Sample analysis method

Abstract

The present disclosure relates to a sample analysis method, comprising: mixing a predetermined volume of a sample with a reagent to form a sample mixture; forming a sample flow without sheath flow in a flow chamber from the sample mixture; measuring an optical signal from the sample flow and analyzing the optical signal to quantify target particles in the sample flow; allowing the sample flow to flow through a flow sensor; and measuring a sensing signal from the sample flow at the flow sensor to detect that the sample flow enters the flow sensor and / or the sample flow leaves the flow sensor.

Description

[0001] This application is filed on November 3, 2017 、Application No. 2017800804705 、The name of the invention is For fine Fluidics cartridges for cell counting and additional analysis A divisional application for a patent application. Technical Field

[0002] The present disclosure relates to drugs and cell counting. Background Art

[0003] All publications cited herein are incorporated by reference in their entirety, just as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the present disclosure or that any publication specifically or implicitly referenced is prior art.

[0004] Flow cytometry is a common tool for cell analysis of biological samples. Typical cell counting analysis includes two parts. The first part is sample preparation. For example, some cell counting analyses use specific fluorophores to mark target cells so that these cells can be detected by optical measurement of fluorescent signals. In another example, some cell counting analyses require selective lysis of cells in the sample, leaving only intact target cells for cell counting measurement. The second part is sample analysis. Usually the sample flow is focused into a narrow stream when flowing through the flow chamber, wherein the optical or other signals of the target cells are measured one by one. This narrow sample flow is usually obtained by hydrodynamic focusing of the sheath flow.

[0005] The signals measured by flow cytometry can be used to evaluate the characteristics of individual target cells, such as cell size and cell surface roughness. With the help of fluorescent markers, additional cell characteristics can also be evaluated, such as the presence of a cell nucleus, the amount of intracellular DNA, antigens on the cell membrane, and many other characteristics. When measuring cells one by one, the total number of target cells detected can also be determined by counting the number of measured signal peaks. In addition, some cell counting analyses also require measuring the particle density in the sample, that is, the number of target particles per unit volume of sample, which is also called the absolute count in cell counting analysis. For this measurement, it is necessary not only to determine the total number of detected particles, but also to determine the volume of the corresponding sample. These two pieces of information can be used together to calculate the number of particles per unit volume of sample, such as the absolute count.

[0006] In conventional flow cytometry analysis, sample preparation steps are often performed manually. For example, preparation steps are often performed in separate containers, such as centrifuge tubes or vials, and only the final prepared sample is loaded into a commercial cytometer for optical or other measurements. These manual sample preparation steps require precise fluid handling by trained technicians and are therefore unsuitable for applications requiring minimal user training.

[0007] Furthermore, for point-of-care applications such as medical diagnostics, cytometry analysis is performed in non-laboratory settings, such as in an emergency room or physician's office. Therefore, it is important that the biological sample is self-contained and not exposed to an environment that could cause biological contamination. To this end, it is advantageous for both the sample preparation and measurement steps to be performed in a self-contained manner, such as within an unexposed container.

[0008] In addition, absolute count measurements require that the total number of target cells detected and the corresponding sample volume be known. In conventional cell counting analysis, a fixed amount of sample of known volume is injected into the system to determine the absolute count. However, fluidic systems often introduce dead volume, meaning that some portion of the sample is not measured by the cell counter. This dead volume causes the actual sample volume measured to be different from the known volume injected into the system, thus introducing inaccuracies into the absolute count.

[0009] In view of the above factors, it is desirable to develop a fluid cartridge that can perform cell analysis, including sample preparation and sample analysis steps, in a self-contained, automated manner. Such a fluid cartridge is also desirable to be capable of not only performing cell analysis and cell counting, but also accurately measuring absolute counts. Summary of the Invention

[0010] The following embodiments and aspects thereof are described and illustrated in conjunction with apparatus, systems, and methods which are intended to be exemplary and illustrative, not limiting in scope.

[0011] The present disclosure provides various fluid cartridges and methods for using and manufacturing such fluid cartridges. These fluid cartridges can be used for sample preparation and cell counting analysis. These fluid cartridges can be used for various types of cell counting analysis. In various embodiments, the fluid cartridges disclosed herein can be used to determine absolute counts. In various embodiments, the fluid cartridges disclosed herein can be used for DNA analysis of cell populations in tumor diagnosis. In various embodiments, the fluid cartridges disclosed herein can be used for CD4+ / CD8+ lymphocyte subtype analysis in AIDS diagnosis. In various embodiments, the fluid cartridges disclosed herein can be used for cell analysis in complete blood counts (CBCs). These fluid cartridges can also be used for other types of analysis, including but not limited to analytes, proteins, enzymes, nucleic acids, and other biomarkers in analytical samples.

[0012] Various embodiments of the present disclosure provide a device for analyzing target particles in a sample. In various embodiments, the device includes a cartridge device. In various embodiments, the cartridge device includes: an inlet configured to receive the sample into the cartridge device; a fluid structure fluidically connected to the inlet and configured to mix at least a portion of the sample with at least a portion of a reagent to form one or more sample mixtures; a flow chamber fluidically connected to the fluid structure and configured to form one or more sample streams from the one or more sample mixtures, wherein the sample stream is formed in the flow chamber without a sheath flow, and the flow chamber includes an optically transparent area configured to measure an optical signal from the sample stream to detect target particles in the sample; and a flow sensor fluidically connected to the flow chamber and configured to measure a sensing signal from the sample stream entering the flow sensor. In various embodiments, the cartridge device disclosed herein also includes a reagent.

[0013] In various embodiments, the apparatus as disclosed herein further comprises a reader device, wherein the reader device is configured to receive, operate and / or drive the cartridge device. In various embodiments, the reader device neither receives any liquid from the cartridge device nor transfers any liquid to the cartridge device.

[0014] Various embodiments of the present disclosure provide a method for analyzing target particles in a sample. The method comprises: applying the sample to a cartridge device as disclosed herein, the cartridge device being configured to collect a predetermined sample volume into the cartridge device; transferring the cartridge device to a reader device as disclosed herein; mixing at least a portion of the collected sample with at least a portion of a reagent to form one or more sample mixtures within the cartridge device; forming one or more sample streams in a flow chamber within the cartridge device from the one or more sample mixtures, wherein the sample streams are formed in the flow chamber without a sheath flow; measuring an optical signal from the sample stream at the flow chamber to detect target particles in the sample stream; and analyzing the measured optical signal using the reader device to quantify the target particles in the sample.

[0015] Various embodiments of the present disclosure provide a method for analyzing particles in a sample. The method comprises: applying the sample to a cartridge device as disclosed herein, the cartridge device being configured to collect a predetermined sample volume into the cartridge device; transferring the cartridge device to a reader device as disclosed herein; mixing at least a portion of the collected sample with at least a portion of a reagent to form one or more sample mixtures within the cartridge device; forming one or more sample streams in a flow chamber within the cartridge device from the one or more sample mixtures, wherein at least two separate sample mixtures are transferred to the same flow chamber to form at least two separate sample streams without a sheath flow; measuring an optical signal from the sample stream at the flow chamber to detect target particles in the sample stream; and analyzing the measured optical signal using the reader device to quantify the target particles in the sample.

[0016] In various embodiments, the method as disclosed herein further includes: passing a sample flow through a flow sensor fluidically connected to a flow chamber; measuring a sensing signal of the sample flow at the flow sensor to detect the sample flow entering the flow sensor and / or the sample flow leaving the flow sensor; and analyzing the measured optical signal and the sensing signal using a reader device to determine the concentration of target particles in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Exemplary embodiments are shown in the accompanying drawings. It is intended that the embodiments and drawings disclosed herein be considered illustrative rather than restrictive.

[0018] Figure 1 One non-limiting example of a basic fluid unit for use in the cartridge devices disclosed herein is shown, in accordance with various embodiments of the present disclosure.

[0019] Figures 2A-2D Several non-limiting examples of passive valves according to various embodiments of the present disclosure are shown.

[0020] Figures 3A-3C Several non-limiting examples of active valves according to various embodiments of the present disclosure are shown.

[0021] Figures 4A-4C One non-limiting example of implementing a passive valve in a basic fluid unit according to various embodiments of the present disclosure is shown.

[0022] Figure 5 A stick diagram representing a basic fluid unit as described herein is shown, according to various embodiments of the present disclosure.

[0023] Figures 6A-6B One non-limiting example of a sheathless flow chamber as described herein and its sticky diagram are shown in accordance with various embodiments of the present disclosure.

[0024] Figures 7A-7B One non-limiting example of a flow sensor as described herein having two sensing regions along the length of a fluid channel and its stick diagram are shown in accordance with various embodiments of the present disclosure.

[0025] Figures 8A-8B Another non-limiting example of a flow sensor as described herein having only one sensing region along the length of a fluid channel and its stick diagram are shown in accordance with various embodiments of the present disclosure.

[0026] Figures 9A-9C An exemplary configuration of a cartridge device as disclosed herein is shown, wherein a basic fluid unit 9001, a sheathless flow chamber 9007, and a flow sensor 9009 having two sensing areas 9011 and 9012 are connected in series via fluid conduits 9006 and 9008, in accordance with various embodiments of the present disclosure.

[0027] Figures 10A-10B Another exemplary configuration of a cartridge device as disclosed herein is shown, wherein a flow sensor 10007 is connected to a microfluidic channel 10004 of a basic fluidic unit 10001 via a fluid conduit 10006 , in accordance with various embodiments of the present disclosure.

[0028] Figures 11A-11B Another exemplary configuration of a cartridge device as disclosed herein is shown, wherein a basic fluid unit 11001, a sheathless flow chamber 11007, and a flow sensor 11009 having one sensing area 11012 are connected in series via fluid conduits 11006 and 11008, in accordance with various embodiments of the present disclosure.

[0029] Figures 12A-12G Another exemplary configuration of a cartridge device as disclosed herein is shown, wherein two basic fluidic units 12101 and 12201 are used in series with a sheathless flow chamber 12301 and a flow sensor 12401 in accordance with various embodiments of the present disclosure.

[0030] Figures 13A-13C Another exemplary configuration of a cartridge device as disclosed herein is shown, wherein three basic fluidic units 13101 , 13201 , and 13301 are used in series with a sheathless flow chamber 13401 and a flow sensor 13501 , in accordance with various embodiments of the present disclosure.

[0031] Figures 14A-14B Another exemplary configuration of a cartridge device as disclosed herein is shown, wherein four basic fluidic units 14101 , 14201 , 14301 , and 14401 are used in series with a sheathless flow chamber 14501 and a flow sensor 14601 , in accordance with various embodiments of the present disclosure.

[0032] Figures 15A-15D Shown are top views (in the xy plane) of some examples of flow chambers as described herein, according to various embodiments of the present disclosure.

[0033] Figures 16A-16B An example of a plurality of particles flowing through a flow chamber for detection according to various embodiments of the present disclosure is shown.

