a colorless fluid portion of blood, lymph, or milk in which blood cells or fat globules are suspended. Thus, plasma is the liquid component of blood and consists of water, proteins, metabolic waste products, minerals, coagulation factors, immunoglobulins, carbon dioxide, and hormones. Methods for separating plasma from blood are well known in the art. In an exemplary embodiment, plasma is generated when whole blood is collected into a tube treated with an anticoagulant. Blood does not clot in the plasma tube, thereby removing cells by centrifugation. The supernatant (labeled as plasma) is carefully removed from the cell clumps using a Pasteur dropper.

[0151] In some embodiments, the sample disclosed herein is serum. As used herein, the term "serum" refers to the aqueous, clear portion of an animal body fluid or plant sap. As used herein, the term "blood serum" refers to the amber-colored, protein-rich liquid that separates when blood clots. In some embodiments, serum includes, but is not limited to, blood serum, serous (or serous membrane) fluid secreted by serous glands, and plant sap. Methods for separating serum from blood are well known in the art. In an exemplary embodiment, blood serum is collected after whole blood has been allowed to clot. Clots are removed by centrifugation, and the resulting supernatant (labeled serum) is carefully removed using a Pasteur dropper.

[0152] In some embodiments, sample delivery includes transferring the sample from a sample collection dish to one or more inlets via a sponge swab sampler. In some embodiments, sample delivery includes transferring the sample from a sample collection dish to one or more inlets via Vacutainer®. In some embodiments, a sample collection device is used to collect a sample from a subject and deliver the sample to the device disclosed herein. In some cases, the sample collection device is inserted directly into the device to deliver the sample. In some cases, the sample in the sample collection dish is poured into an inlet of the device. In exemplary embodiments, one or more sample collection devices include, but are not limited to, syringes, sterile containers, standard urine collection dishes, sponge swab samplers, microsampling devices, microneedles or other minimally invasive painless blood collection devices; one or more blood collection tubes; blood collection needles; capillary blood collection tubes; other single-finger puncture blood collection devices, 16-gauge or other size needles, etc. Several devices are currently available for collecting, processing, and storing whole blood or other bodily fluids. In an exemplary embodiment, the blood collection device includes, for example, a micro-sampling device, a microneedle or other minimally invasive painless blood collection device; one or more blood collection tubes; a blood collection needle; a capillary blood collection tube; other single-finger puncture blood collection devices, etc.

[0153] In some embodiments, the blood collection device includes a venous puncture needle connected to one end of a sample bag via a conduit system. The conduit system is connected to the opposite end of the sample bag and communicates the sample bag with a blood bag. Using this device, blood from the subject passes through a first conduit system, the sample bag, and then through a second conduit system into the blood bag. When the blood bag is full, the conduit system closest to the blood bag is clamped. This is described in U.S. Patent No. 3,654,924, which is incorporated herein by reference. In some cases, the blood collected in the sample bag is transported to the sample holding chamber of the system disclosed herein.

[0154] In some embodiments, the blood collection device may include an integrated double-ended needle well known in the art. For example, a blood collection device including a double-ended needle is discussed by reference to U.S. Patent No. 5,086,780, which is incorporated herein by reference, wherein the double-ended needle is sheathed before use and safely re-sheathed after use, thereby minimizing the risk of accidental needle wounds and resulting infections. The blood collection device also functions as a holder for blood collection tubes during sample acquisition, wherein the blood collection tubes are easily inserted and removed through a rear-port opening of the device. In some embodiments, the collection tube is removed and inserted into the system of this disclosure for analysis. In other embodiments, the blood collection device itself may be removably coupled to the sample fluid analysis system of this disclosure.

[0155] In some embodiments, the sample collection device disclosed herein is a capillary collection device. For example, capillary collection devices include, but are not limited to, blood collection devices / needle devices and finger punctures. Blood collection devices are used to obtain blood samples from a finger or other alternative site on the subject. Exemplary blood collection devices are described in U.S. Patent Nos. 8,152,826 and 8,556,827, which are incorporated herein by reference.

[0156] In some embodiments, the blood collection device described in U.S. Patent No. 8,556,827 includes a blood collection needle and a torsion spring coupled to the blood collection needle via a blood collection needle holder. The torsion spring includes: an inner ring, an intermediate ring, and an outer ring arranged concentrically; a plurality of activation spring arms connecting the intermediate ring and the outer ring; and a plurality of return spring arms connecting the inner ring and the intermediate ring. In use, the plurality of activation spring arms and return spring arms can be independently switched between an energized and de-energized state using a single button-type mechanism. Rotation of this mechanism powers the activation and return spring arms. With the return spring arm in its energized state, pressing the mechanism changes the activation spring arm from its energized state to its de-energized state, which drives the blood collection needle from a retracted position to an extended position. Once the activation spring arm reaches its de-energized state, the return spring arm changes from its energized state to its de-energized state, which pulls the blood collection needle from its extended position back to its retracted position.

[0157] In some embodiments, blood samples are drawn from a subject by medical laboratory scientists, medical practitioners, some emergency medical technicians, paramedics, phlebotomists, and other caregivers. The blood samples are then collected into vacuum tubes. In some embodiments, one or more vacuum tubes containing the blood samples are transported to the system disclosed herein. In some embodiments, the tubes contain various additives, or none at all. For example, whole blood samples need to be mixed with EDTA, which chelates calcium to prevent the whole blood sample from clotting, unless clotting time is the test to be measured, in which case citrate is used. Most biochemical tests are performed on serum and therefore use plain tubes or clotting accelerators. In some cases, some assays may also require whole blood but are interfered with by EDTA, and in such cases, lithium heparin is a suitable alternative. Procedures for sample collection by phlebotomists are well known in the art.

[0158] In some embodiments, the sample disclosed herein is cerebrospinal fluid (CSF). The term "cerebrospinal fluid (CSF)" refers to the clear fluid that surrounds and protects the brain and spinal cord. Analysis of CSF can look for proteins, sugars (glucose), and other substances. Methods for collecting CSF are well known in the art. In exemplary embodiments, CSF is typically obtained via lumbar puncture (spinal puncture). During the procedure, a needle is typically inserted between the 3rd and 4th lumbar vertebrae, and CSF fluid is collected for testing.

[0159] In some embodiments, the sample disclosed herein is saliva. As used herein, the term "saliva" refers to a watery fluid secreted by glands into the oral cavity, which provides lubrication for chewing and swallowing and aids digestion. Saliva consists of 99% water and 1% protein and salt. Methods for collecting saliva are well known in the art. In some embodiments, saliva samples may be refrigerated for up to one week, after which they need to be added to a stable fluid in a tube.

[0160] In some embodiments, the sample disclosed herein is urine. As used herein, the term "urine" refers to a watery, typically pale yellow fluid stored in the bladder and expelled through the urethra. Urine is one of the primary means by which the human body excretes excess water and salt, and also contains nitrogenous compounds (such as urea) and other waste products removed from the blood by the kidneys. Collecting urine samples is well known in the art. In exemplary embodiments, a "first-catch" or "mid-stream" sample of urine is collected in a completely sterile container. The first-catch urine sample is the first portion of the outflowing urine. The mid-stream urine sample is collected to reduce the risk of contamination of the sample with bacteria from the hands or skin around the urethra or from the tubes that carry the urine out of the body. In some embodiments, the collected urine sample may be stored in a sealed plastic bag in a refrigerator at 4°C for no more than 24 hours. In some embodiments, the urine sample is used for infections (such as urinary tract infections (UTIs)), some sexually transmitted infections (STIs) (such as male chlamydia), or kidney damage (such as an ACR test).

[0161] In some embodiments, the sample of this disclosure is interstitial fluid. As used herein, the expressions “interstitial fluid (ISF),” “lymph,” or “tissue fluid” refer to a clear fluid occupying the space between cells in the body or a fluid found in the space surrounding cells. It originates from substances leaking from capillaries. Interstitial fluid helps carry oxygen and nutrients to cells and remove metabolic waste from cells. As new interstitial fluid is produced, it replaces the old fluid, which drains toward the lymphatic vessels. Methods for collecting interstitial fluid are well known in the art. In one embodiment, ISF can typically be collected from the skin using a suction blister by applying suction to the skin at a high temperature for up to 1 hour to create a blister filled with ISF.

[0162] In some embodiments, the sample disclosed herein is intestinal fluid. Intestinal fluid, or gastrointestinal fluid, contains, for example, electrolytes, bile salts, lipids and lipid digestion products, cholesterol, proteins, enzymes, and other components, and may vary depending on anatomical location (stomach vs. small intestine vs. colon). Methods for collecting intestinal fluid samples are well known in the art. In some embodiments, intestinal fluid may be collected via a nasojejunal tube and encapsulated using a lyophilized powder method.

[0163] In some embodiments, the samples of this disclosure are samples collected from nasal swabs. In some embodiments, the samples of this disclosure are samples collected from pharyngeal swabs. In some embodiments, the samples of this disclosure are samples collected from vaginal swabs. Nasal swabs, pharyngeal swabs, and vaginal swabs are well known in the art.

[0164] In some embodiments, the sample includes respiratory specimens. For example, respiratory specimens include, but are not limited to, nasal swabs, throat swabs, sputum, tracheal / bronchial secretions, and bronchoalveolar lavage fluid. In some embodiments, respiratory sampling includes upper respiratory tract materials and lower respiratory tract secretions. In some cases, upper respiratory tract materials include nasal swabs, throat swabs, etc. In other cases, lower respiratory tract secretions include sputum, tracheal / bronchial secretions, bronchoalveolar lavage fluid, etc. In some embodiments, sputum is collected using procedures well known in the art. For example, the steps for collecting sputum are as follows: i) take a deep breath and hold it for 5 seconds; ii) exhale slowly; iii) take another deep breath and cough forcefully until some sputum rises into the mouth; iv) spit the sputum into a sample container. In some embodiments, tracheal / bronchial secretions are collected by inserting a suction catheter as deeply as possible and aspirating the secretions, as is well known in the art. In some embodiments, bronchoalveolar lavage fluid is collected using bronchoscopy, as is well known in the art.

[0165] In some embodiments, the sample includes any tissue obtained from the subject. In other embodiments, the sample includes any cells obtained from the subject. The subject is any living subject, including humans. In some embodiments, the tissue may include, but is not limited to, skeletal muscle tissue, liver tissue, heart tissue, lung tissue, pancreatic tissue, adipose tissue, stomach tissue, gastrointestinal tissue, colon tissue, kidney tissue, myocardial tissue, brain tissue, breast tissue, nerve tissue, bone marrow, cervical tissue, skin, etc. In some embodiments, the cells may include, but are not limited to, skeletal muscle cells, hepatocytes, heart cells, lung cells, pancreatic cells, fat cells, stomach cells, gastrointestinal cells, colon cells, kidney cells, myocardial cells, brain cells, breast cells, nerve cells, bone marrow cells, cervical cells, skin cells, etc. In some cases, the sample is a tumor or cancer cell. For example, samples may include, but are not limited to, brain cancer cells, liver cancer cells, pancreatic cancer cells, lung cancer cells, breast cancer cells, kidney cancer cells, metastatic cancer cells, ovarian cancer cells, colorectal cancer cells, bladder cancer cells, thyroid cancer cells, lymphoma cells, cervical cancer cells, gynecological cancer cells, head and neck cancer cells, mesothelioma cells, myeloma cells, skin cancer cells, prostate cancer cells, uterine cancer cells, vaginal and vulvar cancer cells, etc. In some cases, the sample may be derived from an organ or tissue, such as a biopsy sample, which can be dissolved through tissue disintegration / cell lysis. In some embodiments, the sample may be processed before performing an immunoassay. For example, the sample may be concentrated, diluted, purified, amplified, etc.

