Whole blood monocyte phagocytosis function instant detection kit and application thereof

Abstract

This invention belongs to the interdisciplinary field of in vitro diagnostics and medical devices, and relates to a point-in-time (PIT) assay kit for whole blood mononuclear cell phagocytic function and its application. Specifically, this invention provides a point-in-time assay cartridge for mononuclear cell phagocytic function with a specific structure, comprising suitable phagocytic substrate probes and suitable cell capture probes, and provides a kit for preparing the cartridge and its application. Based on the cartridge of this invention, no separation, purification, centrifugation, or washing is required, and it can rapidly and accurately reflect the lysosome-dependent phagocytic degradation function of mononuclear cells in whole blood.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of in vitro diagnostics and medical devices, specifically relating to a real-time detection kit for whole blood mononuclear cell phagocytic function and its application. Background Technology

[0002] In the dynamic process of tissue debris clearance in tissue damage, infection, and chronic inflammatory diseases, peripheral blood-infiltrated monocyte-derived macrophages and centrally residing microglia play a professional "scavenger" role.

[0003] However, the cellular processing of necrotic debris is not a single phagocytic action, but a complete functional axis encompassing "recognition and uptake - phagosome formation - lysosomal degradation".

[0004] The biogenesis and functional state of intracellular lysosomes (such as the efficiency of lysosomal acidification and degradation under the regulation of key transcription factors and receptors) directly determine whether phagocytes can continuously and efficiently process large-scale myelin sheaths and cell debris.

[0005] Assessing only the number of monocytes or surface markers often fails to accurately reflect the underlying immune metabolic remodeling and actual fragment degradation capacity.

[0006] Therefore, the ability to dynamically and quantitatively assess the true phagocytic-degradation dynamics of monocytes has significant clinical guiding value for disease stratification, prognostic assessment, and target monitoring of immunomodulatory therapy in ischemic stroke. Summary of the Invention

[0007] The purpose of this invention is to provide a real-time detection technology and supporting device that can rapidly and accurately reflect the lysosome-dependent phagocytic degradation function of mononuclear cells in whole blood without separation and purification or centrifugation and washing.

[0008] In a first aspect of the invention, a device for real-time detection of the phagocytic function of a whole blood mononuclear phagocyte system is provided, the device comprising the following modules: (Z1) Sample injection module, which is configured to receive whole blood samples from the subject; (Z2) Substrate incubation module, wherein the substrate incubation module is configured to: provide a pH-sensitive substrate, incubate the whole blood sample with the pH-sensitive substrate, thereby obtaining a pretreated whole blood sample; (Z3) Mononuclear phagocyte system-specific capture module, wherein the mononuclear phagocyte system-specific capture module is configured to: provide a capture probe specific to the mononuclear phagocyte system, incubate a pretreated whole blood sample with the capture probe, thereby enriching the mononuclear phagocyte system from the whole blood sample; (Z4) Detection module, configured to: detect the fluorescence intensity of the mononuclear phagocyte system obtained by module (Z3); and (Z5) Result output module, the result output module is configured to: determine and output the phagocytic function detection result of the mononuclear phagocyte system based on the fluorescence intensity; The mononuclear phagocyte system includes: mononuclear cells, macrophages, or a combination thereof; The pH-sensitive phagocytic substrate has the following characteristics: The fluorescence intensity E1 of the pH-sensitive phagocytic substrate in an acidic environment is significantly higher than that of the pH-sensitive phagocytic substrate in a neutral environment. The fluorescence intensity F1 of the pH-sensitive phagocytic substrate in the mononuclear phagocytic cell system was significantly higher than the fluorescence signal F0 in the whole blood background. The mononuclear phagocytic cell system takes up the pH-sensitive phagocytic substrate through phagocytosis. The device is configured such that, under negative pressure, a whole blood sample flows sequentially from the sample introduction module through the phagocytic substrate incubation module, the mononuclear phagocyte system specific capture module, and the detection module.