[0034] Figures 17A-17D An exemplary design for determining absolute counts of particles according to various embodiments of the present disclosure is shown, wherein an outlet 17103 of a flow chamber 17101 is coupled to an inlet 17202 of a flow sensor 17201 through a fluid conduit 17001 .

[0035] Figures 18A-18B Another exemplary design for determining absolute particle counts according to various embodiments of the present disclosure is shown, wherein an inlet 18102 of a flow chamber 18101 is coupled to an outlet 18203 of a flow sensor 18201 through a fluid conduit 18001 .

[0036] Figure 19 A non-limiting example of an analyzer having a cartridge device and a reader device according to various embodiments of the present disclosure is shown. A cartridge 19101 having a fluid structure 19102 can be inserted into a docking slot 19202 on a reader 19201 .

[0037] Figures 20A-20B An exemplary process for constructing a sheathless flow chamber as described herein is shown, according to various embodiments of the present disclosure. DETAILED DESCRIPTION

[0038] All references cited herein are incorporated by reference in their entirety as if fully set forth herein. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Tabelling, Introduction to Microfluidics (Rev.), Oxford University Press (2010); Hguyen et al., Fundamentals and Applications of Microfluidics (2nd ed.), Oxford University Press (2010); nded.), Artech House Incorporated (2006); Berg et al., Microfluidics for Medical Applications, Royal Society of Chemistry (2014); Gomez et al., Biological Applications of Microfluidics (1st ed.), st ed.), Wiley-Interscience (2008); and Colin et al., Microfluidics (1st ed.). st ed.), Wiley-ISTE (2010) provides those skilled in the art with a general guide to many of the terms used in this application.

[0039] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used to practice the present disclosure. Other features and advantages of the present disclosure will become apparent from the following detailed description in conjunction with the accompanying drawings, which illustrate various features of the embodiments of the present disclosure by way of example. In fact, the present disclosure is in no way limited to the methods and materials described. For convenience, certain terms used herein in the specification, examples, and appended claims are collected here.

[0040] Unless otherwise indicated or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or apparent from the context, the following terms and phrases do not exclude the meanings obtained by the term or phrase in the art to which it belongs. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. It should be understood that the present disclosure is not limited to the specific methods, protocols, reagents, etc. described herein and can therefore vary. The definitions and terms used herein are intended to help describe specific embodiments and are not intended to limit the claims.

[0041] As used herein, the term "comprising" or "comprises" is used to refer to compositions, methods, and their respective components, which can be used in the embodiments but include unspecified elements, whether useful or not. Those skilled in the art will understand that, in general, the terms used herein are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including but not limited to," etc.).

[0042] Unless otherwise stated, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the present application (particularly in the context of the claims) may be interpreted as covering both the singular and the plural. The description of numerical ranges herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise stated herein, each individual value is incorporated into this specification as if it were individually cited herein. All methods described herein may be performed in any suitable order, unless otherwise stated herein or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "such as") provided herein with respect to certain embodiments is intended only to better illustrate the present application and does not limit the scope of the claimed application. The abbreviation "e.g., for example" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "e.g., for example" is synonymous with the term "for example". Any language in the specification should not be interpreted as indicating any unclaimed element necessary to practice the present application.

[0043] Various embodiments of the present disclosure provide a device for analyzing target particles in a sample. In various embodiments, the device includes a cartridge device. In various embodiments, the cartridge device includes: an inlet configured to receive a sample into the cartridge device; a fluid structure fluidically connected to the inlet and configured to mix at least a portion of the sample with at least a portion of a reagent to form one or more sample mixtures; a flow chamber fluidically connected to the fluid structure and configured to form one or more sample streams from the one or more sample mixtures, wherein the sample stream is formed in the flow chamber without a sheath flow, and the flow chamber includes an optically transparent area configured to measure an optical signal from the sample stream to detect target particles in the sample; and a flow sensor fluidically connected to the flow chamber and configured to measure a sensing signal from the sample stream entering the flow sensor.

[0044] In various embodiments, the cartridge device has a range of approximately 0.1-1 cm 3 , 1-5cm 3 , 5-25cm 3 , 25-50cm 3 or 50-200cm 3 size.

[0045] In various embodiments, the device as disclosed herein further includes a reader device, wherein the reader device is configured to receive, operate and / or drive the cartridge device. In various embodiments, the reader device is configured to measure an optical signal at the flow chamber to quantify target particles in the sample. In various embodiments, the reader device is configured to measure a sensing signal at a flow sensor to quantify the volume of the sample flow. In various embodiments, the reader device is configured to measure the optical signal at the flow chamber and the sensing signal at the flow sensor to determine the concentration of target particles in the sample. In various embodiments, the reader device includes a control unit configured to measure the optical signal at the flow chamber. In various embodiments, the reader device includes a control unit configured to measure the optical signal at the flow chamber and the sensing signal at the flow sensor. In various embodiments, the reader device neither receives any liquid from the cartridge device nor transfers any liquid into the cartridge device.

[0046] In various embodiments, the cartridge device as disclosed herein further comprises a reagent. In various embodiments, the reagent comprises a fluorescent labeling agent that selectively labels target particles in the sample with fluorescence, and the optical signal from the sample stream comprises fluorescence.

[0047] In various embodiments, the cartridge device as disclosed herein further comprises: a first reagent that is mixed with a portion of the received sample to form a first sample mixture; and a second reagent that is mixed with another portion of the received sample to form a second sample mixture; and the two sample mixtures are transferred separately to the flow chamber to form two separate sample streams. In various embodiments, the two sample mixtures are formed separately in the chamber or transferred separately to the chamber before being transferred separately to the flow chamber. In various embodiments, the chamber has a volume in the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml.

[0048] In various embodiments, the cartridge device as disclosed herein further comprises a fluid conduit fluidically connected to the inlet and configured to receive or collect a sample. In various embodiments, after the sample is collected in the fluid conduit, the fluid conduit is closed by a valve and / or sealed by an external structure. According to various embodiments of the present disclosure, the collected sample is prevented from leaving the cartridge device by closing the valve and / or sealing by an external structure. In various embodiments, the fluid conduit is configured to collect a predetermined sample volume in the range of approximately 0.1-1 μL, 1-5 μL, 5-10 μL, 10-20 μL, or 20-50 μL. In various embodiments, at least a portion of the reagent is transferred to the fluid conduit to flush a portion of the collected sample into the chamber to form a sample mixture.

[0049] In various embodiments, the sample, reagent, sample mixture, or sample flow is enclosed within the cartridge device to prevent or limit their exposure to the environment outside the cartridge. In various embodiments, a fluid structure is located within the cartridge device to prevent or limit exposure of the sample, reagent, or sample mixture to the environment outside the cartridge. In various embodiments, a flow chamber is located within the cartridge device to prevent or limit exposure of the sample flow to the environment outside the cartridge. In various embodiments, a flow sensor is located within the cartridge device to prevent or limit exposure of the sample flow to the environment outside the cartridge.

[0050] In various embodiments, the fluid structure comprises one or more fluid conduits. In various embodiments, the fluid structure comprises one or more chambers. In various embodiments, each chamber has a volume in the range of approximately 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. In certain embodiments, the fluid structure comprises one or more chambers; each chamber has a volume in the range of approximately 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml; and the fluid structure is configured to transfer the sample mixtures from one of the chambers to the flow chamber to form the sample streams.

[0051] In some embodiments, a cartridge device as disclosed herein comprises one flow chamber. In some embodiments, a cartridge device as disclosed herein comprises two, three, four, five, or more flow chambers. In some embodiments, a cartridge device as disclosed herein comprises a plurality of flow chambers.

[0052] In various embodiments, the flow chamber is configured to allow a flow rate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or 200-1000 μl / min. In various embodiments, the flow chamber has a cross-section that is rectangular, trapezoidal, elliptical, circular, or semicircular, or any other shape or combination thereof. In various embodiments, the flow chamber has a width in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm. In various embodiments, the flow chamber has a depth in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm. In various embodiments, the flow chamber has a length in the range of approximately 1-10 μm, 10-100 μm, 100-1,000 μm, 1,000-10,000 μm, or 10,000-50,000 μm. In various embodiments, the sample flow formed in the flow chamber has a cross-section the same size as the flow chamber.

[0053] In certain embodiments, the flow chamber has a width in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm and a depth in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm; and the sample stream has a cross-section of the same size as the flow chamber.

[0054] In various embodiments, the optically transparent area on the flow chamber has a transmittance of 50-60%, 60-70%, 70-80%, 80-90%, 90-96%, or 96-99.9% for the optical signal from the sample stream. In various embodiments, the optical signal comprises scattered light, reflected light, transmitted light, fluorescence, light absorption, light scattering, or a white light image, or a combination thereof. In certain embodiments, the optically transparent area on the flow chamber has a transmittance of 50-60%, 60-70%, 70-80%, 80-90%, 90-96%, or 96-99.9% for the optical signal from the sample stream, and the optical signal comprises scattered light, reflected light, transmitted light, fluorescence, light absorption, light scattering, or a white light image, or a combination thereof.

[0055] In various embodiments, the optically transparent area on the flow chamber is made of a plastic material. In various embodiments, the plastic material is cyclic olefin copolymer, cyclic olefin polymer, polymethyl methacrylate, polycarbonate, polystyrene, or poly-chloro-trifluoro-ethylene, or a combination thereof.

[0056] In various embodiments, a flow sensor includes a fluid channel and a sensing region on the fluid channel; the fluid channel is fluidically connected to a flow chamber to allow a sample flow therethrough; and a sensed signal is measured when the sample flow enters the sensing region. In various embodiments, the sensed signal includes an optical signal. In certain embodiments, the optical signal includes light transmitted through the sample flow and / or light reflected from the sample flow.

[0057] In various embodiments, the fluid channel in the flow sensor has a channel width in the range of approximately 0.001-0.05 mm, 0.05-1 mm, or 1-5 mm, and a channel depth in the range of approximately 0.001-0.01 mm, 0.01-0.5 mm, 0.5-1 mm, or 1-2 mm. In various embodiments, the flow chamber and the flow sensor are configured to have the same flow rate for the sample flow therethrough. In various embodiments, the fluid connection between the flow chamber and the flow sensor is configured to have the same flow rate for the sample flow through the flow chamber and the flow sensor.

[0058] In various embodiments, the sensing region includes an optically transparent area configured to measure an optical signal that changes level between the absence and presence of a sample flow in the sensing region. In various embodiments, the optically transparent area on the sensing region has a transmittance of 50-60%, 60-70%, 70-80%, 80-90%, 90-96%, or 96-99.9% for the optical signal from the sample flow. In various embodiments, the optical signal includes scattered light, reflected light, transmitted light, fluorescence, light absorption, light dissipation, or a white light image, or a combination thereof. In various embodiments, the optically transparent area on the sensing region is made of a plastic material. In various embodiments, the plastic material is cyclic olefin copolymer, cyclic olefin polymer, polymethyl methacrylate, polycarbonate, polystyrene, or poly-chloro-trifluoro-ethylene, or a combination thereof.