[0166] In some embodiments, the apparatus of this disclosure may be used to measure, detect, or evaluate one or more analytes in a sample. The sample may be any test sample containing or suspected of containing an analyte. As used herein, the terms “analyte,” “target analyte,” and “analyte” are used interchangeably and refer to an analyte measured in the apparatus disclosed herein. The examples of analytes provided herein are for illustrative purposes and are not intended to limit the scope of this disclosure.

[0167] The systems and methods disclosed herein are capable of detecting blood cells or blood cell types. Blood cells and blood cell types that can be detected by the systems, devices, and methods disclosed herein include, but are not limited to, red blood cells, hemoglobin, white blood cells (including neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelets, reticulocytes, and nucleated red blood cells. Various measurements can be performed on different blood components, including, but not limited to, cell count, cell size, cell complexity, granularity, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin level, and mean corpuscular hemoglobin concentration. In some embodiments, the measurements disclosed above can be performed using staining-independent methods in the absence of histological staining.

[0168] In some embodiments, but not limited to, the analyte may be a pathogen, prion protein, cancer cell, blood component, or biomolecule. In some cases, the pathogen is, but is not limited to, a virus, bacteria, fungus, or protozoa. In some cases, the prion protein may be produced in sporadic prion diseases, hereditary prion diseases, or acquired prion diseases. In some cases, the cancer cell may be a cancer cell derived from a tumor or circulating tumor cells. In some cases, the blood component may be red blood cells, white blood cells, platelets, or proteins found in the blood. In some cases, the biomolecule may be a metabolite, a macromolecule, a protein, or a chemical compound. Any combination of analytes can be measured using the methods and systems of this disclosure.

[0169] In some cases, the determinations of this disclosure can be used to determine the presence or absence of an analyte in a sample or to measure the amount of an analyte in a sample to identify or assess a disease or condition. Measurements of the analyte can be used (e.g., but not limited to) to determine the likelihood of having a disease or condition; to diagnose, identify, or classify a disease or condition; to estimate prognosis; to determine the severity of a disease or condition; to determine appropriate treatment; to predict the response of a disease or condition to treatment; to monitor the response of a disease or condition to treatment; to determine treatment effectiveness; and to identify recurrence of a disease or condition.

[0170] The analytes / properties that the sensor responds to can be selected from the following: particles (e.g., blood cells or microparticles), human chorionic gonadotropin (hCG), pH, partial pressure CO2, partial pressure O2, glucose, lactate, creatinine, urea, sodium, potassium, chloride, calcium, magnesium, phosphate, hematocrit, prothrombin time (PT), activated partial thromboplastin time (APTT), activated clotting time (ACT), D-dimer, prostate-specific antigen (PSA), creatine kinase ME (CKMB), and brain natriuretic peptide (BNP). The blood sample contains, but is not limited to, troponin I (Tni), cardiac troponin (cTni), human chorionic gonadotropin (hCG), troponin T, troponin C, myoglobin, neutrophil gelatinase-associated lipotransferase (NGAL), galactoglobin-3, prostate-specific antigen (PSA), parathyroid hormone (PTH), galactoglobin-3, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein, bilirubin, alkaline phosphatase (ALP), and combinations thereof. In various embodiments, an optical sensor is configured to convert light received from cells within a portion of the imaging chamber into an output signal, and a processor connected to the optical sensor is configured to convert the output signal into a count or percentage of each cell type in the blood sample. In some embodiments, a blood cell differential count is a measurement of the number or percentage of each cell type (e.g., white blood cells (WBC)) in a whole blood sample. Cell types include red blood cells, white blood cells, and platelets. Imaging can distinguish various types of white blood cells, including neutrophils, lymphocytes, granulocytes, eosinophils, basophils, and monocytes. Blood cell differential counting can also reveal the presence of any abnormal or immature cells. Preferably, the analyte / property is tested in a liquid sample of whole blood; however, other samples can be used, including blood, serum, plasma, urine, cerebrospinal fluid, saliva, and their modified forms.

[0171] Medical diagnostics often involve the analysis of whole blood samples from patients. One of the more popular diagnostic methods is whole blood counting (referred to as “CBC”), a suite of tests that, in addition to enumeration of cell components, may include red blood cell indices, reticulocyte count, and white blood cell differential count (“LDC”; sometimes referred to as “white blood cell differential”), which identifies and counts the various types of white blood cells (WBCs) present in a blood sample. In some embodiments, blood cell differential counting includes: (i) identifying cells, such as white blood cells, residing within a chamber of the sample; (ii) quantitatively analyzing at least some of the identified cells within an image with respect to one or more predetermined, quantitatively determinable features; and (iii) identifying at least one type of cell from the identified cells using the quantitatively determinable features. For example, to perform blood cell differential counting (such as LDC), an algorithm utilizes a set of identifying features, each of which is distinguishable from other features and each of which is quantitatively determinable from a sample image. Each WBC may be characterized by the presence or absence of certain identifying features and / or quantitative information associated with certain features. In order to provide the disclosure enabling the implementation, the invention is described herein with respect to an exemplary set of identifying features that can be used to selectively identify and distinguish WBCs. This set does not include all possible features, and therefore the invention is not limited to this particular set.

[0172] For WBC analysis, if acridine orange is used, for example, an exemplary set of identifying features includes those that are named: cell, nucleus, leaf number, cell area, nuclear area, large particle ratio, nuclear ratio, red-to-green ratio, nuclear shape, cell shape, nuclear brightness, cytoplasmic brightness, average cell absorption at a given wavelength, nuclear texture, cytoplasmic texture, cell absorption texture at a given wavelength, nuclear indentation, and cytoplasmic indentation; each of the above is described in U.S. Patent Publication No. 2012 / 0034647, which is incorporated herein by reference. In some cases, certain features directly provide information about a particular cell (e.g., nuclear shape). In other cases, a feature (e.g., cell area) may be used to indirectly provide information about a particular cell (e.g., the ratio of nuclear area to cell area—referred to above as the “nuclear ratio”, etc.). Identifying features are based on quantifiable properties such as light intensity, light color, OD, area, and relative position (e.g., shape). As indicated above, color can be generated by incorporating one or more fluorescent colorants into a sample, which emit fluorescence at a specific wavelength associated with a particular color upon excitation. It should be understood that this principle also applies to the detection of non-fluorescent dyes based on absorbance at a specific wavelength associated with a particular color.

[0173] An example of an acceptable staining agent that can be used when performing LDC on whole blood samples is acridine orange (“ACO”). ACO is a fluorescent dye that selectively stains components within a sample when mixed with whole blood; for example, leukocytes, platelets, reticulocytes, and nucleated erythrocytes. With respect to WBCs, ACO permeates through the respective WBC and stains its DNA and RNA. The color emitted by the dye within the WBC depends on several factors, including the amount of RNA and DNA within the dye, the concentration of the dye in the component, and the pH of the component. This invention is not limited to the use of ACO, and other dyes (e.g., Astrazon orange) may be used in place of or in combination with ACO. Using ACO and leukocytes as examples, if a sample is subjected to excitation light at or around 470 nm, ACO bound to material within the nucleus of leukocytes (e.g., DNA) will emit light at approximately 540 nm (which appears green), and ACO bound to material within the cytoplasm of leukocytes (e.g., RNA) will emit light at approximately 660 nm (which appears red).

[0174] In some embodiments, one or more analytes may be cells, such as circulating tumor cells. In other embodiments, the analytes are biological cells (e.g., mammals, birds, reptiles, other vertebrates, insects, yeast, bacteria, cells, etc.). In other embodiments, the analytes may be infectious pathogens, such as bacteria (e.g., Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria, Pseudomonas aeruginosa, Salmonella O8, and Salmonella enteritidis), viruses (e.g., retroviruses (such as HIV), herpesviruses, adenoviruses, lentiviruses, filoviruses (e.g., West Nile virus, Ebola virus, and Zika virus), hepatitis viruses (e.g., hepatitis A, B, C, D, and E), human papillomavirus, parvovirus, etc.), parasites, or fungal spores.

[0175] In exemplary embodiments, one or more analytes are tumors or cancer cells. In some cases, cancer cells can be detected directly, for example, by detecting nucleic acids or antigens specific to cancer cells. In some cases, the presence of cancer cells can be detected by changes or mutations in the nucleic acid sequences of cancer cells, including but not limited to SNPs, insertions, deletions, chromosomal translocations, or gene amplifications. In some cases, cancer cells can be detected by detecting the presence of tumor or cancer markers associated with cancer cells. In some cases, cancer cells can be detected by detecting the expression of receptors associated with cancer cells. In some cases, cancer cells can be detected indirectly, for example, metabolic markers associated with cancer cells can indicate their presence.

[0176] For example, cancer cell types that can be detected by the assays disclosed herein include, but are not limited to, cancer cells, leukemia cells, lymphoma cells, myeloma cells, sarcoma cells, central nervous system cancer cells, and mesothelioma cells. Specific types of cancer include, but are not limited to, bone cancer (including Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brain tumors, breast cancer, cervical cancer, colorectal cancer, endometrial cancer (uterine cancer), esophageal cancer, head and neck cancer, hepatocellular carcinoma (liver cancer), Hodgkin's lymphoma, renal cell carcinoma, gynecological cancer cells, vaginal and vulvar cancer cells, leukemia, lung cancer (non-small cell lung cancer, small cell lung cancer, pleural pulmonary blastoma, myofibroblastic tumor, and tracheobronchial tumor), lymphoma, melanoma, multiple myeloma / plasma cell tumor, neuroblastoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, and thyroid cancer.Cancer biomarkers include, but are not limited to: ALK gene rearrangement and overexpression, alpha-fetoprotein (AFP), B-cell immunoglobulin gene rearrangement, BCL2 gene rearrangement, β-2-microglobulin (B2M), β-human chorionic gonadotropin (β-hCG), bladder tumor antigen (BTA), BRCA1 and BRCA2 gene mutations, BCR-ABL fusion gene (Philadelphia chromosome), RAF V600 mutation, C-kit / CD117, CA15-3 / CA27.29, CA19-9, CA-125, and CA. 27.29, Calcitonin, Carcinoembryonic Antigen (CEA), CD19, CD20, CD22, CD25, CD30, CD33, Chromogranin A (CgA), Chromosomal 17p deletion, Chromosomal 3, 7, 17, and 9p21, Epithelial-derived circulating tumor cells (CELLSEARCH), Cytokeratin fragments21-1, Cyclin D1 (CCND1) gene rearrangement or expression, Degamma-carboxyprothrombin (DCP), DPD gene mutation, EGFR gene mutation, Estrogen receptor (ER) / Progesterone receptor (PR), FGFR2 and FGFR 3. Gene mutations, fibrin / fibrinogen, FLT3 gene mutations, gastrin, HE4, HER2 / neu gene amplification or protein overexpression, 5-HIAA, IDH1 and IDH2 gene mutations, immunoglobulins, IRF4 gene rearrangement, JAK2 gene mutations, KRAS gene mutations, lactate dehydrogenase, microsatellite instability (MSI) and / or mismatch repair deficiency (dMMR), MYC gene expression, MYD88 gene mutations, myeloperoxidase (MPO), neuron-specific enolase (NSE), NTRK gene fusion, nuclear matrix protein 22, PCA3 mRNA, PML / RARα fusion gene, prostatic acid phosphatase (PAP), programmed death-ligand 1 (PD-L1), prostate-specific antigen (PSA), ROS1 gene rearrangement, soluble mesothelin-related peptide (SMRP), somatostatin receptor, T-cell receptor gene rearrangement, terminal transferase (TdT), thiopurine S-methyltransferase (TPMT) enzyme activity or TPMT gene assay, thyroglobulin, UGT1A1*28 variant homozygosity, urinary catecholamines: VMA and HVA, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1), FoundationOneCDx (F1CDx) genome assay, Guardant360 CDx genome assay, 5-protein signature (OVA1), 17-gene signature (Oncotype DX GPS assay), 21-gene signature (Oncotype DX), 46-gene signature (Prolaris), 70-gene signature (Mammaprint).