[0009] In another preferred embodiment, the amount of whole blood sample received from the subject is ≤50 μL.

[0010] In another preferred embodiment, the whole blood sample is selected from the group consisting of venous blood, fingertip blood, arterial blood, or combinations thereof.

[0011] In another preferred embodiment, the detection includes qualitative detection and / or quantitative detection.

[0012] In another preferred embodiment, the judgment includes: qualitative judgment and / or quantitative detection.

[0013] In another preferred embodiment, the quantitative detection refers to relative quantitative detection.

[0014] In another preferred embodiment, "significantly higher than" means E1 / E0 ≥ 20, more preferably ≥ 40, even more preferably ≥ 60, and most preferably ≥ 80.

[0015] In another preferred embodiment, "significantly higher than" means F1 / F0 ≥ 5, preferably ≥ 10, more preferably ≥ 20, even more preferably ≥ 30, and most preferably ≥ 40.

[0016] In another preferred embodiment, the pH-sensitive phagocytic substrate refers to a conjugate of a far-infrared fluorescent group and dextran.

[0017] In another preferred embodiment, the excitation wavelength of the far-infrared fluorescent group is 600 nm - 660 nm, more preferably 630 nm - 650 nm.

[0018] In another preferred embodiment, the far-infrared fluorescent group emits light at a wavelength of 650 nm to 700 nm, more preferably 655 nm to 680 nm.

[0019] In another preferred embodiment, the dextran has a molecular weight of 5 kDa-100 kDa, more preferably 10 kDa-600 kDa, even more preferably 10 kDa-40 kDa, and most preferably 40 kDa.

[0020] In another preferred embodiment, the labeling ratio of the far-infrared fluorescent group to the dextran is 2:1 to 5:1.

[0021] In another preferred embodiment, the marking ratio is a molar ratio.

[0022] In another preferred embodiment, the pH-sensitive phagocytic substrate is pHrodo. ™ A conjugate of a Deep Red fluorescent group and dextran, wherein the molecular weight of the dextran is 10-40 kDa.

[0023] In another preferred embodiment, the mononuclear phagocyte system-specific capture probe comprises: anti-CD14 antibody, anti-CD16 antibody, anti-CD64 antibody, anti-Ly6C antibody, or a combination thereof.

[0024] In another preferred embodiment, the capture probe specific to the mononuclear phagocyte system is an antibody suitable for mouse animal models.

[0025] In another preferred embodiment, the mononuclear phagocyte system-specific capture probe comprises an anti-CD14 antibody.

[0026] In another preferred embodiment, modules (Z1), (Z2), and (Z3) are performed in a cartridge for immediate detection of the phagocytic function of the whole blood mononuclear phagocyte system.

[0027] In another preferred embodiment, in module (Z4), the detection includes: in situ detection in a cartridge for immediate detection of the phagocytic function of the whole blood mononuclear phagocyte system, and / or detection using one or more instruments selected from the group consisting of: flow cytometer, confocal microscope, fully automated high-content screening system, multimode microplate reader, and fluorescence inverted microscope.

[0028] In another preferred embodiment, the in-situ detection is performed in a cartridge for real-time detection of the phagocytic function of the whole blood mononuclear phagocyte system using a far-infrared fluorescence reading device.

[0029] In another preferred embodiment, the far-infrared fluorescence reading device includes: a portable POCT fluorescence immunoassay analyzer, a handheld photomultiplier tube (PMT) fluorescence reader, a miniaturized microfluidic optical detection terminal, or a combination thereof.

[0030] In another preferred embodiment, module (Z5) is performed on the computer.

[0031] In another preferred embodiment, module (Z5) is performed in the far-infrared fluorescence reading device.

[0032] In another preferred embodiment, modules (Z2) and (Z3) are performed simultaneously within the same region.

[0033] In another preferred embodiment, modules (Z2) and (Z3) are performed in any order.