[0059] In some embodiments, the flow sensor comprises one sensing region on the fluid channel. In some embodiments, the flow sensor comprises two, three, four, five or more sensing regions on the fluid channel. In some embodiments, the flow sensor comprises multiple sensing regions on the fluid channel.

[0060] In various embodiments, the fluidic structure comprises at least one basic fluidic unit, the basic fluidic unit comprising: a chamber configured to contain a fluid; a vent connected to the chamber, wherein the vent is connected to a pneumatic pressure source, ambient pressure, or atmospheric pressure; a microfluidic channel connected to the chamber; and a valve on the microfluidic channel. In various embodiments, the cartridge device is configured to transfer a sample mixture from the chamber to a flow chamber to form a sample flow when an external actuation mechanism is applied to the cartridge device.

[0061] In various embodiments, the cartridge device is configured to transfer the sample mixture from the chamber to the flow chamber to form a sample stream when an external actuating mechanism is applied to the cartridge device. In various embodiments, the external actuating mechanism comprises a pneumatic pressure source. In various embodiments, the external actuating mechanism is configured to form a sample stream having a flow rate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or 200-1000 μl / min. In certain embodiments, the cartridge device is configured to transfer the sample mixture from the chamber to the flow chamber to form the sample streams when an external actuating mechanism is applied to the cartridge device and the external actuating mechanism comprises a pneumatic pressure source.

[0062] In various embodiments, the chamber of the basic fluidic unit has a volume in the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. In various embodiments, the microfluidic channel of the basic fluidic unit has a volume in the range of about 0.001-0.01 mm. 2, 0.01-0.1mm 2 , 0.1-0.25mm 2 , 0.25-0.5mm 2 , 0.5-1mm 2 , 1-2mm 2 or 2-10mm 2 In some embodiments, the chamber of the basic fluidic unit has a volume in the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. The microfluidic channel of the basic fluidic unit has a volume in the range of about 0.001-0.01 mm 2 , 0.01-0.1mm 2 , 0.1-0.25mm 2 , 0.25-0.5mm 2 , 0.5-1mm 2 , 1-2mm 2 or 2-10mm 2 cross section.

[0063] In various embodiments, when a cartridge apparatus is used, the chamber of the elementary fluidic unit is positioned such that at least a portion of the fluid within the chamber is pulled toward the microfluidic channel and / or away from the vent by gravity. In various embodiments, when a cartridge apparatus is used, the volume of the chamber of the elementary fluidic unit is greater than the volume of the fluid contained therein, and an air gap exists between the vent and the fluid contained therein.

[0064] In various embodiments, the valve of the basic fluid unit is a passive valve configured to allow fluid flow through the microfluidic channel when pneumatic pressure is applied to the fluid flow and to stop the fluid flow when no pneumatic pressure is applied to the fluid flow. In various embodiments, the valve of the basic fluid unit is a passive valve comprising one of the following structures: (i) a channel having a hydrophilic inner surface with a patch embedded with a hydrophobic surface, (ii) a channel having a hydrophobic inner surface with a patch embedded with a hydrophilic surface, (iii) an enlargement of the channel cross-section along the flow direction in the channel having the hydrophilic inner surface, and (iv) a contraction of the channel cross-section along the flow direction in the channel having the hydrophobic inner surface. In various embodiments, the valve of the basic fluid unit is an active valve operated by an actuation mechanism external to the cartridge device.

[0065] Various embodiments of the present disclosure provide a method for analyzing particles in a sample. The method comprises: providing a cartridge device as disclosed herein and a reader device as disclosed herein; applying a sample to the cartridge device; transferring the cartridge device to a reader device; operating the reader device to actuate the cartridge device; and analyzing target particles in the sample.

[0066] Various embodiments of the present disclosure provide a method for analyzing target particles in a sample. The method comprises: applying a sample to a cartridge device as disclosed herein, the cartridge device being configured to collect a predetermined sample volume into the cartridge device; transferring the cartridge device to a reader device as disclosed herein; mixing at least a portion of the collected sample with at least a portion of a reagent to form one or more sample mixtures within the cartridge device; forming one or more sample streams in a flow chamber within the cartridge device from the one or more sample mixtures, wherein the sample streams are formed in the flow chamber without a sheath flow; measuring an optical signal from the sample stream at the flow chamber to detect target particles in the sample stream; and analyzing the measured optical signal using the reader device to quantify the target particles in the sample.

[0067] Various embodiments of the present disclosure provide a method for analyzing target particles in a sample. The method comprises: applying a sample to a cartridge device as disclosed herein, the cartridge device being configured to collect a predetermined sample volume into the cartridge device; transferring the cartridge device to a reader device as disclosed herein; mixing at least a portion of the collected sample with at least a portion of a reagent to form one or more sample mixtures within the cartridge device; forming one or more sample streams in a flow chamber within the cartridge device from the one or more sample mixtures, wherein at least two separate sample mixtures are transferred to the same flow chamber to form at least two separate sample streams without a sheath flow; measuring an optical signal from the sample stream at the flow chamber to detect target particles in the sample stream; and analyzing the measured optical signal using the reader device to quantify the target particles in the sample.

[0068] In various embodiments, a portion of the collected sample is mixed with a first reagent to form a first sample mixture, and another portion of the collected sample is mixed with a second reagent to form a second sample mixture; and the two sample mixtures are transferred separately to the flow chamber to form two separate sample streams. In various embodiments, the two sample mixtures are formed separately in the chamber or transferred separately to the chamber before being transferred separately to the flow chamber. In various embodiments, the chamber has a volume in the range of about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml.

[0069] In various embodiments, the sample is collected into a fluid conduit. In various embodiments, after the sample is collected into the fluid conduit, the fluid conduit is closed by a valve and / or sealed by an external structure. According to a number of different embodiments of the present disclosure, the collected sample is prevented from leaving the cartridge device by closing the valve and / or sealing by an external structure. In various embodiments, the fluid conduit is configured to collect a predetermined sample volume in the range of approximately 0.1-1 μL, 1-5 μL, 5-10 μL, 10-20 μL, or 20-50 μL. In various embodiments, at least a portion of the reagent is transferred to the fluid conduit to flush a portion of the collected sample into the chamber to form a sample mixture.

[0070] In various embodiments, the method as disclosed herein further includes: passing the sample flow through a flow sensor fluidically connected to the flow chamber; measuring a sensing signal from the sample flow at the flow sensor to detect the sample flow entering the flow sensor and / or the sample flow leaving the flow sensor; and analyzing the measured optical signal and the sensing signal using a reader device to determine the concentration of target particles in the sample.

[0071] In various embodiments, the methods disclosed herein further include: flowing the sample flow through a flow sensor fluidically connected to the flow chamber; measuring a sensing signal from the sample flow at the flow sensor to detect the sample flow entering and / or leaving the flow sensor; and analyzing the measured optical signal and the sensing signal using a reader device to determine the concentration of target particles in the sample. In various embodiments, the sample flow in the flow chamber and the flow sensor has the same flow rate.

[0072] In various embodiments, the methods disclosed herein further include: flowing the sample flow through a flow sensor fluidically connected to the flow chamber; measuring a sensing signal from the sample flow at the flow sensor to quantify the volume of the sample flow; and analyzing the measured optical signal and the sensing signal using a reader device to determine the concentration of target particles in the sample. In various embodiments, the sample flow in the flow chamber and the flow sensor has the same flow rate.

[0073] In various embodiments, the optical signal and the sensing signal are measured by a reader device.

[0074] In various embodiments, the collected samples, reagents, sample mixtures, or sample streams are enclosed within the cartridge device to prevent or limit their exposure to the environment external to the cartridge.

[0075] In various embodiments, the mixing step is performed in a fluid structure. In various embodiments, the fluid structure comprises one or more fluid conduits. In various embodiments, the fluid structure comprises one or more chambers. In various embodiments, each chamber has a volume ranging from about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. In certain embodiments, the mixing step is performed in a fluid structure comprising one or more chambers, and each chamber has a volume ranging from about 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml.

[0076] In various embodiments, a fluidic structure is located within the cartridge device to prevent or limit exposure of a sample, reagent, or sample mixture to the environment external to the cartridge. In various embodiments, a flow chamber is located within the cartridge device to prevent or limit exposure of a sample flow to the environment external to the cartridge. In various embodiments, a flow sensor is located within the cartridge device to prevent or limit exposure of a sample flow to the environment external to the cartridge.

[0077] In various embodiments, the flow chamber has a width in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm. In various embodiments, the flow chamber has a depth in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm. In various embodiments, the flow chamber has a length in the range of approximately 1-10 μm, 10-100 μm, 100-1,000 μm, 1,000-10,000 μm, or 10,000-50,000 μm. In various embodiments, the sample flow formed in the flow chamber has a cross-section that is the same size as the flow chamber.

[0078] In certain embodiments, the flow chamber has a width in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm and a depth in the range of approximately 1-10 μm, 10-40 μm, 40-100 μm, or 100-200 μm; and the sample stream has a cross-section of the same size as the flow chamber.

[0079] In various embodiments, when measuring the optical signal from the sample flow, the sample flow in the flow cell has a flow rate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or 200-1000 μl / min. In various embodiments, the optical signal measured from the sample flow at the flow cell comprises scattered light, reflected light, transmitted light, fluorescence, light absorption, light scattering, or a white light image, or a combination thereof.

[0080] In various embodiments, when measuring the sensed signal from the sample flow, the sample flow in the flow sensor has a flow rate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200, or 200-1000 μl / min. In various embodiments, the sensed signal measured from the sample flow at the flow sensor comprises an optical signal. In various embodiments, the optical signal comprises light transmitted through the sample flow and / or light reflected from the sample flow.

[0081] In various embodiments, the sample flow has the same flow rate in the flow chamber and the flow sensor.

[0082] In various embodiments, the reagent comprises a fluorescent labeling agent that selectively labels target particles in the sample with fluorescence, and the optical signal from the sample stream comprises fluorescence.

[0083] In various embodiments, each sample stream is formed and measured separately in the flow chamber. In various embodiments, at least two separate sample mixtures are transferred to the same flow chamber to form at least two separate sample streams. In some embodiments, the at least two separate sample streams are formed continuously (i.e., one after the other). In other embodiments, the at least two separate sample streams are formed non-continuously (i.e., not immediately after one another). In various embodiments, at least one sample stream contains white blood cells as target particles detected in the flow chamber, and at least another sample stream contains red blood cells and / or platelet cells as target particles detected in the flow chamber.

[0084] In various embodiments, the fluid channel in the flow sensor has a channel width in the range of approximately 0.001-0.05 mm, 0.05-1 mm, or 1-5 mm, and a channel depth in the range of approximately 0.001-0.01 mm, 0.01-0.5 mm, 0.5-1 mm, or 1-2 mm; and the sample flow in the flow chamber and the flow sensor has the same flow rate.