[0177] In addition, cancer cell types that can be detected by the assays disclosed herein include: gastric cancer cells (e.g., HGC-27 cells); non-small cell lung cancer (NSCLC) cells; colorectal cancer cells (e.g., DLD-1 cells); H23 lung adenocarcinoma cells; Ramos cells; T-cell acute lymphoblastic leukemia (T-ALL) cells; CCRF-CEM cells; acute myeloid leukemia (AML) cells (e.g., HL60 cells); small cell lung cancer (SCLC) cells (e.g., NCI-H69 cells); human glioblastoma cells (e.g., U118-MG cells); prostate cancer cells (e.g., PC-3 cells); HER-2 overexpressing human breast cancer cells (e.g., SK-BR-3 cells); and pancreatic cancer cells (e.g., Mia-PaCa-2 cells).

[0178] In exemplary embodiments, one or more analytes are viruses. In some cases, viruses are detected directly, for example, by detecting virus-specific nucleic acids or antigens. In other cases, viruses are detected indirectly, for example, by detecting antiviral antibodies produced by the subject that may indicate the presence of a virus, or by detecting a virus that induces hemagglutination in the blood. For example, viruses detectable by the assays of this disclosure include animal, plant, fungal, and bacterial viruses. In other embodiments, viruses detectable by the assays of this disclosure include viruses that affect animals, particularly mammals, especially humans and livestock. In other embodiments, viruses detectable by the methods described in this disclosure include, but are not limited to: papillomaviruses, such as polyomavirus and SV40; poxviruses, such as vaccinia virus and smallpox (smallpox); adenoviruses, such as human adenovirus; herpesviruses, such as human herpes simplex virus type I and II; parvoviruses, such as adeno-associated virus (AAV); reoviruses, such as rotavirus and human reovirus; picornaviruses, such as poliovirus; cloacal viruses, including group A viruses, such as Sindbis virus and Semlee forest virus (SFV), and group B viruses, such as dengue virus, yellow fever virus, and St. Louis encephalitis virus; retroviruses, such as lentiviruses and HIV. Viruses I and II, Rous sarcoma virus (RSV) and mouse leukemia virus; rod-shaped viruses, such as vesicular stomatitis virus (VSV) and rabies virus; paramyxoviruses, such as mumps virus, measles virus and Sendai virus; arenaviruses, such as Lassa virus; bunyaviruses, such as bunyawere (encephalitis); coronaviruses, such as common cold virus, gastrointestinal upset virus; orthomyxoviruses, such as influenza; caliciviruses, such as norovirus, hepatitis E virus; filoviruses, such as Ebola virus and Marburg virus; and astroviruses, such as astrovirus, etc. Specific examples of viruses include, but are not limited to, sinobovirus, influenza (especially H5N1 influenza), herpes simplex virus (HSV1 and HSV-2), Coxsackievirus, human immunodeficiency virus (I and II), Andean virus, dengue virus, Ebola virus (monocytosis), smallpox (small pox) and other poxviruses, West Nile virus, hepatitis viruses (e.g., A, B, C, D and E), HPV, SARS-CoV-2 (COVID-19), CMV, parvovirus B19, chlamydia, gonorrhea, Zika virus, chikungunya virus, babesi, malaria and Usutu virus.

[0179] In some embodiments, one or more analytes may be bacteria. In some cases, bacteria are detected directly, for example, by detecting bacterial-specific nucleic acids or antigens. In other cases, bacteria are detected indirectly, for example, by detecting antimicrobial antibodies produced by the subject, or by detecting the presence of bacterial enzyme activity products. For example, bacteria that can be detected by the methods described in this disclosure include, but are not limited to: denitrifying achromobacterium, xylose-oxidizing achromobacterium, Acinetobacter baumannii, calcium acetate Acinetobacter, Actinomyces isatis, Balloonellarella kerensiensis, Aeromonas hydrophila, Aeromonas sobrio, Actinomyces sympathomimeticus, Alcaligenes faecalis, Allergan serotoninus, vaginal anaerobic cocci, geminidia anaerobic cocci, Cryptococcus hemolyticus, Cryptococcus pyogenes, Arthrobacter cammings, Atobacillus vaginalis, Bacillus anthracis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus spheroides, Bacillus subtilis, Bacteroides davidiana, Bacteroides sclerotiorum, Bacteroides fragilis, Bacteroides dauricum, Bacteroides salils, Bacteroides polymorpha, Bacteroides monomorpha, Bacteroides succinate, Bartonella henselae, Bartonella pentaphyllum, Bifidobacterium bifidum, Bifidobacterium breve. *Bacillus vortexi*, *Bordetella pertussis*, *Borrelia burgdorferi*, *Treponema pallidum*, *Bacillus lateralis*, *Brucella abortus*, *Brucella melitensis*, *Brucella suis*, *Burkholderia cepacia*, *Burkholderia melitensis*, *Burkholderia pseudomelitensis*, *Campylobacter coli*, *Campylobacter flexneri*, *Campylobacter jejuni*, *Campylobacter rectum*, *Caucasian gingivalis*, *Caucasian granulosum*, *Caucasian hemolyticus*, *Caucasian sputum*, *Humanoidobacterium*, *Citrobacter meningitidis*, *Citrobacter amalgam*, *Citrobacter freundii*, *Citrobacter kohlii*, *Clostridium butyricum*, *Clostridium difficile*, *Clostridium histolyticum*, *Clostridium helemonii*, *Clostridium putrefactive*, *Clostridium perfringens*, *Clostridium perfringens*, *Clostridium septicum*, *Clostridium sporogenes*, *Clostridium proximalense*, *Clostridium triquetrum*, *Clostridium tetanus*, *Corynebacterium amylopectin*, *Corynebacterium spp.* (confusum), Corynebacterium diphtheriae, Corynebacterium glucuronide, Corynebacterium jejuni, Corynebacterium krusei, Corynebacterium micranthum, Corynebacterium minimus, Corynebacterium pseudodiphtheriae, Corynebacterium pseudotuberculosis, Corynebacterium reesei, Corynebacterium tuberculosis stearate, Corynebacterium ulcerans, Corynebacterium xerosis, Edwardsiella tarda, Egeria profusely, Eikelium ergium, Elizabethan bacillus meningitidis, Sterilobacterium shortness of breath, Enterobacter aerogenes, Enterobacter aerogenes, Dark Enterobacter sakazakii, Enterococcus avianus, Enterococcus bovis, Enterococcus typhimurium, Enterococcus cecum, Enterococcus atypical, Enterococcus durableis, Enterococcus faecium, Enterococcus flavus, Enterococcus rota, Enterococcus foetida, Enterococcus hydatopsus, Enterococcus helix, Enterococcus italicum, Enterococcus stoloniferus, Enterococcus montelukast, Enterococcus pallidus, Enterococcus pseudoavianus, Enterococcus raffinis, Enterococcus hemorrhagicus, Erysipelothrix aureus, Escherichia coli, Escherichia coli, Myxobacterium mucosae, and Bacillus venereum.*Fusobacterium tularemia*, *Fusobacterium necrophorum*, *Fusobacterium nucleatum*, *Fusobacterium periodontium*, *Fusobacterium proteoglycanum*, *Gardnerella vaginalis*, *Twincocephalus measlese*, *Bacillus stearothermophilus*, *Streptococcus septembrittleus*, *Haemophilus ducreyi*, *Haemophilus influenzae*, *Haemophilus parainfluenzae*, *Haffniella vesicella*, *Haemophilus virgaurea*, *Helicobacter pylori*, *Grinella aureus*, *Granularia granulomatosa*, *Klebsiella pneumoniae*, *Klebsiella pneumoniae*, *Lactobacillus acidophilus*, *Lactobacillus curvularia*, *Lactobacillus delbrueckii*, *Lactobacillus jenny*, *Lactococcus globosum*, *Legionella pneumophila*, *Leptospira taeniorhynchus*, *Listeria monocytogenes*, *Micrococcus luteus*, *Morganella catarrhalis*, *Mycoplasma genitalium*, *Mycoplasma hominis*, *Neisseria gonorrhoeae*, *Cerebrospinal fluid* Neisseria meningitidis, Nocardia gailles of Gelsenkirchen, Visceral Odor Bacterium, Pantotheca clumps, Parabacterium difficile, Micromonas microsporum, Pasteurella multocida, Pediococcus toxicans, Peptidoglycan, Peptidoglycan, Anaerobic Peptostreptococcus, Shigella-like Porphyromonas, Porphyromonas gingivalis, Prevotella bisporus, Prevotella bisporus, Prevotella humanis, Prevotella intermedia, Prevotella melaninogenica, Prevotella melanogaster, Prevotella timoninii, Prevotella propria, Propionibacterium acnes, Propionibacterium greaseriformis, Propionibacterium granulosum, Proteus mirabilis, Proteus vulgaris, Providencia reticularis, Providencia szepus, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Fouleiformis. *Pseudomonas aeruginosa*, *Rochetomyces cariolatus*, *Rochetomyces villosa*, *Salmonella enterica*, *Serratia marcescens*, *Serratia marcescens*, *Shigella boydii*, *Shigella dysenteriae*, *Shigella flexneri*, *Shigella sonnei*, *Symplocos spp.*, *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus capitis*, *Staphylococcus carinatum*, *Staphylococcus caudatus*, *Staphylococcus epidermidis*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus ludens*, *Staphylococcus pasteurellii*, *Staphylococcus precatorius*, *Staphylococcus suis*, *Staphylococcus saprophyticus*, *Staphylococcus stearothermiae*, *Staphylococcus mimicus*, *Staphylococcus waleckii*, *Staphylococcus xylose*, *Stenotrophomonas maltophilia*, *Streptococcus candida*, *Streptococcus agalactiae*, *Streptococcus pharyngitis*, *Bovine tadalafil*, *Staphylococcus aureus*, *Staphyl ... Streptococci, including *Streptococcus canis*, *Streptococcus constellans*, *Streptococcus galactiae*, *Streptococcus equi*, *Streptococcus gallolyticus*, *Streptococcus globulus*, *Streptococcus infantis*, *Streptococcus dolphinus*, *Streptococcus intermedia*, *Streptococcus Paris*, *Streptococcus proteus*, *Streptococcus stomatum*, *Streptococcus pasteurellosis*, *Streptococcus pneumoniae*, *Streptococcus hognatus*, *Streptococcus pyogenes*, *Streptococcus salivarius*, *Streptococcus salivarius*, *Streptococcus salivarius*, *Streptococcus vestibulum*, *Streptococcus waleckii*, *Treponema pallidum*, *Ureaplasma microsporum*, *Lycoccus fluvialis*, *Veillonella atypical*, *Veillonella septemlobus*, *Vibrio alginolyticus*, *Vibrio cholerae*, *Vibrio flexneri*, *Cryptolytica parahaemolyticus*, *Vibrio vulnificus*, *Yersinia enterocolitica*, *Yersinia pestis*, and *Yersinia pseudotuberculosis*.