[0034] In another preferred embodiment, the device includes the following modules: (Z1) Sample injection module, which is configured to receive whole blood samples from the subject; (Z2') Mononuclear phagocyte system-specific capture module, wherein the mononuclear phagocyte system-specific capture module is configured to: provide a capture probe specific to the mononuclear phagocyte system, incubate the whole blood sample with the capture probe, thereby enriching the mononuclear phagocyte system from the whole blood sample; (Z3') Phagocytic substrate incubation module, the incubation module being configured to: provide a pH-sensitive phagocytic substrate, incubate the enriched mononuclear phagocyte system with the pH-sensitive phagocytic substrate, thereby obtaining an incubated mononuclear phagocyte system; (Z4) Detection module, configured to: detect the fluorescence intensity of the mononuclear phagocyte system obtained by module (Z3'); and (Z5) Result output module, the result output module is configured to: determine and output the phagocytic function detection result of the mononuclear phagocyte system based on the fluorescence intensity; The device is configured such that, under negative pressure, a whole blood sample flows sequentially from the sample introduction module through the mononuclear phagocyte system specific capture module, the phagocytic substrate incubation module, and the detection module.

[0035] In a second aspect of the invention, a cartridge for the immediate detection of phagocytic function of a whole blood mononuclear phagocyte system is provided, the cartridge comprising the following regions: Inlet; An in-situ incubation chamber containing a pH-sensitive phagocytic substrate; A specific capture and in situ detection chamber, wherein the specific capture and in situ detection chamber contains: a capture probe specific to the mononuclear phagocyte system; The pH-sensitive phagocytic substrate has the following characteristics: The fluorescence intensity E1 of the pH-sensitive phagocytic substrate in an acidic environment is significantly higher than that of the pH-sensitive phagocytic substrate in a neutral environment. The fluorescence intensity F1 of the pH-sensitive phagocytic substrate in the mononuclear phagocytic cell system was significantly higher than the fluorescence signal F0 in the whole blood background. The mononuclear phagocyte system takes up the pH-sensitive phagocytic substrate through phagocytosis.

[0036] In another preferred embodiment, the specific capture is located on one or more sides of the in-situ detection cavity via a transparent optical window.

[0037] In another preferred embodiment, the mononuclear phagocyte system-specific capture probe comprises an anti-CD14 antibody.

[0038] In another preferred embodiment, the pH-sensitive phagocytic substrate is a conjugate of a far-infrared fluorescent group and dextran.

[0039] In another preferred embodiment, the term "included" in the in-situ incubation cavity refers to: an intracavitary load.

[0040] In another preferred embodiment, the specific capture and in-situ detection cavity includes a microarray, wherein the microarray surface is coated with a capture probe specific to the mononuclear phagocyte system.

[0041] In another preferred embodiment, the far-infrared fluorescent group comprises: pHrodo ™ The Deep Red fluorescent group, wherein the dextran has a molecular weight of 10-40 kDa.

[0042] In another preferred embodiment, the cartridge further includes an anticoagulant mixing chamber containing an anticoagulant.

[0043] In another preferred embodiment, the cartridge further includes a waste liquid collection chamber that provides a stable passive fluid negative pressure throughout the cartridge.

[0044] In another preferred embodiment, the card holder also includes a vent.

[0045] In another preferred embodiment, the card holder sequentially comprises the following areas: Inlet; An anticoagulant mixing chamber, wherein the anticoagulant mixing chamber contains an anticoagulant; In situ incubation chamber; Specific capture and in-situ detection chamber; Waste liquid collection chamber; End vent.

[0046] In another preferred embodiment, the inclusion in the specific capture and in-situ detection chamber and / or the anticoagulation mixing chamber refers to: inner surface coating.

[0047] In another preferred embodiment, the anticoagulant comprises EDTA K2.

[0048] In another preferred embodiment, the concentration of the anticoagulant is 1-5 mg / mL, more preferably 1-3 mg / mL, and most preferably 1.5-2 mg / mL.

[0049] In another preferred embodiment, the waste liquid collection chamber contains a highly absorbent polymer medium.