[0085] In certain embodiments, mixing is performed in at least one basic fluidic unit comprising: a chamber configured to contain a fluid; a vent connected to the chamber, wherein the vent is connected to a pneumatic pressure source, ambient pressure, or atmospheric pressure; a microfluidic channel connected to the chamber; and a valve on the microfluidic channel. In various embodiments, the chamber of the basic fluidic unit has a volume in the range of approximately 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml. In various embodiments, the microfluidic channel of the basic fluidic unit has a volume in the range of approximately 0.001-0.01 mm 2 , 0.01-0.1mm 2 , 0.1-0.25mm2 , 0.25-0.5mm 2 , 0.5-1mm 2 , 1-2mm 2 or 2-10mm 2 cross section.

[0086] In some embodiments, the mixing is performed in at least one basic fluid unit comprising: a chamber configured to contain a fluid, wherein the chamber has a volume in the range of approximately 0.01-0.1 ml, 0.1-0.2 ml, 0.2-0.4 ml, 0.4-0.8 ml, 0.8-2 ml, or 2-10 ml; a vent connected to the chamber, wherein the vent is connected to a pneumatic pressure source, ambient pressure, or atmospheric pressure; a microfluidic channel connected to the chamber, wherein the microfluidic channel has a volume in the range of approximately 0.001-0.01 mm 2 , 0.01-0.1mm 2 , 0.1-0.25mm 2 , 0.25-0.5mm 2 , 0.5-1mm 2 , 1-2mm 2 or 2-10mm 2 a cross section of a microfluidic channel; and a valve on a microfluidic channel.

[0087] In various embodiments, when the external actuating mechanism is applied to the cartridge device, the sample mixture is transferred from the chamber to the flow chamber to form a sample stream. In various embodiments, the external actuating mechanism comprises a pneumatic pressure source. In various embodiments, the external actuating mechanism is configured to form these sample streams with a flow rate in the range of 0.001-0.01, 0.01-0.1, 0.1-1, 1-50, 50-200 or 200-1000 μl / min. In certain embodiments, when the external actuating mechanism is applied to the cartridge device and the external actuating mechanism comprises a pneumatic pressure source, the sample mixture is transferred from the chamber to the flow chamber to form a sample stream.

[0088] In various embodiments, the target particles have a size in the range of 0.1-1 μm, 1-10 μm, 10-15 μm, 15-30 μm, 30-50 μm, or 50-100 μm. In various embodiments, the target particles have a concentration in the range of 1-100, 100-1000, 1000-5000, 5000-20,000, or 20,000-50,000 target particles per μl of sample vapor. In certain embodiments, the target particles have a size in the range of 0.1-1 μm, 1-10 μm, 10-15 μm, 15-30 μm, 30-50 μm, or 50-100 μm; and the target particles have a concentration in the range of 1-100, 100-1000, 1000-5000, 5000-20,000, or 20,000-50,000 target particles per μl of sample vapor.

[0089] In various embodiments, the target particles comprise cells, plant cells, animal cells, blood cells, white blood cells, red blood cells, platelet cells, viruses, bacteria, fungi, yeast, beads, fluorescent beads or non-fluorescent beads; or other particles of proteins, enzymes, nucleic acids, polysaccharides or polypeptides; or other particles associated with biomarkers; or combinations thereof.

[0090] Figure 1 A non-limiting example of a basic fluid unit used in a cartridge is shown. Basic fluid unit 1001 has a chamber 1002, a vent 1003, and at least one microfluidic channel 1004, which enters the chamber and has a valve 1005 on the microfluidic channel. The operation of basic fluid unit 1001 depends on gravity or any other force used as a gravity substitute (e.g., centrifugal force) to hold the fluid in place. In addition, basic fluid unit 1001 uses another force, such as pneumatic pressure, to transfer fluid. More information about the design, operation, and manufacture of fluid unit 1001 can be found in U.S. application Ser. No. 15 / 176,729 and PCT application PCT / US16 / 36426, the entire contents of which are incorporated herein by reference as if fully set forth. In some embodiments, valve 1005 can be a passive valve. In other embodiments, valve 1005 can be an active valve. In certain embodiments, valve 1005 can be a mixture or combination of passive and active valves. In other embodiments, valve 1005 can be of any design known to those of ordinary skill in the art.

[0091] Figures 2A-2D Several non-limiting examples of passive valves are shown. Other passive valve designs known to those skilled in the art may also be used. Figure 2A It is a passive valve design with a channel with a hydrophilic inner surface and a hydrophobic surface patch. Figure 2BIt is a passive valve design with a channel with a hydrophobic inner surface and a hydrophilic surface patch. Figure 2C It is a passive valve design with an enlargement of the channel cross section in the flow direction and the channel has a hydrophilic surface. Figure 2D It is a passive valve design with a narrow channel cross section along the flow direction and the channel has a hydrophobic surface.

[0092] Figures 3A-3C Several non-limiting examples of active valves are shown. Other active valve designs known to those skilled in the art may also be used. Figure 3A A valve design is shown that includes a flexible membrane 3001 and a substrate 3002. When the flexible membrane 3001 is bent away from the substrate 3002, the valve is in an "open" state, allowing fluid to flow through. When the flexible membrane 3001 is bent toward the substrate 3002 without leaving a gap, the valve is in a "closed" state, and fluid cannot flow through. Figure 3B A valve design is shown with a movable membrane 3003 and a substrate 3004. When the movable membrane 3003 is away from the substrate 3004, a fluid path 3005 exists between the inlet and outlet, and the valve is in the "open" state. When the movable membrane is close to the substrate without leaving a gap, there is no fluid path between the inlet 3006 and the outlet 3007, and the valve is in the "closed" state. Figure 3C A valve design is shown with a plug 3008 in a channel. When the plug 3008 is pulled away from the channel and away from the base 3009, the channel is in an "open" state, allowing fluid to flow from the inlet 3010 to the outlet 3011. When the plug 3008 is inserted into the channel and contacts the base 3009, the channel is in a "closed" state, and there is no fluid path between the inlet 3010 and the outlet 3011. The plug 3008 can be made of a solid material, a polymer, an elastomer, a gel, a wax, a silicone oil, or other materials. When an active valve is used, an additional actuation mechanism can be used to operate the valve.

[0093] Figure 4A Another non-limiting example of implementing a passive valve in a basic fluid unit 4001 is shown. In one embodiment, a transition region 4005 from the chamber 4002 to the channel 4004 provides a cross-sectional reduction in the flow channel. When the channel inner surface and the chamber inner surface in the transition region 4005 are both hydrophobic, as shown in FIG. Figure 4B As shown, the transition region 4005 is equivalent to a sudden narrowing of the hydrophobic channel and acts as a passive valve to prevent the fluid in the chamber 4002 from entering the channel 4004. When the inner surface of the channel in the transition region 4005 and the inner surface of the chamber are both hydrophilic, as shown in FIG. Figure 4C As shown, the transition region corresponds to an abrupt expansion of the hydrophobic channel and acts as a passive valve to prevent fluid in channel 4004 from entering chamber 4002. Other designs of passive valves known to those skilled in the art may also be implemented.

[0094] Figure 5 A symbolic diagram representing a basic fluidic unit as described herein is shown, wherein a basic fluidic unit 5001 includes a chamber 5002, a vent 5003, and at least one microfluidic channel 5004 that enters the chamber and has a valve 5005 on the microfluidic channel. The valve 5005 can be a passive valve, an active valve, or a hybrid or combination of the two. In some embodiments, the basic fluidic unit 5001 can have one or more microfluidic channels (each having a valve) that enter the chamber 5002 (see, for example, U.S. application Ser. No. 15 / 176,729 and PCT application PCT / US16 / 36426, the entire contents of which are incorporated herein by reference as if fully set forth).

[0095] In various embodiments, the present disclosure provides a fluid cartridge having at least one basic fluid unit as described herein. In various embodiments, the fluid cartridge may have additional fluid structures. An example of an additional fluid structure is one or more flow chambers for cell counting analysis. For conventional flow cytometers, the flow chamber typically has a core diameter of several hundred microns. In order to obtain a sample flow with a smaller core diameter (e.g., a few microns to tens of microns), the flow chamber utilizes a sheath flow to focus the sample flow. In some embodiments, the fluid cartridge as described herein includes a conventional flow chamber with a sheath flow.

[0096] In other embodiments, the fluid cartridges described herein include a flow chamber with sheathless flow, rather than a conventional flow chamber with sheath flow. The sheathless flow chamber has a fluid channel with a core diameter selected based on the target sample flow diameter. For example, a target sample flow with a diameter of 30 μm can be achieved using a fluid channel with a diameter of 30 μm. Additionally, the channel of the flow chamber can be transparent to certain excitation and emission light wavelengths, thereby enabling measurement of optical signals (e.g., wavelengths of light) from the sample in the flow chamber. Figure 6A ). Figure 6B A stick figure representing a sheathless flow chamber as described herein is shown. Because the flow chamber does not utilize sheath flow, the sample flow has a cross section of the same dimensions as the flow chamber.

[0097] Another example of an additional fluidic structure is one or more flow sensors for detecting sample flow. This document describes the design and operation of such flow sensors, which have one or more sensing regions on a channel to detect the presence of liquid in the channel and / or measure fluid displacement volume, fluid plug volume, flow rate or velocity, etc. Further information regarding the design, operation, and manufacture of flow sensors can be found in U.S. application Ser. No. 15 / 209,226 and PCT application No. PCT / US16 / 42089, the entire contents of which are incorporated herein by reference as if fully set forth. Figure 7AA non-limiting example of a flow sensor is shown, which has two sensing regions along the length of a fluid channel. The sensor detects the presence of fluid within the channel that overlaps the sensing regions. The volume of fluid filling the channel between the two sensing regions can be determined from the known geometry of the channel. Figure 7B is a symbolic diagram representing this design. Figure 8A Another non-limiting example of a flow sensor is shown having only one sensing region along the length of the fluid channel. Figure 8B is a symbolic diagram representing this design.

[0098] Described herein are various fluidic units and additional fluidic structures that can be used together in various configurations to perform flow cytometric analysis. This function Integrates sample preparation and absolute counting in a self-contained cartridge.

[0099] Figure 9A An exemplary configuration is shown in which a basic fluidic unit 9001, a sheathless flow chamber 9007, and a flow sensor 9009 having two sensing areas 9011 and 9012 are connected in series via fluid conduits 9006 and 9008. In some embodiments, the upstream end of the flow chamber 9007 is connected to the microfluidic channel 9004 of the basic fluidic unit 9001, and the downstream end of the flow chamber 9007 is connected to the flow sensor. In a specific configuration, the sample in the chamber 9002 of the unit 9001 will first pass through the flow chamber and then through the flow sensor for cytometry analysis.