[0180] In exemplary embodiments, one or more analytes may be fungi. In some cases, fungi are detected directly, for example, by detecting fungal-specific nucleic acids or antigens. In some cases, fungi are detected indirectly, for example, fungal cell wall components released into the bloodstream may indicate the presence of fungi. In some embodiments, the fungi that can be detected by the assays of this disclosure include, but are not limited to, fungi from genera selected from the group consisting of: Candida, Aspergillus, Rhizopus, Cryptococcus, Histoplasma, Pneumocystis, Staphylococcus, Sporothrix, Trichophyton, Microsporum, Blastomyces, Mucormycetes, Coccidioides, Helicobacter, Cladosporium, Hemosporium spheroidum, and Cerebrospinal fluid. The genera *Endoprotozoa*, *Ceretoprotozoa*, *Fusarium*, *Pterocytospora*, *Malassezia*, *Protosporum*, *Pythium*, *Rhodotorula*, *Fusarium*, *Rhizopus*, *Verticillium*, *Gastrodia*, *Sclerotinia*, *Ustilago*, *Rhizopus*, *Pseudomonas*, *Armillaria*, *Gyromyces*, *Pyromyces*, *Coccidioidomyces*, *Anthracis*, *Scenedesmus*, *Saprolegnia*, *Fish Sporozoa*, *Exophyllum*, *Gillobryctomyces*, and *Penicillium*. Specific examples of fungal species that can be detected by the determinations in this disclosure include, but are not limited to, *Candida albicans*, *Candida glabrata*, *Candida parapsilosis*, *Candida tropicalis*, and *Candida auris*; *Cryptococcus neoformans* and *Cryptococcus gattii*; *Coccidioides immitis* and *Coccidioides posadas*; *Histoplasma capsulatum*; *Blastomyces dermatitidis*; and *Pneumocystis yew*.

[0181] In some embodiments, one or more analytes may be protozoa. In some cases, protozoa are detected directly, for example, by detecting protozoan-specific nucleic acids or antigens. In other cases, protozoa are detected indirectly, for example, by protozoan metabolites indicating their presence. In some cases, the classes of protozoa that can be detected by the assays of this disclosure include, but are not limited to, Plasmodium (malaria), Leishmania (leishmaniasis), Trypanosoma (sleeping sickness and Chagas disease), Cryptosporidium, Giardia lamblia, Toxoplasma gondii, Babesia, small giardia, and endoamoeba. Specific examples of protozoa that can be detected by the assays of this disclosure include, but are not limited to, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, Leishmania donovani, Trypanosoma brucei, Trypanosoma krusei, Toxoplasma gondii, and Babesia microsauria.

[0182] In exemplary embodiments, one or more analytes may be prion proteins. In some cases, prions are detected directly, for example, by detecting prion-specific nucleic acids or antigens. In other cases, the presence of prions or the potential for prion formation is detected by identifying mutations in the nucleic acid sequence. In some cases, the presence of structures formed by prions can indicate the presence of prions. In some cases, prions are detected indirectly, for example, biochemical changes induced by prion formation can indicate the presence of prions. In some cases, prions are amplified prior to detection using methods such as protein misfolding cycle amplification (PMCA) or real-time vibration-induced transformation (RT-QUIC). Exemplary prion proteins include, but are not limited to: scrapie in sheep (sheep and goats), transmissible mink encephalopathy (TME), chronic wasting disease (CWD) in mule deer and elk, bovine spongiform encephalopathy (BSE) in cattle, feline spongiform encephalopathy (FSE) in cats, ectopic ungulate encephalopathy (EUE), human kuru, human Creutzfeldt-Jakob disease (CJD), human lethal familial insomnia (FFI), and human Gerstmann-Straussler-Scharink syndrome (GSS).

[0183] In some embodiments, one or more analytes measured by the methods and systems of this disclosure may be blood components. Examples of blood components that can be detected by the measurements of this disclosure include, but are not limited to, red blood cells, hemoglobin, white blood cells (including neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelets, reticulocytes, and nucleated red blood cells. Various measurements can be performed on different blood components, including but not limited to cell count, cell size, cell complexity, granularity, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin level, and mean corpuscular hemoglobin concentration.

[0184] In some embodiments, one or more analytes may be biomolecules. Non-limiting examples of biomolecules include macromolecules, such as, for example, proteins, lipids, and carbohydrates. In some cases, analytes may be hormones, antibodies, growth factors, cytokines, electrolytes (e.g., sodium, potassium, and chloride), enzymes (e.g., alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and amylase), receptors (e.g., nerve, hormone, nutrient, and cell surface receptors) or ligands thereof, cancer markers (e.g., PSA, TNF-α), markers of myocardial infarction (e.g., troponin, creatine kinase, etc.), toxins, drugs (e.g., therapeutic drugs, addictive drugs), metabolites (e.g., including vitamins and minerals), metabolites (e.g., glucose, urea nitrogen, triglycerides, uric acid), nutrients, etc. Non-limiting embodiments of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, etc. In some embodiments, analytes may be post-translational modified proteins (e.g., phosphorylated, methylated, glycosylated proteins). In some embodiments, the analyte is a nucleic acid. In other embodiments, the analyte is a protein or a small molecule.

[0185] A non-limiting list of analytes that can be analyzed by the apparatus presented herein includes Aβ42 amyloid β-protein, fetoglobulin-A, tau, secretory granulin II, prions, α-synuclein, tau protein, neurofilament light chains, pergenin, PTEN-induced putative kinase 1, DJ-1, leucine-rich repeat kinase 2, mutant ATP13A2, Apo H, ceruloplasmin, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), thyroxine transporter, vitamin D-binding protein, pro-apoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3 (sperm), p14 endothelial cell-specific molecular fragment, serum, ACE2, autoantibodies against CD25, hTERT, CAI25 (MUC) 16) VEGF, sIL-2, osteopontin, human epididymal protein 4 (HE4), alpha-fetoprotein, albumin, albuminuria, microalbuminuria, neutrophil gelatinase-associated lipotransferase (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), liver fatty acid-binding protein (L-FABP), LMP1, BARF1, IL-8, carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, LZTS1, α-amylase, carcinoembryonic antigen, CA 125, IL-8, thioredoxin, β-2 microglobulin levels – monitoring viral activity, tumor necrosis factor-α receptor – monitoring viral activity, CA15-3, follicle-stimulating hormone (FSH), luteinizing hormone (LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin, EC39, bimodalin, dUTPase, secretory coagulant protein (pGSN), PSA (prostate-specific antigen), thymosin β15, insulin, plasma C-peptide, glycosylated hemoglobin (HBA1c), C-reactive protein (CRP), interleukin-6 (IL-6), ARHGDIB (Rho GDP dissociation inhibitor 2), CFL1 (filoprotein-1), PFN1 (inhibitor-1), GSTP1 (glutathione S-transferase P), S100A11 (protein S100-A11), PRDX6 (peroxidase-6), HSPE1 (10 kDa heat shock proteinmitochondria), LYZ (lysozyme C precursor), GPI (glucose-6-phosphate isomerase), HIST2H2AA (histone H2A) 2-A type), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HSPG2 (precursor to heparan sulfate proteoglycan core protein of basement membrane), LGALS3BP (precursor to galactoglobin-3-binding protein), CTSD (precursor to cathepsin D), APOE (precursor to apolipoprotein E), IQGAP1 (RasGTPase activator-like protein IQGAP1), CP (precursor to ceruloplasmin) and IGLC2 (IGLC1 protein), PCDGF / GP88, EGFR, HER2, MUC4, IGF-IR, p27 (kip1), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1 (phosphoinositol-dependent protein kinase-1), TSC1, TSC2, mTOR, MIG-6 (ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEK mitogen-activated protein kinase 1, cMYC, TOPO II. Topoisomerase (DNA) II α 170 kDa, FRAP1, NRG1, ESR1, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1, FOXO3A, PPP1R1B, PXN, ELA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1, PITX2, MKI67, PHLPP, Adiponectin (ADIPOQ), Fibrinogen α chain (FGA), Leptin (LEP), Advanced Glycosylation Receptor (AGER, also known as RAGE), α-2-HS-glycoprotein (AHSG), Angiopoietin (ANG), CD14 molecule (CD14), Ferritin (FTH1), Insulin-like growth factor binding protein 1 (IGF) BP1), interleukin-2 receptor, α (IL2RA), vascular cell adhesion molecule 1 (VCAM1) and von Willebrand factor (VWF), myeloperoxidase (MPO), IL1α, TNFα, perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilms' tumor-1 protein, aquaporin-1, MLL3, AMBP, VDAC1, Escherichia coli enterotoxin (heat-sensitive exotoxin, heat-stable enterotoxin), influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum toxin, Shiga toxin, Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxin A and B, etc.

[0186] Exemplary targets for nucleic acid aptamers that can be measured in samples (such as environmental samples, biological samples obtained from patients or subjects) using the subject device when needed include: drugs of abuse (e.g., cocaine), protein biomarkers (including but not limited to nucleolin, nuclear factor-κB essential regulator (NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoform, immunoglobulin μ heavy chain (IGHM), immunoglobulin E, αvβ3 integrin, α-thrombin, HIV) gp120, NF-κB, E2F transcription factor, HER3, plasminogen activator inhibitor, tendinin C, CXCL12 / SDF-1, prostate-specific membrane antigen (PSMA), gastric cancer cells, HGC-27; cells (including but not limited to non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF-CEM, acute myeloid leukemia (AML) HL60 cells, small cell lung cancer (SCLC) cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells, HER-2 overexpressing human breast cancer cells, SK-BR-3, pancreatic cancer cell line (Mia-PaCa-2)); and infectious pathogens (including but not limited to Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria, Pseudomonas aeruginosa, Salmonella O8, and Salmonella enteritidis).

[0187] Exemplary targets of protein or peptide aptamers that can be measured in samples obtained from patients or subjects using the subject device when needed include, but are not limited to: HBV core capsid protein, CDK2, E2F transcription factor, thymidine synthase, Ras, EB1, and receptor for advanced glycation end products (RAGE). Aptamers and their uses and methods of production have been reviewed in, for example, Shum et al., J Cancer Ther. 2013 4:872; Zhang et al., Curr Med Chem. 2011;18:4185; Zhu et al., Chem Commun (Camb). 2012 48:10472; Crawford et al., Brief Funct GenomicProteomic. 2003 2:72; Reverdatto et al., PLoS One. 2013 8:e65180.