[0050] In another preferred embodiment, the end vent contains a microporous filter membrane.

[0051] In another preferred embodiment, the concentration of the pH-sensitive phagocytic substrate is 1-100 μg / mL, more preferably 1-60 μg / mL, even more preferably 5-50 μg / mL, and most preferably 10-30 μg / mL.

[0052] In another preferred embodiment, the concentration of the antibody is 1-100 μg / mL, more preferably 1-60 μg / mL, even more preferably 5-50 μg / mL, and most preferably 10-30 μg / mL.

[0053] In a third aspect of the invention, a combination of the card holder and the far-infrared fluorescent reading device described in the second aspect of the invention is provided.

[0054] In a fourth aspect of the invention, the use of the cartridge described in the second aspect of the invention or the combination described in the third aspect of the invention in the preparation of a point-of-care detection device for the phagocytic function of a whole blood mononuclear phagocyte system is provided.

[0055] In another preferred embodiment, the device is used for one or more purposes selected from the group consisting of: (a) Prognostic assessment and / or immune status monitoring for stroke; (b) Prognostic assessment of traumatic brain injury (TBI); (c) Tissue repair and / or prognostic assessment of acute myocardial infarction (AMI) and other solid organ ischemia-reperfusion injuries; (d) Assessment of the peripheral immune microenvironment and debris clearance capacity after central nervous system injury; (e) Assessment of immunosuppressive status (immune paralysis) in systemic severe infections and / or early warning of the risk of secondary infections; (f) Monitoring disease activity and / or assessing the function of apoptotic cell clearance (Efferocytosis) in autoimmune diseases.

[0056] In another preferred embodiment, the systemic severe infection includes sepsis and severe pneumonia.

[0057] In another preferred embodiment, the stroke includes: ischemic stroke and hemorrhagic stroke.

[0058] In another preferred embodiment, the central nervous system injury includes: spinal cord injury (SCI).

[0059] In another preferred embodiment, the autoimmune disease includes: systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

[0060] In a fifth aspect of the invention, a kit is provided, the kit comprising: (i) First component: pHrodo ™ A conjugate of a Deep Red fluorescent group and dextran, or a combination of reagents forming said conjugate; and (ii) Second component, which contains: anti-CD14 antibody.

[0061] In another preferred embodiment, the kit further comprises: a cross-linking agent, a blocking agent, an anticoagulant, or a combination thereof.

[0062] In another preferred embodiment, the reagent combination forming the coupling comprises pHrodo ™ Deep Red fluorescent group, dextran, and succinimide.

[0063] In another preferred embodiment, the kit also includes instructions for guiding the preparation of the cartridges.

[0064] In a sixth aspect of the invention, the use of the kit described in the fifth aspect of the invention is provided in the preparation of a cartridge for the point-of-care detection of phagocytic function of a whole blood mononuclear phagocyte system.

[0065] In another preferred embodiment, the cartridge for immediate detection of the phagocytic function of the whole blood mononuclear phagocyte system is the cartridge described in the second aspect of the present invention.

[0066] In a seventh aspect of the present invention, a method for real-time detection of the phagocytic function of a whole blood mononuclear phagocyte system is provided, the method comprising the following steps: (S1) Provide the subject with a whole blood sample; (S2) Add the whole blood sample of the test subject to the cartridge described in the second aspect of the present invention and incubate it; (S3) Detect the fluorescence intensity of the specific capture and in-situ detection chambers to obtain the phagocytic function detection results of the mononuclear phagocyte system.

[0067] In another preferred embodiment, the method includes the following steps: (S1) Provide the subject with a whole blood sample; (S2) A whole blood sample of the subject is added to the apparatus described in the first aspect of the present invention to obtain the phagocytic function detection results of the mononuclear phagocyte system.

[0068] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.

[0069] In another preferred embodiment, the method is an in vitro method.