[0100] When using this configuration for cell counting analysis, such as Figure 9B As shown, a fluid sample 9101 can first be loaded into chamber 9002. Pneumatic pressure can then be applied to the vent 9003 of the basic fluidic unit 9001 and the outlet 9010 of the flow sensor. When the pneumatic pressure at the vent 9003 is higher than the pneumatic pressure at the port 9010, a pressure differential is created, pumping the sample 9101 from chamber 9002 into a flow chamber 9007 for cell counting analysis and then into a flow sensor 9009 for volume measurement. When valve 9005 is a passive valve, a sufficiently high pressure differential can pump the fluid sample through valve 9005. When valve 9005 is an active valve, valve 9005 can be switched to an open state before pressure can pump the fluid sample 9101 through valve 9005.

[0101] After the pneumatic pressure is applied, data is continuously recorded for cytometry analysis in the flow chamber 9007. The recorded data includes the physical signal A (light emission, electrical impedance, etc.) measured along time T as an array (A, T). Figure 9CAn example of recorded data is shown, where the amplitude of signal A is plotted against time T. The number of particles detected in the cell counter is determined by the number of peaks in signal A. Simultaneously, as the sample continues to pass through the flow sensor 9009, the time point T1 at which the sample reaches the first sensing region 9011 is recorded, and the time point T2 at which the sample reaches the second region 9012 is also recorded. Figure 9C As shown in the example of , the number of particles N detected between T1 and T2 can be determined based on the recorded signals (A, T). The volume of fluid V0 used to fill the channel between sensing regions 9011 and 9012 is a known parameter from the design of flow sensors (see, for example, U.S. application Ser. No. 15 / 209,226 and PCT application PCT / US16 / 42089, the entire contents of which are incorporated herein by reference as if fully set forth). Because the sheathless flow chamber contains only the fluid sample for analysis (without sheath flow), the volume of fluid between the two sensing regions can be used to determine the volume of the sample analyzed in flow chamber 9007. Therefore, the absolute count can then be calculated as:

[0102] Absolute count = N / V0 [1]

[0103] In addition to the ability to perform cell counting analysis using absolute counts, Figures 9A-9C The examples in the invention also have the following features: the entire fluid structure can be implemented in a self-contained cartridge device. The cartridge device can be a plastic molded part with an additional sealing layer.

[0104] Figure 10A Another exemplary configuration is shown, in which a flow sensor 10007 is connected to the microfluidic channel 10004 of the basic fluidic unit 10001 via a fluid conduit 10006. At the same time, a sheathless flow chamber 10009 is connected downstream of the flow sensor 10007 via a fluid conduit 10008. In this example, the number of cells counted, N, is determined by the signal (A, T) between time points T1+ΔT and T2+ΔT, as shown in FIG. Figure 10B As shown, ΔT can be any empirical value to compensate for the time delay between the sample reaching the first sensing region 10011 and the sample reaching the flow chamber 10009. Figure 9A Operation, Figure 10A The configuration can also be used for cell counting analysis using absolute counts:

[0105] Absolute count = N / V0 [2]

[0106] Figure 11AAnother exemplary configuration is shown in which a basic fluidic unit 11001, a sheathless flow chamber 11007, and a flow sensor 11009 having one sensing area 11012 are connected in series via fluid conduits 11006 and 11008. In some embodiments, the upstream end of the flow chamber is connected to the microfluidic channel 11004 of the basic fluidic unit 11001, and the downstream end of the flow chamber is connected to the flow sensor. In this exemplary configuration, the sample in the chamber 11002 of the unit 11001 first passes through the flow chamber and then passes through the flow sensor for cell counting analysis. In this example, as shown in FIG. Figure 11B As shown, time points T1 = 0 (when the flow chamber begins detecting particles) and T2 (when the sample reaches sensing region 11012) are used to determine the total particle count N based on the recorded signal (A, T). Additionally, the fluid volume V1 used to obtain the particle count N includes the total fluid conduit volume between flow sensor 11007 and sensing region 11012 of flow sensor 11009. Fluid volume V1 is a known parameter from the fluidics design. Similar to the operation of FIG. 9 discussed above, the configuration of FIG. 10 can also be used for cell counting analysis using absolute counts:

[0107] Absolute count = N / V1 [3]

[0108] Figure 12A Another exemplary configuration is shown in which two basic fluidic units 12101 and 12201 are used in series with a sheathless flow chamber 12301 and a flow sensor 12401. Fluid conduit 12001 connects the microfluidic channel 12104 (with valve 12105) of basic fluidic unit 12101 with the microfluidic channel 12204 (with valve 12205) of basic fluidic unit 12201. Unit 12201 has a second microfluidic channel 12206 (with valve 12207) connected to the upstream end of flow chamber 12301 via fluid conduit 12002. The downstream end of flow chamber 12301 is also connected to flow sensor 12401 via fluid conduit 12003. In this example, flow sensor 12401 has two sensing regions 12402 and 12403.

[0109] For cytometry analysis, pneumatic pressure is applied to three ports, including vent 12103 of unit 12101 (P1), vent 12203 (P2) of unit 12201, and downstream port 12404 (P3) of flow sensor 12401. By controlling the applied pneumatic pressure (P1, P2, and P3), a fluid sample can be transferred between chamber 12102 and chamber 12202, and further transferred to the flow chamber for cytometry analysis using absolute counting.

[0110] Figure 12BAn exemplary method of controlling pneumatic pressures (P1, P2, and P3) and corresponding fluid transfer is shown in the illustration. When a fluid sample is in the first chamber (chamber 12102), by applying pneumatic control (P1 > P0 and P2 = P3 = P0), the fluid sample can be transferred from the first chamber to the second chamber (chamber 12202). Similarly, by applying pneumatic control (P1 < P0 and P2 = P3 = P0), the fluid sample can be transferred from the second chamber to the first chamber. When the fluid sample is in the second chamber, by applying pneumatic control (P1 = P2 = P0 and P3 < P0), the fluid sample can be transferred from the second chamber to the flow chamber and flow sensor for cell counting analysis. In this exemplary pneumatic control method, during operation, the vent hole of the second chamber P2 remains at a constant pressure P2 = P0. Following the teachings of the present disclosure and those previously disclosed by the applicant (see, for example, U.S. application US15 / 176,729 and PCT application PCT / US16 / 36426, the entire contents of which are incorporated herein by reference as if fully set forth), other methods can also be used to control fluid transfer in the configuration. For example, by applying pneumatic control (P1 = P2 > P0 and P3 = P0), the fluid sample in the second chamber can be transferred from the second chamber to the flow chamber and flow sensor for cell counting analysis. In some embodiments, P0 is the atmospheric pressure at which the fluid configuration is operated.

[0111] Using the pneumatic control method, a sample can be transferred in a fluid configuration for cell counting analysis using absolute counting. For example, a fluid sample can first be introduced into the first chamber (chamber 1210 [2]). Then the sample is transferred to the second chamber (chamber 12202). Then the sample can be driven through the flow chamber and flow sensor for cell counting analysis of absolute counting as described above. In another example, a fluid sample can first be introduced into the second chamber and then driven through the flow chamber and flow sensor for cell counting analysis using absolute counting. In another example, fluid sample A can first be introduced into the first chamber, while fluid sample B can first be introduced into the second chamber, and then the two samples can be transferred between the two chambers for multiple mixing cycles before being delivered to the flow chamber for counter analysis using absolute counting. The mixing action includes the fluid sample moving from the first chamber to the second chamber in one direction and then from the second chamber to the first chamber in the opposite direction, and vice versa. In some embodiments, fluid sample A has a predetermined volume. In certain embodiments, fluid sample B has a predetermined volume.

[0112] In another embodiment, fluid sample A can be first introduced into the first chamber 12102 and fluid sample C can be first loaded into the fluid conduit 12001. By transferring the fluid samples between the two chambers, fluids A and C can be mixed together and then transported to the flow chamber for absolute cell counting analysis. The sample exiting the flow sensor outlet can be placed or collected in a reservoir. In embodiments where a reservoir is used to collect the outflow sample, the fluid configuration of this embodiment implements sample preparation, cell counting analysis, and absolute counting functions in a self-contained manner, without requiring the exchange of fluid samples between the fluid structure and the external environment. This embodiment can be used for cell counting analysis of different biological samples. For example, fluid sample C can be a biological sample, such as whole blood from a human. Fluid sample A can be a reagent containing a fluorophore-conjugated antibody that targets a specific cell type, for example, CD4+ lymphocytes in whole blood. By mixing the two fluids and then measuring the mixture in the flow chamber, an absolute count of CD4+ lymphocytes is achieved. In another example, fluid sample C can be human whole blood, while fluid sample A can be a lysis solution that selectively targets red blood cells (RBCs). After mixing the two fluids together and incubating the mixture for a period of time, the mixture is measured in a flow cell for cytometry analysis, such as absolute white blood cell (WBC) counts.

[0113] Figure 12C An exemplary fluid configuration is shown in which an additional fluid conduit 12004 is connected to the fluid conduit 12001 to introduce an initial sample C. The sample can be introduced into the fluid conduit 12001 via port 12006. Valve 12005 can then be closed to seal the conduit 12004, preventing the sample from leaving port 12006. In some embodiments, the fluid conduit 12001 can be used to collect a predetermined volume of the initial sample. In some embodiments, valve 12005 can be a blood clotting valve (see, e.g., U.S. Patent No. 8,845,979, the entire contents of which are incorporated herein by reference as if fully set forth herein). Other methods known to those skilled in the art can also be used to introduce the initial sample.

[0114] In some embodiments, a reservoir chamber can be connected to the outlet of a flow sensor to collect a fluid sample after cytometry analysis. Figure 12D As shown in the exemplary fluid configuration of FIG, a reservoir 12501 having a vent 12502 is connected to the outlet 12404 of the flow sensor via a fluid conduit 12503. With this additional reservoir, the pneumatic pressure P3 can be adjusted by controlling the pneumatic pressure P4 at the vent 12502 of the reservoir. The exemplary operation of this fluid configuration is Figure 12E Shown in.

[0115] In other embodiments, additional fluidic structures may be connected to one or more basic fluidic units to implement additional operational functions. Figure 12F An example of these embodiments is shown. Figure 12A Compared to the example of FIG5 , the second basic fluid unit 12201 has a third microfluidic channel 12208 (with a valve 12209), which is connected to a reservoir structure 12601 having a vent 12602 via a fluid conduit 12007. The vent 12602 corresponds to the pneumatic pressure P5. The fluid sample D is first stored in the reservoir 12601. Figure 12G The pneumatic controls for operating this configuration are shown. By applying the pneumatic controls (P5>P0 and P2=P0), the sample D first stored in the reservoir 12601 is transferred to the second chamber 12202. The remaining pneumatic operations for cell counting analysis can be the same as Figure 12B Same as the example in .