[0188] In some cases, biological samples containing or suspected of containing target nucleic acids (e.g., human blood samples) may undergo preparation / processing before being detected by the sample fluid analysis system of the system disclosed herein. In some embodiments, preparation / processing may include the following steps: i) isolating total nucleic acids containing target nucleic acids from the sample; ii) optionally, enriching the target nucleic acids; iii) amplifying the target nucleic acids; and iv) processing the amplified target nucleic acids. Each step may be performed manually, automatically, or by a combination of both. In some embodiments, the analyte is not amplified (i.e., the copy number of the analyte is not increased) before measurement. For example, in the case of the analyte being DNA or RNA, the analyte is not replicated to increase its copy number.

[0189] In some cases, the method involves using one or more reference standards to quantify the analyte. These reference standards can be used to establish a standard curve for interpolating and / or extrapolating analyte concentrations. In other embodiments, the system of this disclosure may include reference standards that vary with respect to concentration levels. For example, the apparatus may include one or more reference standards having high, medium, or low concentration levels. This can be optimized for each assay in terms of the concentration range of the reference standards. Exemplary concentration ranges for the reference standards include, but are not limited to, for example: approximately 10 fg / mL, approximately 20 fg / mL, approximately 50 fg / mL, approximately 75 fg / mL, approximately 100 fg / mL, approximately 150 fg / mL, approximately 200 fg / mL, approximately 250 fg / mL, approximately 500 fg / mL, approximately 750 fg / mL, approximately 1000 fg / mL, approximately 10 pg / mL, approximately 20 pg / mL, approximately 50 pg / mL, approximately 75 pg / mL, approximately 100 pg / mL, approximately 150 pg / mL, approximately 200 pg / mL, approximately 250 pg / mL, approximately 500 pg / mL, approximately 750 pg / mL, approximately 1 ng / mL, approximately 5 ng / mL, approximately 10 ng / mL, and approximately 12 ng / mL.5 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL, approximately 55 ng / mL, approximately 60 ng / mL, approximately 75 ng / mL, approximately 80 ng / mL, approximately 85 ng / mL, approximately 90 ng / mL, approximately 95 ng / mL, approximately 100 ng / mL, approximately 125 ng / mL, approximately 150 ng / mL, approximately 165 ng / mL, approximately 175 ng / mL, approximately 200 ng / mL, approximately 225 ng / mL, approximately 250 ng / mL, approximately 275 ng / mL, approximately 300 ng / mL, approximately 400 ng / mL, approximately 425 ng / mL, approximately 450 ng / mL, approximately 465 ng / mL, approximately 475 ng / mL, approximately 500 ng / mL, approximately 525 ng / mL, approximately 550 ng / mL, approximately 575 ng / mL, approximately 600 ng / mL, approximately 700 ng / mL, approximately 725 ng / mL, approximately 750 ng / mL, approximately 765 ng / mL, approximately 775 ng / mL, approximately 800 ng / mL, approximately 825 ng / mL, approximately 850 ng / mL, approximately 875 ng / mL, approximately 900 ng / mL, approximately 925 ng / mL, approximately 950 ng / mL, approximately 975 ng / mL, approximately 1000 ng / mL, approximately 2 µg / mL, approximately 3 µg / mL, approximately 4 µg / mL, approximately 5 µg / mL, approximately 6 µg / mL, approximately 7 µg / mL, approximately 8 µg / mL, approximately 9 µg / mL, approximately 10 µg / mL, approximately 20 µg / mL, approximately 30 Approximately 40 µg / mL, approximately 50 µg / mL, approximately 60 µg / mL, approximately 70 µg / mL, approximately 80 µg / mL, approximately 90 µg / mL, approximately 100 µg / mL, approximately 200 µg / mL, approximately 300 µg / mL, approximately 400 µg / mL, approximately 500 µg / mL, approximately 600 µg / mL, approximately 700 µg / mL, approximately 800 µg / mL, approximately 900 µg / mL, approximately 1000 µg / mL, approximately 2000 µg / mL, approximately 3000 µg / mL, approximately 4000 µg / mL, approximately 5000 µg / mL, approximately 6000 µg / mL, approximately 7000 µg / mL, approximately 8000 µg / mL, approximately 9000 µg / mL, or approximately 10000 µg / mL.

[0190] In some embodiments, the system disclosed herein may optionally include quality control components (e.g., a sensitivity panel, a calibrator, and a positive control). The preparation of quality control reagents is well known in the art and is described in the insert sheets for various immunodiagnostic products. The sensitivity panel members may optionally be used to establish assay performance characteristics and may also optionally serve as useful indicators of the integrity of the device reagents and the standardization of assays.

[0191] In some embodiments, the system disclosed herein may optionally include other reagents required for diagnostic assays or to facilitate quality control assessments, such as buffers, salts, enzymes, enzyme cofactors, substrates, assay reagents, etc. Other components (such as buffers and solutions (e.g., pretreatment reagents) for isolating and / or processing test samples) may also be included in the device. The device may additionally include one or more other controls. One or more of the components of the device may be lyophilized, in which case the device may also include reagents suitable for reconstructing the lyophilized components. One or more of these components may be in liquid form.

[0192] In some embodiments, the various components of the apparatus may optionally be disposed in suitable containers as needed. In other embodiments, the apparatus may also include containers for containing or storing samples (e.g., containers or boxes for urine, saliva, plasma, cerebrospinal fluid, or serum samples, or suitable containers for storing, transporting, or processing tissue to produce tissue aspirates). Where appropriate, the apparatus may also optionally include reaction dishes, mixing dishes, and other components to facilitate the preparation of reagents or test samples.

[0193] In some embodiments, the analysis of the sample fluid includes the analysis of nucleic acids in the sample fluid. Nucleic acid assays may include, but are not limited to, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), real-time quantitative PCR (RT-qPCR), isothermal PCR, thermal cycling-based PCR, hot-start PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), lateral flow immunoassay (NAFLIA), helicase-dependent amplification (HAD), rolling circle amplification (RCA), nickase amplification reaction (NEAR), CRISPR-Cas detection methods (e.g., SHERLOCK (specific high-sensitivity enzyme reporter unlocking)), DETECTR (DNA endonuclease targeting CRISPR trans reporter gene), and HOLMES (one-hour... Low-cost, versatile, and efficient systems, nucleic acid hybridization detection and probes (e.g., dot blot, Southern blotting, in situ hybridization, sequence-specific probes (TaqMan probes), bead- and microarray-based oligonucleotide probes), fluorescence in situ hybridization (FISH), peptide-nucleic acid fluorescence in situ hybridization (PNA-FISH), chromogenic in situ hybridization (CISH), nucleic acid sequencing, high-throughput sequencing, next-generation sequencing, deep sequencing, whole-genome sequencing, whole-exome sequencing, Northern blotting, nuclease protection assay (NPA), oligonucleotide probes on chips (e.g., ViroChip), and other detection methods (such as fluorophore probes, enzyme or fluorescently labeled probes, turbidity, colorimetry, etc.). These techniques are well known in the art. In some embodiments, amplification is used to increase the amount of nucleic acid used to perform the assay and in the process of detecting and labeling nucleic acid sequences. For example, amplification is performed in PCR and RT-PCR.

[0194] In some cases, the analytical sample fluid includes an immunoassay (IA). An immunoassay typically involves: contacting an antigen with an antibody specific to the antigen to form an antibody-antigen complex; and detecting the antibody-antigen complex. In some embodiments, the antibody-antigen complex is an antibody-analyte complex. In other embodiments, the analyte is an antigen. In other embodiments, antigens that can be bound by antibodies include, but are not limited to, proteins, peptides, polysaccharides, lipids, or nucleic acids.

[0195] The kit can be designed to perform various types of immunoassays. In some embodiments, the immunoassay can be a labeled immunoassay. In a labeled immunoassay, an antibody-analyte complex can be detected using a detectably labeled antibody. The detectable label can be selected from a variety of such labels known in the art, but is typically a radioisotope, fluorophore, enzyme (e.g., horseradish peroxidase), or other portion or compound that emits a detectable signal (e.g., radioactivity, fluorescence, color) or emits a detectable signal after the label has been exposed to its substrate. Additional labels can include, but are not limited to, DNA probes and reporter genes, electrochemiluminescent tags, and magnetic particles. Various detectable label / substrate pairs (e.g., horseradish peroxidase / diaminobenzidine, avidin / streptavidin, luciferase / luciferin), methods for labeling antibodies, and methods for detecting antigens using labeled antibodies are well known in the art. In other embodiments, the immunoassay can be an unlabeled immunoassay. Unlabeled immunoassays are performed without labeling and include, but are not limited to, techniques such as immunodiffusion and turbidimetry.

[0196] In some embodiments, the immunoassay may be a heterogeneous immunoassay. A heterogeneous immunoassay requires the separation of the antibody-analyte complex from other components of the immunoassay prior to analysis. In other embodiments, the immunoassay may be a homogeneous immunoassay. A homogeneous immunoassay does not require the separation of the antibody-analyte complex from other components of the immunoassay prior to analysis.

[0197] In some embodiments, the immunoassay may be a competitive immunoassay. In a competitive immunoassay, the analyte competes with a specific amount of labeled antigen for an antibody. In other embodiments, the immunoassay may be a non-competitive immunoassay. In a non-competitive immunoassay, an excess of labeled antibody is used to bind to the analyte.

[0198] The antibodies and antigens in an immunoassay can be arranged in various configurations. In some embodiments, the antibodies and antigens in the immunoassay are in solution. In other embodiments, the antibody or antigen binds to a solid surface. In yet another embodiment, the antibody or antigen from the sample binds to a solid surface. In some embodiments, the antibody is labeled. In some embodiments, the antigen is labeled. In some embodiments, more than one antibody may be used to detect the analyte. In other embodiments, two or more antibodies may bind to the same antigen. In other embodiments, two or more antibodies may bind to different epitopes of the same antigen. In other embodiments, two or more antibodies may bind to different antigens of the analyte. In other embodiments, a first antibody binds to the antigen, and a second antibody binds to the first antibody. In other embodiments, two antibodies compete to bind to the antigen. In some embodiments, a known amount of identifiable antigen or analyte competes with the antigen or analyte for binding to the antibody.

[0199] Any suitable immunoassay can be used. Examples of well-known immunoassay variants include, but are not limited to: immunoassays such as sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays); enzyme assays such as enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA) (e.g., direct, indirect, competitive, and sandwich ELISA); competitive inhibition immunoassays (e.g., forward and reverse); enzyme multiplication immunoassay (EMIT); competitive binding assays; bioluminescent resonance energy transfer (BRET); one-step antibody assays; homogeneous assays; heterogeneous assays; point-of-care capture assays, etc.

[0200] In some embodiments, immunoassays can be used to detect nucleic acid sequences. Once the target nucleic acid sequence has been amplified to the desired extent, an immunoassay can be used to detect the amplification product. Various assay formats can be employed. For example, an immunoassay can be performed to capture the amplified nucleic acid sequence using a tag incorporated into the amplified target nucleic acid. Specifically, the capture object (such as beads, e.g., magnetic beads) includes one binding member of a specific binding pair, and the amplified target nucleic acid is captured via the interaction of that member with another member of the binding pair, which has been introduced into the amplified target nucleic acid during amplification. The surface of the capture object is not coated with nucleic acids that can bind to the amplified target nucleic acid.