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0071] Figure 1 The optical properties and biological mechanisms of far-infrared pH-sensitive smart phagocytic substrates were screened and validated. in, Figure 1 Figure A shows a superimposed comparison of the fluorescence histograms of the optimal substrate group (group 5) and control group 2 (group 2) in whole blood under a wash-free environment; Figure 1 B shows a bar chart comparing the signal-to-noise ratio (SNR) of different orthogonal combinations of probe backbones and fluorescent groups in whole blood washless detection; Figure 1 Bar chart of the inhibition of cytochalasin D (Cyto D) by specific uptake mechanism in groups 3 and 5.

[0072] Figure 2 A two-dimensional structural diagram of the POCT cartridge for mononuclear cell phagocytosis in ischemic stroke is shown. Specifically, module A represents the sample inlet for receiving 1-2 drops of peripheral whole blood samples; module B represents the anticoagulation mixing chamber, with an internal volume adapted to 50 μL of blood, and the inner wall pre-coated with a 1.5-2.0 mg / mL EDTA-K2 freeze-dried coating; module C represents the substrate premixing area (incubation channel), which contains 20 μg / mL far-infrared luminescent phagocytic substrate (Deep Red), with a macromolecular carrier coupled to an acid-sensitive probe, forming the fluid basis for whole blood wash-free detection; module D represents the mononuclear cell-specific capture and in situ detection area, with a channel width of 800 μm and a depth of 50 μm, and an internal array of micropillars with a diameter of 20 μm and a spacing of 30 μm, coated with 15 μg / mL anti-CD14 antibody, and a transparent optical window at the bottom of this area for adapting to external instruments for 37°C isothermal heating and in situ reading of 647 nm fluorescence signals; module E... The "F" indicates the waste liquid collection chamber, which contains a highly absorbent polymer medium to provide stable passive fluid negative pressure and waste liquid collection for the entire cartridge; "F" indicates the end vent, which contains a microporous filter membrane to balance the air pressure inside and outside the cartridge.

[0073] Figure 3 The fluorescence emission spectra of the pH-sensitive smart phagocytic substrate used in this invention under different pH buffer conditions are shown. The figures show that the substrate exhibits fluorescence quenching (extremely low background signal) in the neutral or alkaline microenvironment (pH 7-10) simulating extracellular fluid and plasma; however, as the environmental pH decreases to the acidic microenvironment (pH 4-6) simulating intracellular lysosomes, its fluorescence emission intensity undergoes a sharp, non-linear increase. This result confirms the substrate's "acid-activated" characteristic from a physicochemical perspective, which is the core foundation for the microfluidic cartridge of this invention to achieve wash-free, high signal-to-noise ratio in-situ detection of whole blood.

[0074] Figure 4 The microfluidic cartridge demonstrated the purity of whole blood mononuclear cell capture, maintenance of in situ biological activity, and clinical relevance. in, Figure 4 Figure A shows the target mononuclear cells (CD14) before whole blood samples are input into the chip and after specific capture by the microcolumn array. + A bar scatter plot comparing purity was used to quantitatively demonstrate the high-purity in-situ enrichment effect of the cartridge of this invention on target cells. Figure 4Figure B shows a dynamic line graph (including a 90% viability reference line) of cell bioactivity maintenance during in situ isothermal incubation of captured monocytes at 37°C on the micropillar surface. The kinetic line graph shows the percentage of viable cells (%Live) after incubation in the 37°C isothermal channel for 0 min, 15 min, and 30 min. Throughout the in situ incubation, cell viability was maintained at high levels of 97.8%, 96.4%, and 95.8% (well above the 90% safety baseline), confirming that the low-shear microenvironment did not cause mechanical damage to target cell function. Figure 4 C shows a linear regression plot between the relative fluorescence intensity (MFI) measured by the wash-free POCT method of the present invention and the phagocytic percentage (%) measured by the classical flow cytometry washing method, which conclusively demonstrates the consistency and high accuracy of the point-of-care testing method of the present invention compared with the clinical gold standard method. Detailed Implementation

[0075] Through extensive and in-depth research, and after numerous screenings and experiments, the inventors have, for the first time, constructed a cartridge with a cleverly partitioned structure containing specific phagocytic substrate probes and specific monocyte capture probes. Based on this cartridge, the phagocytic function of monocytes can be directly detected by simulating their ability to phagocytose debris, without the need for separation, purification, centrifugation, or washing. This allows for rapid, timely, accurate, safe, and efficient in-situ detection of monocyte phagocytic function in whole blood samples. This invention is based on this achievement.