[0116] In yet another exemplary configuration, Figure 13A As shown, there are three basic fluidic units 13101, 13201, and 13301. Units 13101 and 13201 have microfluidic channels 13104 (with valve 13105) and 13204 (with valve 13205), respectively. Unit 13301 has three microfluidic channels 13304 (with valve 13305), 13306 (with valve 13307), and 13308 (with valve 13309). Fluid conduit 13001 connects channels 13104 and 13304, while another fluid conduit 13002 connects channels 13204 and 13306. Fluidic unit 13302 is also connected to the upstream port of a sheathless flow chamber 13401 via fluid conduit 13003, while the downstream port of flow chamber 13401 is connected to a flow sensor 13501 via fluid conduit 13004. The fluid configuration is operated by controlling the pneumatic pressure at the vents of the elementary fluidic units 13103 (P1), 13203 (P2) and 13303 (P3), and by controlling the pneumatic pressure at the outlet port 13504 of the flow sensor (P4).

[0117] Figure 13BAn exemplary method for controlling pneumatic pressures (P1, P2, P3, and P4) and corresponding fluid transfers is shown. By applying pneumatic control (P1 > P0 and P2 = P3 = P4 = P0), a fluid sample in the first chamber (13102) can be transferred to the third chamber (13302), and by applying pneumatic control (P1 < P0 and P2 = P3 = P4 = P0), a fluid sample in the third chamber can be transferred to the first chamber. Similarly, a fluid sample in the second chamber (13202) can be transferred to the third chamber by applying pneumatic control (P2 > P0 and P1 = P3 = P4 = P0), and a fluid sample in the third chamber can be transferred to the second chamber by applying pneumatic control (P2 < P0 and P1 = P3 = P4 = P0). Meanwhile, by applying pneumatic control (P4 < P0, P1 = P2 = P3 = P0), a fluid sample in the third chamber can be transferred to the flow chamber and flow sensor for cell counting analysis. In this exemplary pneumatic control method, the vent hole of the third chamber P3 remains at a constant pressure P3 = P0 during operation. Following the teachings of the present disclosure and those previously disclosed by the applicant (see, for example, U.S. Application 15 / 176,729 and PCT Application PCT / US16 / 36426, the entire contents of which are incorporated herein by reference as if fully set forth herein), other methods can also be used to control fluid transfers in this configuration.

[0118] This fluid configuration and fluid transfer diagram can be used for more complex sample preparation and cell counting analysis. For example, as Figure 13CAs shown, fluid sample A1 is first introduced into the first chamber, and fluid sample A2 is first introduced into the second chamber. At the same time, fluid samples B1 and B2 are introduced into fluid conduits 13001 and 13002, respectively. By applying pneumatic control (P1 > P0 and P2 = P3 = P4 = P0), fluid samples A1 and B1 are transferred into the third chamber. By applying pneumatic control (P4 < P0 and P1 = P2 = P3 = P0), the sample mixture of A1 and B1 is transferred into the sheathless flow chamber and the flow sensor for absolute counting in cell counting analysis. After the cell counting analysis, if any residue of the sample mixture remains in the third chamber, it can be transferred back into the first chamber by applying pneumatic control (P1 < P0 and P2 = P3 = P4 = P0), and thus the third chamber is emptied to prepare for analyzing other samples. Before the cell counting analysis, if the mixing uniformity of the sample mixture is a design consideration, pneumatic control (P1 < P0 and P2 = P3 = P4 = P0) and (P1 > P0 and P2 = P3 = P4 = P0) can be applied sequentially to move the mixture from the third chamber into the first chamber and then back into the third chamber from the first chamber. The mixing effect of the introduced samples. This step can be repeated to obtain the desired mixing uniformity. In these fluid transfer steps, fluid samples A2 and B2 do not move. This is achieved by maintaining the pneumatic pressure (P2 = P3). Next, fluid samples A2 and B2 can be transferred into the third chamber by applying pneumatic control (P2 > P0 and P1 = P3 = P4 = P0). Then, the mixture of samples A2 and B2 is transferred into the flow chamber for the cell counter by applying pneumatic control (P4 < P0 and P1 = P2 = P3 = P0). If the mixing uniformity is ideal, the repeated transfer steps between the second and third chambers can also be carried out similar to the repeated transfer steps between the first and third chambers. In some embodiments, fluid sample A1 has a predetermined volume. In some embodiments, fluid sample A2 has a predetermined volume. In some embodiments, fluid sample B1 has a predetermined volume. In some embodiments, fluid sample B2 has a predetermined volume.

[0119] This fluid configuration and fluid transfer diagram can be used for implementing the cell counting analysis of various biological samples.For example, fluid sample A1 can be the dilution buffer for RBC analysis, and sample B1 can be whole blood, and sample A2 can be the lysis buffer for WBC analysis, and sample B2 can be whole blood.A1 and B1 can be transferred in the third chamber to form a mixture, then enter into the flow chamber to count and analyze the RBC and the platelet in the blood.Then A2 and B2 are transferred in the third chamber to form a mixture, then transferred into the flow chamber to count and analyze the WBC in the blood.Can use different dilution buffers and lysis buffer known to those skilled in the art of hematology analyzer field.Like this, fluid configuration can be used for realizing the complete blood count (CBC) analysis that is widely used in clinical trials.

[0120] Figures 12A-12G and Figures 13A-13C Examples with two and three basic fluid units are shown, respectively. In other embodiments, more basic fluid units can be used in this configuration to achieve additional complexity. Figures 12A-12G and Figures 13A-13C In the example of FIG, the fluid conduits (eg, fluid conduit 12001, fluid conduit 13001, and fluid conduit 13002) used to connect the basic fluid units are fluid channels. In other embodiments, fluid structures with additional complexity can be used as fluid conduits.

[0121] Figure 14AAn example is shown having four basic fluid units 14101, 14201, 14301, and 14401. Each of the four units 14010, 14201, and 14401 has a microfluidic channel with a valve. Unit 14301 has four microfluidic channels, including channel 14304 (with valve 14305), channel 14306 (with valve 14307), channel 14308 (with valve 14309), and channel 14310 (with valve 14311). Basic fluid unit 14101 is connected to basic fluid unit 14301 via fluid conduit 14001. Basic fluid unit 14201 is connected to basic fluid unit 14301 via fluid conduit 14002. Basic fluid unit 14401 is connected to basic fluid unit 14301 via fluid conduit 14003. The upstream side of the unsheathed flow chamber 14501 is connected to the basic fluid unit 14301 via a fluid conduit 14004, while the downstream side of the flow chamber 14501 is connected to a flow sensor 14601 having two sensing areas 14602 and 14603 via a fluid conduit 14005. The flow sensor 14601 is then connected to a reservoir chamber 14701 having a vent 14702. Simultaneously, sample A1 can be initially stored in chamber 14102 of the basic fluid unit 14101, sample A2 can be initially stored in chamber 14202 of the basic fluid unit 14201, and sample A3 can be initially stored in chamber 14402 of the basic fluid unit 14401. Additionally, sample B1 can be introduced into the fluid conduit 14001 via an inlet 14801 through a fluid conduit 14801 having a valve 14803. Valve 14803 can be closed after the sample is introduced. In some embodiments, fluid conduit 14001 can be used to collect a predetermined volume of sample. Similarly, sample B2 can be introduced into fluid conduit 14002 via inlet 14901 through fluid conduit 14902 having valve 14903. Valve 14903 can be closed after the sample is introduced. In some embodiments, fluid conduit 14002 can be used to collect a predetermined volume of sample.

[0122] An exemplary method of controlling the pneumatic pressures (P1, P2, P3, P4, and P5) is described below and is Figure 14BThe corresponding fluid transfer is shown in the illustration. When there is a pneumatic path between the two ports (e.g., when there is an air path in reservoir 14701 to balance vent 14702 and port 14604), the pneumatic pressure P5 at vent 14702 of reservoir 14701 balances the pressure P5’ at the downstream port 14604 of flow sensor 14601. By applying pneumatic control (P1 > P0 and P2 = P3 = P4 = P5 = P0), the fluid sample in the first chamber (14102) can be transferred to the third chamber (14302), and by applying pneumatic control (P1 > P0 and P2 = P3 = P4 = P5 = P0), the fluid sample in the third chamber can be transferred to the first chamber, where P0 is the atmospheric pressure. The fluid sample in the second chamber (14202) can be transferred to the third chamber by applying pneumatic control (P2 > P0 and P1 = P3 = P4 = P5 = P0), and the fluid sample in the third chamber can be transferred to the second chamber by applying pneumatic control (P2 < P0 and P1 = P3 = P4 = P5 = P0). Similarly, by applying pneumatic control (P4 > P0 and P1 = P2 = P3 = P5 = P0), the fluid sample in the fourth chamber (14402) can be transferred to the third chamber, and by applying pneumatic control (P4 < P0 and P1 = P2 = P3 = P5 = P0), the fluid sample in the third chamber can be transferred to the fourth chamber. Meanwhile, by applying pneumatic control (P5 < P0, P1 = P2 = P3 = P4 = P0), the fluid sample in the third chamber can be transferred to the flow chamber and the flow sensor for cell counting analysis. In this exemplary pneumatic control method, the vent of the third chamber P3 remains at a constant pressure P3 = P0 during operation. Following the teachings of the present disclosure and those previously disclosed by the applicant (see, e.g., U.S. Application 15 / 176,729 and PCT Application PCT / US16 / 36426, the entire contents of which are incorporated herein by reference as if fully set forth), other methods can also be used to control fluid transfer in this configuration.

[0123] In various embodiments, the sheathless flow chamber is where target particles in a fluid sample stream are detected and measured by different signals such as fluorescence, light scattering, light absorption and light extinction, white light imaging, etc. The excitation light (EL) beam from a light source can be shaped and used to illuminate a designated sensing area of the fluid flow chamber and trigger the above signals from the target particles.

[0124] The sheathless flow chamber can be a fluid channel with various geometries. Figures 15A-15DShown are top views (in the xy plane) of several examples of flow chambers. The top view (xy plane) is defined as the plane perpendicular to the excitation light direction (z-axis). Length is defined as the channel dimension along the sample flow (x-axis), width is defined as the dimension along the y-axis, and depth is defined as the channel dimension along the z-axis. Figure 15B An example of a flow chamber with a gradually decreasing width is shown, where the maximum width is W1 and the minimum width is W2. In other embodiments, the flow chamber can have a gradually increasing width. Figure 15C An example of a flow chamber having a non-gradually varying width is shown, where the maximum width is W1 and the minimum width is W2. Figure 15D An example of a flow chamber with a fixed width (W1=W2=W0) at different positions along the length of the channel is shown. In some embodiments, the difference between the maximum width W1 and the minimum width W2 is within a specified difference. A non-limiting example of a width difference range is (W1-W2) / W2≤20%. The range of channel width and depth is selected to be large enough so that target particles (e.g., cells in a biological sample) can pass through the flow chamber without blocking the flow chamber. At the same time, they are selected to be small enough to minimize the coincidence error in flow cytometry analysis. The minimum width W1 can be in the range of 1-10 μm, 10-40 μm, 40 to 100 μm, or 100 to 200 μm. The depth of the channel can be in the range of 1 μm to 10 μm, 10 μm to 40 μm, 40 μm to 100 μm, or 100 μm to 200 μm. The cross section of the channel (in the yz plane) can have a rectangular, trapezoidal, circular, semicircular shape, or any other shape. The length of the flow chamber should be long enough to optically detect particles in the sample stream, yet short enough to reduce flow resistance to the sample stream flowing therethrough. In various embodiments, the length of the flow chamber can be in the range of approximately 1-10 μm, 10-100 μm, 100-1,000 μm, 1,000-10,000 μm, or 10,000-50,000 μm.