[0201] Various methods can be used to analyze current methods and devices for immunoassays to detect antibody-analyte complexes. These detection methods may vary depending on the immunoassay format and may include, but are not limited to, radiation detection, enzyme product detection, fluorescence detection, color changes, turbidity changes, electrical impedance changes, optical property changes, agglutination reactions, and so on.

[0202] In other embodiments, the assay for measuring biomolecules or clinical chemistry tests in a sample includes enzymatic methods, i.e., reacting an analyte (such as electrolytes, CO2, serum creatinine, blood urea nitrogen) with an enzyme and detecting the reaction products. In still other embodiments, the assay for measuring biomolecules or clinical chemistry tests in a sample includes chemical reaction methods similar to enzymatic methods, but utilizing chemical reagents and employing spectrophotometry. In still other embodiments, the assay for measuring biomolecules or clinical chemistry tests in a sample includes changes in pH levels. In still other embodiments, the assay for measuring biomolecules or clinical chemistry tests in a sample includes the use of turbidimetry. Turbidimetry is used to measure the amount of turbidity or cloudiness by measuring scattered light and can be used in conjunction with immunoassays. In still other embodiments, the assay for measuring biomolecules or clinical chemistry tests in a sample includes the use of photometry, which measures absorbed light (UV, visible, IR) to determine the amount of analyte in a solution or liquid. In other embodiments, the assay for measuring biomolecules or clinical chemistry tests in a sample includes coagulation assays. In coagulation assays, reagents are added to blood to measure coagulation / clotting time. In yet another embodiment, assays for measuring biomolecules or clinical chemistry assays in a sample include the use of electrophoresis.

[0203] Computer-controlled systems This disclosure also includes computer-controlled systems, wherein such systems include one or more computers for full or partial automation. In some embodiments, the system includes a computer operatively connected to a memory storing instructions that, when executed, cause the computer to perform one or more methods of the invention (e.g., as discussed above). For example, the computer may be configured to calculate the absorbance of each of the plurality of detection chambers to analyze a sample fluid. As discussed above, this may include: calculating the average intensity of incident light from a light source; calculating the average intensity of emitted light from each of the plurality of detection chambers; deactivating the light source and calculating the dark-image average intensity of the emitted light from each of the plurality of detection chambers; and calculating the absorbance of each of the plurality of detection chambers based on the average intensity of incident light from the light source, the average intensity of emitted light from each of the plurality of detection chambers, and the dark-image average intensity of the emitted light from each of the plurality of detection chambers.

[0204] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access memory on which instructions for performing steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are available or will become available. The processor executes the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor's coordination and execution of the functions of various computer programs written in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system coordinates and executes the functions of other components of the computer, typically in cooperation with the processor. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0205] System memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media (such as resident hard disks or magnetic tapes), optical media (such as compact optical discs for reading and writing), flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including compact optical disc drives, magnetic tape drives, or floppy disk drives. This type of memory storage device typically reads from and / or writes to a program storage medium (not shown) (such as a compact optical disc). Any of these program storage media, or other program storage media now in use or that may be developed later, can be considered a computer program product. As will be understood, these program storage media typically store computer software programs and / or data. Computer software programs (also known as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0206] In some embodiments, a computer program product is described, comprising a computer-usable medium storing control logic (a computer software program, including program code). When executed by a computer's processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. It will be apparent to those skilled in the art that implementing a hardware state machine to perform the functions described herein is appropriate.

[0207] The memory can be any suitable device in which a processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic disks or magnetic tapes or optical discs or optical tapes, or RAM, or any other suitable device (fixed or portable)). The processor may include a general-purpose digital microprocessor that can be suitably programmed by a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel, or it can be previously stored in a computer program product (such as memory or some other portable or fixed computer-readable storage medium) in conjunction with any of those devices. For example, a magnetic disk or optical disc may carry the programming and can be read by a disk writer / reader. The system of the present invention also includes programming, for example, in the form of a computer program product or algorithm for practicing the methods described above. The programming according to the invention can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROMs; portable flash drives; and mixtures of these categories, such as magnetic / optical storage media.

[0208] The processor can also access communication channels to communicate with users at remote locations. "Remote location" means that the user does not have direct contact with the system, but instead relays input information from an external device to the input manager. This external device can be a computer, including mobile phones (i.e., smartphones), connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel.

[0209] In some embodiments, the system according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., RFID, Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM)).

[0210] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as USB ports, USB-C ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject system and other external devices, such as computer terminals configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0211] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that a user may use in conjunction with them.

[0212] In one embodiment, the communication interface is configured to provide connectivity for data transfer using the Internet Protocol (IP) via: a mobile network, a short message service (SMS), a personal computer (PC) wirelessly connected to a local area network (LAN) (which is connected to the Internet), or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0213] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, such as using 802.11 or Bluetooth. ® Common standards such as RF protocols or IrDA infrared protocols. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer; or a larger device, such as a desktop computer, home appliance, etc. In some embodiments, the server device has a display (such as a liquid crystal display (LCD)) and input devices (such as buttons, keyboard, mouse, or touch screen).

[0214] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the subject system (e.g., optional data storage unit) to a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0215] The output controller may include a controller for any of a variety of known display devices used to present information to a user (whether human or machine, local or remote). If one of the display devices provides visual information, that information is typically logically and / or physically organized as an array of image elements. The graphical user interface (GUI) controller may include any of a variety of known or future software programs used to provide a graphical input and output interface between the system and the user, and for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be implemented using networks or other types of telecommunications in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location, such as via the Internet, telephone, or satellite networks, according to known techniques. The presentation of data by the output manager may be implemented according to a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, emails or other files, or data in other forms. The data may include Internet URLs that allow the user to retrieve additional SQL, HTML, XML, or other documents or data from a remote source. The one or more platforms present in the subject system can be any type of known computer platform or a type to be developed in the future, though they will generally fall into the category of computers commonly referred to as servers. However, they can also be mainframes, workstations, or other computer types. They can be connected (networked or otherwise) via any known or future type of cabling or other communication system (including wireless systems). They can be located in the same location or they can be physically separated. Depending on the type and / or brand of the computer platform chosen, various operating systems may be used on any of the computer platforms. Suitable operating systems include Windows. ® NT, Windows ® XP, Windows ® 7. Windows ® 8. Windows ® 10. iOS ® macOS ®® Linux ® Ubuntu ® Fedora ® OS / 400 ® i5 / OS ® IBM i ® Android™, SGI IRIX ® Oracle Solaris ® And others.

[0216] Reagent test kit Various aspects of this invention additionally include a kit. In some embodiments, the kit includes one or more sample analysis systems of the present invention. For example, the kit may include one or more sample analysis systems, such as two or more sample analysis systems, such as three or more sample analysis systems, and including five or more sample analysis systems. Alternatively or additionally, the kit may include one or more boxes of the present invention. For example, the kit may include a number of boxes ranging from 1 to 100, such as 2 to 50, and including 3 to 10. In some cases, components of the subject kit are provided in packaging (such as sealed packaging). In some cases, the sealed packaging is sterile packaging.

[0217] In addition to the components described above, the subject kit may also include (in some embodiments) instructions for carrying out the methods of the invention (e.g., performing sample fluid dilution). These instructions may exist in various forms within the subject kit, one or more of these forms. One form of these instructions may be printed information, such as on a suitable medium or substrate (e.g., one or more sheets of paper on which information is printed), in the kit packaging, in the instruction manual, etc. Another form of these instructions is a computer-readable medium on which information is recorded, such as a floppy disk, a compact optical disc (CD), a portable flash drive, etc. Yet another form of these instructions may be a website address, which can be used via the Internet to access information on a remote website.

[0218] The following content is presented through explanation rather than through limitation.

[0219] experimental Example 1: Generate based on Figures 7A-7CThe optical interrogation system comprises a photodiode array sensor consisting of a SONY Pregius Gen3, IMX428, 14.4 x 9.9 mm (Pixelink PL-D797MU). Received in the system is a 1536 microwell plate (1.7 mm square, 2.2 mm aperture space, 10 μL volume) consumable / kit. It also includes a first pinhole plate and a second pinhole plate (5 x 7 pinholes totaling 35 pinholes, corresponding to 35 apertures, Φ0.8 mm pinhole pairs, 2.2 mm space), and an emission bandpass filter to minimize the effects of autofluorescence. The light source is configured to emit eight wavelengths from UV to IR and includes a light guide plate. The light guide plate is either 1) readily available with minor modifications (Advanced Illumination, 8 wavelengths, no UV), or 2) custom-made (e.g., UV LGP, LED (especially 340 nm), light distribution, material with UV durability).

[0220] like Figure 7A As shown, 35 Φ0.8 mm pinholes and 2.25 mm spaces on the front and rear plates match the hole size. The plate height is 25 mm and it is constructed from black Delrin®. A CRA <2° keeps the light within the holes. A SONY IMX428 1.1” image sensor is mounted on the rear plate and covers all pinholes. An LGP illuminator is mounted on the front plate. Additionally, a Thermosphere 1536 microplate is used for 13 μL liquid samples. Figure 7B The dimensions of the prototype pinhole plate are depicted. The distance between pinholes is 2.2 mm, Φ0.8 mm for a high-density 1536-well plate, and there are 35 micro-cubicles in an area the size of a penny. Figure 7C The distance between pinholes is shown to be 4 mm, which is Φ1 mm for a 1 cm spectrophotometer.

[0221] Figures 7A-7C The prototype was used to illuminate consumables. Figure 8 The image presented shows the light collected from the rear pinhole plate, demonstrating that the system is well calibrated because the pinhole positions match their calculated locations. Figure 8 As shown, each pinhole acts as a super photodiode. Pinhole masks (circular outlines) mark their locations for intensity measurements.

[0222] Example 2: Stray light testing was conducted, which involved blocking one pinhole of the device and assessing the crosstalk to the blocked pinhole. Figure 9A The experimental setup shown depicts a blocked pinhole. First, the test was performed on an unblocked pinhole at 0.1 ms exposure, 470 nm, and 6% power. Figure 9B -C). Subsequently, the test was performed on the obscured pinhole at 0.1 ms exposure, 470 nm, and 6% power. Figure 9D-9E Finally, the test was repeated for the obscured pinhole at 10 ms exposure, 470 nm, and 6% power. Figure 9F-9G ).