[0125] Because the sheathless flow chamber is used for optical measurements, at least one surface of the channel is transparent to the wavelength of light involved in the measurement. The material used to form the channel surface can be any transparent material, such as glass, quartz, and plastic, including but not limited to cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), and poly-chloro-trifluoroethylene (PCTFE) materials such as Aclar.

[0126] The fluid sample for analyzing in the flow chamber can be a fluid suspension of multiple particles. For example, the fluid sample can be a blood sample containing different cells, such as white blood cells, red blood cells and platelets. In another example, the fluid sample can be a blood sample in which certain types of cells, such as white blood cells, remain intact, while other types of cells, such as red blood cells, have been dissolved. In another example, the fluid sample can be a blood sample in which certain types of cells have been labeled with a fluorophore. In another example, the fluid sample can be a mixture of cells and other particles, such as non-fluorescent beads and / or fluorescent beads. In other examples, the fluid sample can also be other biological samples, such as cerebrospinal fluid, urine, saliva, semen, etc.

[0127] As particles flow through the sheathless flow chamber, various optical signals can be measured to detect and characterize the particles. Measurable signals include, but are not limited to, fluorescence, light scattering, light absorption, light discrimination, and the like. Figure 16A An example of multiple particles flowing through a flow cell for detection is shown. All particles in the sample flow through one by one. Under illumination with excitation light (EL), each cell can be characterized for optical signals including, but not limited to, fluorescence (FL) and light scattering (LS). Figure 16B Another example is shown in which multiple particles flow through a flow chamber for detection. Some particles flow through while overlapping each other. However, if only target particles are considered, they still flow through one by one without overlapping with other target particles. In this case, light scattering from the target particles can be blocked by other particles that overlap with them. However, if these particles are pre-treated with specific fluorophores to distinguish them from other particles, other signals including but not limited to fluorescence signals can still be measured to detect these target particles. In some embodiments, the other particles can be non-fluorescent or treated with a fluorophore different from the target particles.

[0128] In one embodiment, the fluid sample can be a blood sample in which white blood cells are labeled with a fluorophore and red blood cells are not. As the labeled white blood cells pass through the flow chamber one by one, corresponding fluorescence signals can be measured to detect and characterize the white blood cells, even when overlapping red blood cells are present. In another embodiment, the fluid sample can be a blood sample in which the white blood cells are labeled with a fluorophore and the red blood cells are lysed. As the labeled white blood cells pass through the flow chamber one by one, corresponding fluorescence signals and light scattering signals can be measured simultaneously from these cells for detection and characterization. In another embodiment, the fluid sample can be a blood sample in which fluorophore-labeled white blood cells and fluorescent beads are present. As these white blood cells and beads pass through the flow chamber one by one, they can be detected by corresponding fluorescence signals. Other cells that do not fluoresce or have different fluorescence wavelengths do not interfere with the measurement. In another embodiment, the fluid sample can be a blood sample in which red blood cells and beads are present, along with other cells. As the cells pass through one by one, light scattering signals can be measured to detect and characterize the red blood cells and beads. In another embodiment, the beads can be labeled with a fluorophore so that they can be distinguished from the red blood cells by light scattering signals, fluorescence signals, or both.

[0129] The combination of a sheathless flow chamber and a flow sensor achieves the desired functionality of cytometry analysis using absolute particle counting. Figure 17A An exemplary design is shown in which the outlet 17103 of the flow chamber 17101 is coupled to the inlet 17202 of the flow sensor 17201 via the fluid conduit 17001. In certain embodiments, the outlet of the flow chamber 17101 can be directly coupled to the inlet 17202 of the flow sensor 17201 without the need for an additional fluid conduit. The flow sensor 17201 has two sensing regions 17204 and 17205. The fluid sample flows into the inlet 17102 of the flow chamber 17101 and then flows out of the outlet 17203 of the flow sensor 17201. The signal measured in the flow chamber 17101 is recorded, as shown in FIG. Figure 17B As shown. Time T0 is when the sample begins to be detected in the flow chamber 17101, T1 is when the fluid sample passes through the sensing area 17204, and T2 is when the fluid sample passes through the sensing area 17205. From time T1 to T2, the total number of target particles detected in the flow chamber 17101 is N. The fluid volume V0 between the two sensing areas 17204 and 17205 is a known parameter from the flow sensor design. Because the flow chamber 17101 has a sheathless design, the volume of fluid flowing through the flow chamber 17101 is only the fluid sample. Therefore, the sample volume measured in the flow chamber 17101 between T1 and T2 is equal to V0. In this design, the absolute count is determined as:

[0130] Absolute count 1 = N / V0 [4]

[0131] Figure 17C Another exemplary design is shown in which the flow sensor 17201 has only one sensing region 17205 . Figure 17D is the signal measured from this design, where time T0 is when sample detection begins in the flow chamber and T2 is when the fluid sample passes through sensing region 17205. From time T0 to T2, the total number of target particles detected in the flow chamber is N'. Volume V0' is the total volume of fluid filling the fluid conduit from flow chamber 17101 to sensing region 17205. In this design, the absolute count is determined as:

[0132] Absolute count 2 = N' / V0' [5]

[0133] Figure 18A Another exemplary design is shown, in which the inlet 18102 of the flow chamber 18101 is connected to the outlet 18203 of the flow sensor 18201 through the fluid conduit 18001. The flow sensor 18201 has two sensing areas 18204 and 18205. The fluid sample flows into the inlet 18102 of the flow sensor 18201 and then flows out of the outlet 18203 of the flow sensor 18201. The signal measured in the flow chamber 18101 is recorded, as shown in FIG. Figure 18B As shown. T1 is when the fluid sample passes through the sensing area 18204, and T2 is when the fluid sample passes through the sensing area 18205. In this example, the number of counted units N" is determined by the signal (A, T) between time points T1+ΔT and T2+ΔT, as shown in FIG. Figure 18B As shown, where ΔT can be any empirical value to compensate for the time delay between the sample reaching the first sensing region 18204 and the sample reaching the flow chamber 18101. From time T1 + ΔT to T2 + ΔT, the total number of target particles detected in the flow chamber is N". The fluid volume V0 between the two sensing regions 18204, 18205 is a known parameter from the design of the flow sensor 18201. In this design, the absolute count is determined as:

[0134] Absolute count 3 = N" / V0 [6]

[0135] This combination of flow chamber 18101 and flow sensor 18201 can be used to measure particles or cells. The size of the target particle depends on the size of the flow chamber 18101, which can be in the range of 0.1-1 μm, 1-10 μm, 10-15 μm, 15-30 μm, 30-50 μm or 50-100 μm. To minimize the risk of clogging the sheathless channel, the size of the particles being measured should be smaller than the size of the flow chamber 18101, and the size difference can be in the range of 1-5 μm, 5-10 μm, 10-20 μm or 20-50 μm. To minimize the coincidence error of the cell counting analysis, the concentration of the target particles in the fluid sample can be in the range of 1-100, 100-1000, 1000-5000, 5000-20,000 or 20,000-50,000 particles or cells per μl sample.

[0136] When the target particles are biological cells, a flow rate that is too fast in the sheathless flow chamber may introduce shear forces and may lyse the cells. Because the sheathless flow chamber has a size similar to that of the target particles, this places a limit on the flow rate of the sample. The flow rate can be in the range of 0.001-1, 1-50, 50-200, or 200-1000 μl / min. In certain embodiments, these ranges can be 1-50 or 50-200 μl / min. For size considerations when implemented in an independent cartridge, the range of the fluid sample volume can be limited by the cartridge size. The volume of the flow sensor and the total volume of the sample can be in the range of 0.1-1 μl, 1-200 μl, 200-1000 μl, 1-5 ml, or 5-30 ml. In certain embodiments, the range can be 1-200 μl, 200-1000 μl, or 1-5 ml. In certain embodiments, by taking into account the sample volume and flow rate, the measurement is completed in less than 10 minutes.

[0137] like Figures 9A-14B As shown in the illustrated embodiment, by further integrating the sheathless flow chamber and flow sensor combination with the basic fluidic unit, the sample preparation step can be further integrated with cytometry analysis, including absolute counting. The integration of the above functions enables the fluidic structure to operate as a standalone structure for cytometry analysis without fluid exchange with the external environment after the fluid sample has been loaded into the cartridge.

[0138] In various embodiments of the fluid configuration, pneumatic pressure is applied to the vents of the basic fluid unit and additional vents of other fluid structures such as reservoirs (see, e.g., Figure 12D and Figure 14A). The higher the pressure differential between the two vents, the higher the flow rate of fluid transferred in the microfluidic channel. When the fluid sample is a biological sample containing cells, the high flow rate in the restricted channel can induce large shear forces to lyse the cells. Taking this limitation into account, the pressure differential between any two applied pressures can be in the range of 0-1, 1-5, 5-15, or 15-30 psi. In certain embodiments, the range can be 0-1, 1-5, or 5-15 psi. In certain embodiments, at least one vent can be connected to the ambient atmospheric pressure. When at least one vent is connected to atmospheric pressure, another pressure higher than the applied atmospheric pressure introduces a positive pressure differential compared to the atmospheric pressure. This positive pressure differential can be in the range of 0-1, 1-5, 5-15, or 15-30 psi. In certain embodiments, the range can be 0-1, 1-5, or 5-15 psi. When at least one vent is connected to atmospheric pressure, another pressure lower than the applied atmospheric pressure introduces a negative pressure differential. This negative pressure differential can be in the range of 0-1, 1-5, 5-15, or 15-30 psi. In some embodiments, the range can be 0-1, 1-5, or 5-15 psi. The flow rate achieved for transferring a sample through a channel between any two basic fluidic units can be in the range of 0-1, 1-50, 50-200, or 200-1000 μl / min, or 1-10 ml / min. In some embodiments, the range can be 1 microliter to 1-50, 50-200, or 200-1000 μl / min.