[0223] Example 3: Construction basis Figure 6C The optical interrogation system is characterized by a 2.2 mm aperture space, a 1.78 mm square cuvette, a depth of 5.1 mm, and a height of 7.4 mm (d3). The front pinhole has a diameter of 0.8 mm (Φ1) and a thickness of 23 mm (d1). The rear pinhole has a diameter of 1.0 mm (0.04") (Φ2) and a thickness of 23 mm (d2). The optical diameter (d') at the cuvette outlet is 1.4 mm. Figure 10A The resulting pinhole array image is shown in the figure. Figure 10B The image shows the resulting 470 nm beam after the front pinhole. The entire system is characterized by 35 pinholes (x7), corresponding to 35 pinholes, Φ0.8 mm pinhole pairs, and a 2.2 mm space. The system features an emission 7-bandpass filter (Chroma JC27395). The center wavelengths of the narrow transparency bands are 340, 405, 467, 500, 550, 600, and 660 nm, with an FWHM of 10 nm and a transmittance of 90%. The high-pass band is >800 nm with a transmittance of 90%. The system features a SONY IMX4281.1” image sensor (14.4 x 9.9 mm) with the following specifications: (HxV) 3208 x 2200, 4.5 μm pixels; QE of 22% at 340 nm, 77% at 530 nm, and 20% at 850 nm; readout noise of 5.5 e-; full-well noise of 24.8 Ke- (9.7E8 electrons for a Φ1 mm pinhole); and dark current (25°C) of 2.8 e- / s / pixel. The light source consists of seven LEDs: 405 nm, 470 nm, 500 nm, 530 nm, 590 nm, 660 nm, and 850 nm. It also includes a custom-made edge-lit LGP with a 50 x 50 mm illumination area. The light source is characterized by a continuously adjustable drive current from 0 to 1 A. Figure 2A ).

[0224] CMP absorption calibration and OD calculation were then performed. Images of absorbed incident light and camera dark current were captured under the following settings: 470 nm: 500 mA, 290 ms exposure, gain x1; 660 nm: 500 mA, 175 ms exposure, gain x1; 405 nm: 500 mA, 600 ms exposure, gain x1; and 850 nm: 500 mA, 175 ms exposure, gain x1. Sample images were captured under these settings, and the mOD value for each pixel was calculated as follows: Each pinhole was masked, and the average value of the masked area was taken as the absorbance of the cuvette. Figure 11A-11B The results are described in the text. For example... Figure 11A As shown, the average pinhole area OD is the absorbance of the cuvette (100mOD of the neutral density filter). Figure 11B The absorbance image of the lemon yellow 1536-well plate is shown.

[0225] Then, a UV1800 spectrometer was used at 0.1 OD, 0.4 OD, 1.2 OD, 2.0 OD and a blank target; neutral density glass filters (Edmund Absorption Neutral Density Filter Kit #63-470) were measured at 35 pinholes using four wavelengths: 403 nm, 465 nm, 657 nm and 850 nm. Figure 12 The target OD spectrum of the Shimatzu UV1800 spectrometer is shown. The OD of the 35 pinholes was measured, calculated, and evaluated in terms of the linearity of the average OD to the UV1800 reading and the repeatability (cv%) of the OD of the 35 pinholes. Figures 13A-13D The results are shown in the figure. The linearity gain is between 0.97 and 1.01, and the R² is 0.9995 or better.

[0226] Example 3: The effect of notch filter placement on stray light in an optical interrogation system was investigated. The notch filter was placed in front of the sensor background ( Figure 6A Or before the rear pinhole plate ( Figure 6B Images were acquired at 600 ms exposure, 470 nm illumination, and 500 mA current. Visualization was characterized by 0–44 ADUs. 13,000 to 15,000 pixels were tested in each region. Figure 14A and Figure 14B The images shown are those obtained before and after the notch filter is placed against the sensor background. Table 1 below shows the images obtained before and after the notch filter is placed against the sensor background. Figures 14A-14B Data collected in regions 1-4 shown: Table 1

[0227] Therefore, moving the filter in front of the emission plate (just behind the micro cuvette) reduces stray light.

[0228] Example 4: structure Figures 15A-15C The light interrogation system shown. Figure 15A The LGP light source, front pinhole plate, 1536 microplate, and rear pinhole plate are shown from bottom to top. Figure 15B The light pattern behind the front pinhole plate is depicted. Figure 15C The exit pupil of the illuminator was depicted. The power distribution of the Consun LGP was then measured. Figures 16A-16B The spectrum of Consun LGP is shown in the figure, and the low autofluorescence LGP material reduces the spectral shift by 405 nm. Figure 18 The LGP outlet surface was masked, featuring Φ3.17 mm openings in 5 mm increments. Power was measured at each opening using a Thorlabs PM100USB power meter coupled to a Φ400 μm fiber. Average power and non-uniformity were measured in a central 25 x 25 mm region. A summary of the results is shown in Table 2 below. Table 2 LED wavelength (nm) <![CDATA[Average power (uW / mm 2 )]]> Non-uniformity (%) 409 0.44 25% 534 0.34 18% 470 0.54 25% 509 0.43 15% 595 0.47 23% 656 0.32 17% 850 0.70 23%

[0229] according to Figure 17 The specifications (from top to bottom) are adjusted for the optical interrogation system to give it the configuration described in Table 3 below: Table 3 LED angle (θ°) 67 45 Boundary (d1, mm) 4 LGP width (mm) 50 LGP operating width (d2, mm) 42 LGP to prism (d3, mm) 30 Prism width (d4, mm) 10

[0230] The power efficiency of the Hexatron LPG + HoneyComb illuminator was measured using a Thorlabs PM100USB power meter with a Luxeon LED LHUV-0405 (cw 509 nm, current 51 mA) coupled to a Φ400 μm fiber. Efficiency was measured at the LGP edge (incident light) and at the top of the LGP in 5 mm increments (i.e., from the incident light). Figure 18 (From top to bottom). The following is presented in Table 4 and... Figure 19 The results are plotted in the image.

[0231] Table 4 Location <![CDATA[Power (uW / mm 2 )]]><![CDATA Incident 106.25 1 2.50 2 1.63 3 1.38 4 1.13 5 1.06 6 1.00 7 0.94 8 0.94 9 1.13

[0232] The LGP power efficiency is ~1 / 100 for incident light. A current of 500 mA and an exposure time of 10 x 10 (<100 ms) can be used. With optimization of LED placement and LGP size, 1 / 4 to 1 / 5 efficiency is considered feasible.

[0233] Hybrid LGP ( Figure 16B The emission surface (shown in the figure) was masked, with Φ3.17 mm openings in 5 mm increments, and power was measured at each opening using a Thorlabs PM100USB power meter coupled to a Φ400 μm fiber. Average power and non-uniformity were measured in a central 25 x 25 mm region. The results are shown below in Table 5: Table 5 LED wavelength (nm) <![CDATA[Average power (uW / mm 2 )]]> Non-uniformity (%) 409 0.65 30% 534 0.65 26% 470 1.11 28% 509 0.72 29% 595 0.82 29% 656 0.65 24% 850 1.10 31%

[0234] Example 5: The following steps are performed using an optical interrogation system comprising a first pinhole plate and a second pinhole plate: 1. Before loading the cuvette array, expose it to an exposure time of, for example, 100 µs. 入射 The average intensity (I) of the illuminator pinhole region of the image of each cuvette is measured below. 入射 ).

[0235] 2. Run the sample preparation steps, fill each cuvette, and wait a specific time for it to react with the reagent to produce a color change.

[0236] 3. Load the cuvette array and record the average intensity of the cuvettes over a series of longer exposure times (e.g., 1 ms, 10 ms, 100 ms, 500 ms). Find the intensity close to 80% of the trap capacity of the pinhole region: I abs_i And record the corresponding exposure time: T abs_i .

[0237] 4. Cut off the LED, capture the dark image, and at T 入射 and T abs_i The average strength (I) was measured at the pinhole area. 暗_入射 and I 暗_abs_i ).

[0238] 5. Calculate the absorbance of each cuvette: A = log((I 入射 – I 暗_入射 ) / T 入射 ÷( I abs_i –I 暗_abs_i ) / T abs_i ).

[0239] Experimental and simulation models show that the above algorithm produces a low cv% of less than 0.5% when measuring absorbance from 0.05 OD to 1.8 OD. Measuring transmitted light power at multiple longer exposure times extends this range to 4 OD while maintaining a low cv%. The algorithm uses absorbance measured in empty cuvettes and cuvettes filled only with diluent to correct for material and buffer bias.

[0240] Example 6: Simulation and bench testing were used to implement the... Figure 5A Evaluation of the optical interrogation system. It was found that a Φ1 mm super photodiode exhibits 6-9 x 10⁻⁶ Ω·cm² performance. 8 The electron trap capacity was increased, thereby improving the SNR and reducing the cv% at high OD measurements. Both simulations and bench tests showed that the cv% was less than 0.5% at up to 1.8 OD. Using a multi-exposure algorithm, the measurement range was extended to 4 OD at low cv%.

[0241] Although the invention described above has been described in some detail by way of illustration and examples for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that some changes and modifications may be made therein without departing from the spirit or scope of the appended claims.

[0242] Therefore, the foregoing merely illustrates the principles of the invention. It will be understood that those skilled in the art will be able to devise various arrangements, although not explicitly described or shown herein, that embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language described herein are primarily intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to advance the art, and are to be construed as not being limited to such explicitly described examples and conditions. Moreover, all statements herein describing the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover both their structural and functional equivalents. Additionally, it is intended that such equivalents include both currently known equivalents and future-developed equivalents, i.e., any element developed to perform the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly mentioned in the claims.

[0243] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is explicitly defined as being invoked only against such limitation in the claims if the exact phrase “device for…” or the exact phrase “step for…” is recited at the beginning of the limitation in the claims; if such an exact phrase is not used in the limitation in the claims, then 35 USC §112(f) or 35 USC §112(6) is not invoked.

Claims

1. An optical interrogation system, comprising: A first pinhole plate includes one or more pinholes, each pinhole being configured for optical alignment with one of a plurality of detection chambers; A light source is configured to illuminate each of the plurality of detection chambers through a pinhole in the first pinhole plate, the pinhole being optically aligned with the detection chamber; The second pinhole plate includes one or more pinholes, each pinhole being optically aligned with a pinhole of the first pinhole plate and configured for optical alignment with one of the plurality of detection chambers; as well as An optical sensor is configured to collect light from each of the plurality of detection chambers through a pinhole in the second pinhole plate that is optically aligned with the detection chamber.

2. The optical interrogation system according to claim 1, wherein, The first pinhole plate and the second pinhole plate each include a pinhole array.

3. The optical interrogation system according to claim 2, wherein, The number of pinholes in each array ranges from 2 to 50.

4. The optical interrogation system according to claim 2 or 3, wherein, The distance between adjacent pinholes in the first pinhole plate and the second pinhole plate ranges from 2 mm to 2.5 mm.

5. The optical interrogation system according to any one of claims 2 to 4, wherein, The light source is composed of an optical fiber array.

6. The optical interrogation system according to claim 5, wherein, The fiber optic array includes optical tubes.

7. The optical interrogation system according to claim 5 or 6, wherein, Each fiber in the fiber array is optically aligned with a different pinhole in the first pinhole plate.

8. The optical interrogation system according to claim 7, wherein, The light source is optically coupled to a fiber array board, which has holes for mounting the fiber array.

9. The optical interrogation system according to any one of the preceding claims, further comprising a small lens array positioned between the light source and the first pinhole plate, wherein, Each small lens of the array is optically aligned with a pinhole in the first pinhole plate.

10. The optical interrogation system according to claim 9, wherein, The microlens array is arranged inside the microlens plate.

11. The optical interrogation system according to any one of claims 1 to 4, wherein, The light source includes a miniature light-emitting diode (LED) array.

12. The optical interrogation system according to claim 11, wherein, The light source includes a side-lit micro light-emitting diode (LED).

13. The optical interrogation system according to claim 12, wherein, The light source includes a light guide plate (LGP).

14. The optical interrogation system according to claim 13, wherein, The LGP is composed of casting grade polymethyl methacrylate (PMMA).

15. The optical interrogation system according to any one of the preceding claims, further comprising a diffuser positioned along an optical path between the light source and the first pinhole plate.

16. The optical interrogation system according to any one of the preceding claims, further comprising a folding mirror or folding prism positioned along an optical path between the light source and the first pinhole plate.

17. The optical interrogation system according to any one of the preceding claims, further comprising a collimating lens positioned along an optical path between the light source and the first pinhole plate.

18. The optical interrogation system according to any one of the preceding claims, further comprising a tapered optical mixing rod positioned between the light source and the first pinhole plate.

19. The optical interrogation system according to any one of the preceding claims, further comprising a bandpass filter positioned between the second pinhole plate and the optical sensor.

20. The optical interrogation system according to any one of the preceding claims, further comprising a notch filter.

21. The optical interrogation system according to claim 20, wherein, The notch filter is positioned between the first pinhole plate and the second pinhole plate.

22. The optical interrogation system according to any one of the preceding claims, wherein, The optical sensor is a CMOS sensor.

23. The optical interrogation system according to any one of claims 1 to 21, wherein, The optical sensor is a spectrometer.

24. The optical interrogation system according to any one of the preceding claims, further comprising a processor operatively connected to the optical sensor, the light source, and a memory, the memory storing instructions that, when executed by the processor, cause the processor to calculate the absorbance of each of the plurality of detection chambers.

25. The optical interrogation system according to claim 24, wherein, The processor is configured to: Calculate the average intensity of the incident light from the light source; Calculate the average intensity of the emitted light from each of the plurality of detection chambers; The light source is deactivated and the average intensity of the emitted light from each of the plurality of detection chambers in the dark image is calculated; as well as The absorbance of each of the plurality of detection chambers is calculated based on the average intensity of the incident light from the light source, the average intensity of the emitted light from each of the plurality of detection chambers, and the dark image average intensity of the emitted light from each of the plurality of detection chambers.

26. The optical interrogation system according to claim 25, wherein, The processor is configured to calculate the absorbance of each detection chamber as follows: in: A is the absorbance; I 入射 It is the average intensity of the incident light from the light source; I abs_i It is the average intensity of the emitted light from each of the plurality of detection chambers; and I 暗 It is the dark image average intensity of the emitted light from each of the plurality of detection chambers.

27. The optical interrogation system according to claim 26, wherein, The processor is configured to repeatedly calculate the absorbance of each detection chamber until a change in absorbance is measured.

28. The optical interrogation system according to claim 27, wherein, The processor is configured to calculate the analyte concentration based on the rate of change of absorbance.

29. The optical interrogation system according to claim 28, wherein, The processor is configured to calculate the rate of change as follows: in: ROC is the rate of change; A 结束 The absorbance was calculated at time point T2 when the significant absorbance change was measured; and A 开始 The first absorbance is calculated at time point T1.

30. The optical interrogation system according to any one of the preceding claims, wherein, The first pinhole plate and the second pinhole plate have one or more pinholes with a diameter ranging from 0.5 mm to 1.5 mm.

31. The optical interrogation system according to any one of the preceding claims, wherein, The distance between the first pinhole plate and the second pinhole plate ranges from 5 mm to 15 mm.

32. The optical interrogation system according to any one of the preceding claims, wherein, The first pinhole plate and the second pinhole plate have a thickness ranging from 20 mm to 50 mm.

33. The optical interrogation system according to any one of the preceding claims, further comprising a housing configured as a receiving box, the box including a detection chamber among the plurality of such chambers.

34. A method for analyzing a sample fluid, the method comprising: (a) Introducing the sample fluid into a cartridge comprising multiple detection chambers; (b) Inserting the box into an optical interrogation system, the optical interrogation system comprising: (i) A first pinhole plate comprising one or more pinholes, each pinhole being configured for optical alignment with one of the plurality of detection chambers; (ii) A light source configured to illuminate each of the plurality of detection chambers through a pinhole in the first pinhole plate, the pinhole being optically aligned with the detection chamber; (iii) A second pinhole plate comprising one or more pinholes, each pinhole being optically aligned with a pinhole of the first pinhole plate and configured for optical alignment with one of the plurality of detection chambers; and (iv) An optical sensor configured to collect light from each of the plurality of detection chambers through a pinhole in the second pinhole plate optically aligned with that detection chamber; and (c) Irradiate the plurality of detection chambers with the light source to analyze the sample fluid.

35. The method according to claim 34, wherein, The method includes: optically aligning the detection chamber of the box with one or more pinholes of the first pinhole plate and the second pinhole plate.

36. The method according to claim 34 or 35, wherein, The first pinhole plate and the second pinhole plate each include a pinhole array.

37. The method of claim 36, wherein, The number of pinholes in each array ranges from 2 to 50.

38. The method according to claim 36 or 37, wherein, The distance between adjacent pinholes in the first pinhole plate and the second pinhole plate ranges from 2 mm to 2.5 mm.

39. The method according to any one of claims 36 to 38, wherein, The light source is optically coupled to the fiber optic array.

40. The method according to claim 39, wherein, The fiber optic array includes optical tubes.

41. The method according to claim 39 or 40, wherein, Each fiber in the fiber array is optically aligned with a different pinhole in the first pinhole plate.

42. The method according to claim 41, wherein, The light source also includes a fiber array board having holes for mounting the fiber array.

43. The method according to any one of claims 34 to 42, wherein, The optical interrogation system further includes a small lens array positioned between the light source and the first pinhole plate, wherein each small lens in the array is optically aligned with a pinhole in the first pinhole plate.

44. The method according to claim 43, wherein, The microlens array is arranged inside the microlens plate.

45. The method according to any one of claims 34 to 38, wherein, The light source includes a miniature light-emitting diode (LED) array.

46. ​​The method according to claim 45, wherein, The light source includes a side-lit micro light-emitting diode (LED).

47. The method according to claim 46, wherein, The light source includes a light guide plate (LGP).

48. The method according to claim 47, wherein, The LGP is composed of casting grade polymethyl methacrylate (PMMA).

49. The method according to any one of claims 34 to 48, wherein, The optical interrogation system also includes a diffuser positioned along an optical path between the light source and the first pinhole plate.

50. The method according to any one of claims 34 to 49, wherein, The optical interrogation system also includes a folding mirror or folding prism positioned along the optical path between the light source and the first pinhole plate.

51. The method according to any one of claims 34 to 50, wherein, The optical interrogation system also includes a collimating lens positioned along the optical path between the light source and the first pinhole plate.

52. The method according to any one of claims 34 to 51, wherein, The optical interrogation system also includes a tapered optical mixing rod positioned between the light source and the first pinhole plate.

53. The method according to any one of claims 34 to 52, wherein, The optical interrogation system also includes a bandpass filter positioned between the second pinhole plate and the optical sensor.

54. The method according to any one of claims 34 to 53, wherein, The optical interrogation system also includes a notch filter.

55. The method according to claim 54, wherein, The notch filter is positioned between the first pinhole plate and the second pinhole plate.

56. The method according to any one of claims 34 to 55, wherein, The optical sensor is a CMOS sensor.

57. The method according to any one of claims 34 to 55, wherein, The optical sensor is a spectrometer.

58. The method according to any one of claims 34 to 57, further comprising: Calculate the absorbance of each of the plurality of detection chambers.

59. The method according to claim 58, wherein, The method includes: Calculate the average intensity of the incident light from the light source; Calculate the average intensity of the emitted light from each of the plurality of detection chambers; The light source is deactivated and the average intensity of the emitted light from each of the plurality of detection chambers in the dark image is calculated; and The absorbance of each of the plurality of detection chambers is calculated based on the average intensity of the incident light from the light source, the average intensity of the emitted light from each of the plurality of detection chambers, and the dark image average intensity of the emitted light from each of the plurality of detection chambers.

60. The method according to claim 59, wherein, The method includes calculating the absorbance of each detection chamber as follows: in: A is the absorbance; I 入射 It is the average intensity of the incident light from the light source; I abs_i It is the average intensity of the emitted light from each of the plurality of detection chambers; and I 暗 It is the dark image average intensity of the emitted light from each of the plurality of detection chambers.

61. The method according to claim 60, wherein, The method includes: repeatedly calculating the absorbance of each detection chamber until a change in absorbance is measured.

62. The method according to claim 61, wherein, The method includes: calculating the analyte concentration based on the rate of change of absorbance.

63. The method according to claim 62, wherein, The method includes calculating the rate of change as follows: in: ROC is the rate of change; A 结束 The absorbance was calculated at time point T2 when the significant absorbance change was measured; and A 开始 The first absorbance is calculated at time point T1.

64. The method according to any one of claims 34 to 63, wherein, The first pinhole plate and the second pinhole plate have one or more pinholes with a diameter ranging from 0.5 mm to 1.5 mm.

65. The method according to any one of claims 34 to 64, wherein, The distance between the first pinhole plate and the second pinhole plate ranges from 5 mm to 15 mm.

66. The method according to any one of claims 34 to 65, wherein, The first pinhole plate and the second pinhole plate have a thickness ranging from 20 mm to 50 mm.

67. The method according to any one of claims 34 to 66, wherein, The sample fluid is a blood sample.

68. The method according to claim 67, wherein, The sample fluid is a whole blood sample.

69. The method according to any one of claims 34 to 68, wherein, The analysis of the sample fluid includes performing a full suite of metabolic assays (CMP).

70. The method according to claim 69, wherein, Performing the full set of metabolic tests (CMP) includes assessing the following: serum glucose, calcium, blood urea nitrogen (BUN), creatinine, sodium, potassium, chloride, carbon dioxide (CO2), serum total protein (TP), serum albumin, bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase, and alanine aminotransferase.

71. An optical interrogation system, comprising: A pinhole plate comprising one or more pinholes, each pinhole being configured for optical alignment with one of a plurality of detection chambers; A light source is configured to illuminate each of the plurality of detection chambers through a pinhole in the pinhole plate, the pinhole being optically aligned with the detection chamber; A reflector configured to reflect light from each detection chamber onto the pinhole plate; as well as An optical sensor is configured to collect light from each of the plurality of detection chambers through a pinhole in the second pinhole plate that is optically aligned with the detection chamber.

72. A method for analyzing a sample fluid, the method comprising: (a) Introducing the sample fluid into a cartridge comprising multiple detection chambers; (b) Inserting the box into an optical interrogation system, the optical interrogation system comprising: (i) A pinhole plate comprising one or more pinholes, each pinhole being configured for optical alignment with one of a plurality of detection chambers; (ii) A light source configured to illuminate each of the plurality of detection chambers through a pinhole in the pinhole plate, the pinhole being optically aligned with the detection chamber; (iii) A reflector configured to reflect light from each detection chamber onto the pinhole plate; and (iv) An optical sensor configured to collect light from each of the plurality of detection chambers through a pinhole in the second pinhole plate optically aligned with that detection chamber; and (c) Irradiate the plurality of detection chambers with the light source to analyze the sample fluid.

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