[0139] Various fluidic configurations of combinations of multiple basic fluidic units and multiple sheathless flow chambers and flow sensors can be implemented in various manufacturing configurations to form a fluidic cartridge. In some embodiments, the cartridge can be inserted into a reader for operation, such as Figure 19As shown in the example of . A cartridge 19101 having a fluidic structure 19102 can be inserted into a docking slot 19202 on a reader 19201. In some embodiments, the control unit of the reader records signals from the cell counting analysis. Some examples of signals include, but are not limited to, optical signals, such as fluorescence, light scattering, light absorption, and the like. In some embodiments, the reader has alignment mechanisms and features to align the sheathless flow chamber with the optics in the instrument for optical signal measurement. In some embodiments, the control unit of the reader also detects signals from a flow sensor to determine absolute counts. In some embodiments, the control unit of the reader also applies a pneumatic pressure source to the cartridge to drive fluid transfer. In some embodiments, the control unit of the reader also supports additional actuation, such as opening or closing valve structures in the cartridge fluidics. In some embodiments, the cartridge is self-contained and there is no exchange of liquid sample between the cartridge and the reader. In some embodiments, the cartridge is not self-contained and the reader has an onboard liquid reservoir and there is liquid exchange between the reader and the cartridge, such as liquid injection from the reader into the cartridge.

[0140] In some embodiments, the cartridge remains stationary after insertion into the reader, while an interface for external connection (e.g., a pneumatic pressure source) moves to contact the cartridge. In other embodiments, the cartridge may be movable after insertion into the reader and moved to contact an interface for external connection (e.g., a pneumatic pressure source).

[0141] The sheathless flow chamber in the fluid structure can be constructed using various manufacturing processes. In some embodiments, the open fluid channel for the flow chamber can be constructed by injection molding, embossing, etching, CNC, laser cutting or die cutting. A cover can then be added to the pattern to form a closed fluid channel as the flow chamber. The cover can be added by various manufacturing processes, such as hot melt bonding, thermal lamination, adhesive bonding, solvent assisted bonding, laser welding and ultrasonic welding. Non-limiting examples of constructing a sheathless flow chamber are described herein. In some embodiments, an optical signal is detected from particles flowing inside the sheathless flow chamber. The smooth surface of the flow chamber is beneficial for obtaining an acceptable optical signal. Figure 20A An example of a sheathless flow chamber 20101 with two workpieces is shown. The cross-sectional view (yz plane) is perpendicular to the sample flow direction (x-axis). The bottom piece 20102 forms three sides of the channel without a cover. The top piece 20103 adds a cover to the channel, which then forms a closed channel. The bottom surface 20104 and top surface 20105 can achieve smoothness for optical measurement in the two pieces 20102 and 20103, respectively. Figure 20BAnother example of constructing a sheathless flow chamber 20201 with three components is shown. Middle piece 20202 forms the sides of the channel, without the top and bottom sides. Then, bottom piece 20203 and top piece 20204 are added separately. Together, these three components form a closed channel that serves as the flow chamber. Surfaces 20205 and 20206 can achieve smoothness for optical measurement in the two components 20203 and 20204, respectively.

[0142] The cartridge device for cell counting analysis can be of any size. In certain embodiments, the cartridge device is housed in a reader device for measurement and analysis and has a range of about 0.1-1 cm 3 , 1-5cm 3 , 5-25cm 3 , 25-50cm 3 or 50-200cm 3 size.

[0143] Many variations and alternative elements have been disclosed in the embodiments of the present disclosure. Additional variations and alternative elements will be apparent to those skilled in the art. Among these variations are, but not limited to, the fluid units, components, and structures selected for use in the apparatus and methods of the present disclosure, as well as the samples that can be analyzed therewith. Various embodiments of the present disclosure may specifically include or exclude any of these variations or elements.

[0144] In some embodiments, the amount, properties, such as concentrations, reaction conditions, etc., of the components used to describe and claim certain embodiments of the present disclosure should be understood to be modified by the term "about" in some cases. As a non-limiting example, a value difference (increase or decrease) of no more than 10% is generally considered by those of ordinary skill in the art to be the meaning of the term "about". Therefore, in some embodiments, the numerical parameters set forth in the written specification and the appended claims are approximate values, which may vary according to the desired properties sought to be obtained by a particular embodiment. In some embodiments, numerical parameters should be interpreted according to the number of reported significant figures and by applying conventional rounding techniques. Although the numerical ranges and parameters of the wide range of some embodiments of the present disclosure are approximate values, the numerical values ​​set forth in the specific embodiments are reported as accurately as possible. The numerical values ​​presented in some embodiments of the present disclosure may include certain errors that are inevitably caused by the standard deviation found in their respective test measurements.

[0145] The grouping of the alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group element can be quoted and claimed individually or in any combination with other elements of the group or other elements found herein. For convenience and / or patentability, one or more elements of a group may be included in the group or deleted from the group. When any such inclusion or deletion occurs, this specification is considered to comprise the group modified at this, thereby meeting the written description of all Markush groups used in the appended claims.

[0146] The present disclosure is explained by various examples, which are intended to be purely examples of the present disclosure and should not be considered to limit the present disclosure in any way. Various examples are provided to better illustrate the claimed disclosure and should not be interpreted as limiting the scope of the present disclosure. With regard to the specific materials mentioned, they are only for illustrative purposes and are not intended to limit the present disclosure. Those skilled in the art can develop equivalent means or reactants without practicing inventive ability and without departing from the scope of the present disclosure.

[0147] The various methods and techniques described above provide a variety of ways to implement the application. Of course, it will be understood that not all of the objects or advantages described can necessarily be achieved according to any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other objects or advantages taught or suggested herein. Various alternatives are mentioned herein. It will be understood that some preferred embodiments specifically include one, another, or several features, while other embodiments specifically exclude one, another, or several features, and still other embodiments reduce specific features by including one, another, or several advantageous features.

[0148] Furthermore, those skilled in the art will recognize the applicability of various features from various embodiments. Similarly, the various elements, features, and steps discussed above, as well as other known equivalents of each such element, feature, or step, can be used in various combinations by those skilled in the art to perform methods according to the principles described herein. Among the various elements, features, and steps, some will be specifically included and others specifically excluded in different embodiments.

[0149] While the present application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the present application extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0150] The preferred embodiments of the present application are described herein, including the best mode known to the inventor for implementing the present application. By reading the foregoing description, variations of those preferred embodiments will become apparent to those of ordinary skill in the art. It is conceivable that those skilled in the art may appropriately adopt such variations, and the present application may be implemented in a manner different from that specifically described herein. Therefore, many embodiments of the present application include all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradicted by the context, the present application encompasses any combination of the above-mentioned elements in all possible variations thereof.

[0151] All patents, patent applications, patent application publications, and other materials, such as articles, books, specifications, publications, documents, things, and / or the like, cited herein are hereby incorporated by reference in their entirety for all purposes, except any prosecution document history related thereto, any prosecution document history that is inconsistent or conflicting with this document, or any claims that may have a limiting effect on the broadest scope of the claims now or later associated with this document. For example, if there is any inconsistency or conflict between the description, definitions, and / or use of a term associated with any incorporated material and the use of a term in this document, the description, definition, and / or use of the term in this document shall control.

[0152] It should be understood that the embodiments of the present application disclosed herein illustrate the principles of the embodiments of the present application. Other modifications that may be adopted may be within the scope of the present application. Therefore, by way of example and not limitation, alternative configurations of the embodiments of the present application may be utilized according to the teachings herein. Therefore, the embodiments of the present application are not limited to the embodiments precisely shown and described.

[0153] Various embodiments of the present disclosure have been described above in the detailed description. Although these descriptions directly describe the above-described embodiments, it should be understood that modifications and / or variations of the specific embodiments shown and described herein may be conceivable to those skilled in the art. Many such modifications or variations falling within the scope of this specification are also included therein. Unless otherwise indicated, it is the inventor's intention that the words and phrases in this specification and claims are given the ordinary and customary meanings given to those of ordinary skill in the art.

[0154] The above description of various embodiments of the present disclosure known to the applicant at the time of filing this application has been presented and is intended for the purposes of illustration and description. This description is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed, and many modifications and variations are possible in light of the above teachings. The described embodiments serve to explain the principles of the present disclosure and its practical application, and to enable those skilled in the art to utilize the present disclosure in various embodiments and with various modifications suitable for the specific use contemplated. Therefore, it is intended that the present disclosure be not limited to the specific embodiments disclosed for practicing the present disclosure.

[0155] While particular embodiments of the present disclosure have been shown and described, it will be obvious to those skilled in the art that, based on the teachings herein, changes and modifications may be made without departing from this disclosure and its broader aspects, and, therefore, the appended claims are intended to include within their scope all such changes and modifications as are within the true spirit and scope of this disclosure.

Claims

1. A sample analysis method comprising: mixing a predetermined volume of sample with a reagent to form a sample mixture; forming a sample flow without a sheath flow in a flow chamber from the sample mixture; measuring an optical signal from the sample stream and analyzing the optical signal to quantify target particles in the sample stream; allowing the sample flow to flow through a flow sensor connected in series with the flow chamber, so that the sample flow in the flow chamber and the sample flow in the flow sensor have the same flow rate; and A sensing signal of the sample flow from the flow sensor is measured to detect the sample flow entering the flow sensor and / or the sample flow leaving the flow sensor. 2 . The sample analysis method according to claim 1 , further comprising: determining the concentration of the target particles in the sample by analyzing the optical signal and the sensing signal.

3. The sample analysis method according to claim 1, wherein: The reagent comprises a fluorescent labeling agent that selectively labels the target particles in the sample with fluorescence, and the optical signal from the sample stream comprises fluorescence.

4. The sample analysis method according to claim 1, wherein The optical signal includes scattered light, reflected light, transmitted light, fluorescence, light absorption, light scattering or a white light image or a combination thereof.

5. The sample analysis method according to claim 1, wherein: Mixing is performed in a basic fluid unit comprising: a chamber configured to contain a fluid; a vent connected to the chamber, wherein the vent is connected to a pneumatic pressure source, ambient pressure, or atmospheric pressure; A microfluidic channel is connected to the chamber.

6. The sample analysis method according to claim 5, wherein: The sample flow is formed by applying an external actuation mechanism to transfer the sample mixture from the chamber into the flow chamber, and the external actuation mechanism includes a pneumatic pressure source.

7. The sample analysis method according to claim 5, wherein: When performing an analysis, the chamber is positioned such that at least a portion of the fluid within the chamber is pulled by gravity toward the microfluidic channel and / or away from the vent.

8. The sample analysis method according to claim 5, wherein: When analysis is performed, the volume of the chamber is larger than the volume of the fluid contained therein, and an air gap exists between the vent and the fluid contained therein.

9. The sample analysis method according to claim 1, wherein: At least two separate sample mixtures are transferred into the same flow cell to form at least two separate sample streams.

10. The sample analysis method according to claim 1, wherein: The sample is applied to a cartridge device to form the sample mixture with the reagent within the cartridge, and the flow chamber and the flow sensor are within the cartridge.

11. The sample analysis method according to claim 10, wherein: The cartridge device is transferred to a reader device, and the optical signal is analyzed using the reader device to quantify the target particles in the sample. 12 . The sample analysis method according to claim 1 , wherein the flow chamber comprises an optically transparent region configured to measure the optical signal from the sample flow to detect target particles in the sample.

Images