[0076] Specifically, this invention provides an integrated microfluidic cartridge comprising a sample anticoagulation mixing zone, a mononuclear cell-specific capture microchannel based on antibody modification (anti-CD14), and a 37°C isothermal reaction chamber. This invention also provides an intelligent detection substrate suitable for the integrated microfluidic cartridge: a phagocytic target labeled with a pH-sensitive fluorescent probe. This probe is fluorescence-quenched in a neutral environment (pH 7.2-7.4) and is excited by light upon entering the acidic environment of mononuclear cell lysosomes (pH 4.5-5.0), thereby achieving wash-free quantification of live cell function. Furthermore, this invention provides a detection method: a drop of whole blood is added to the cartridge, and cell capture and substrate co-incubation are automatically completed through a microfluidic program. Finally, the fluorescence signal intensity is read by the optical module of a matching instrument, and the phagocytic index is calculated using a preset algorithm.

[0077] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0078] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0079] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0080] As used in this article, the term "or a combination thereof" means "or any combination thereof".

[0081] Myeloid cells and debris clearance Myelin is a lipid structure formed by oligodendrocytes that encapsulates axons. After injury, a large amount of myelin breaks down, and its fragments contain axonal growth inhibitors (such as Nogo-A and MAG). If these fragments are not cleared in time, they form a strong inhibitory microenvironment, directly blocking neural repair and regeneration. Therefore, brain injury (whether traumatic, ischemic, or hemorrhage) produces a special and troublesome type of fragment—myelin fragments. If the ability to clear these fragments is impaired, they will accumulate in large quantities, indicating poor neurological recovery.

[0082] Ischemic stroke is a leading neurological disease worldwide, causing high rates of disability and mortality. Following cerebral ischemia and hypoxia, a severe cascade of neuroinflammatory responses rapidly erupts, producing a large amount of necrotic tissue and myelin fragments. Recent neuroimmunological studies have shown that the "capacity for endogenous inflammation to subside" in the early stages of the disease is a crucial factor determining the evolution of infarct volume, neural remodeling, and the patient's ultimate neurological prognosis. Timely and efficient removal of microscopic tissue debris from the ischemic area is a prerequisite for breaking the vicious cycle of inflammation and promoting tissue repair.

[0083] When acute hemolysis occurs, a large number of red blood cells rupture, releasing hemoglobin and cell membrane fragments. If these free hemoglobin and fragments are not cleared, they can clog renal tubules and consume nitric oxide, leading to vasospasm. Monocytes and macrophages (especially in the spleen and liver) are responsible for engulfing these fragments. Examining whole blood monocyte phagocytic function can determine whether the body's clearance capacity is sufficient to cope with the current hemolytic load. If function is impaired, the patient is at extremely high risk of acute kidney injury and multiple organ failure. Acute hemolysis can occur in situations including, but not limited to, mechanical hemolysis (such as after mechanical heart valve replacement) and severe burns.

[0084] Rhabdomyolysis syndromes (such as crush syndrome, earthquake or car accident trauma, high-intensity training, etc.) also produce a large amount of debris. Extensive necrosis of muscle tissue occurs due to trauma, ischemia, drugs, etc., releasing large amounts of intracellular myoglobin, creatine kinase, and other substances into the bloodstream. Monocytes and macrophages need to phagocytose and clear these muscle cell debris. Assessing monocyte phagocytic function helps determine the body's ability to clear myoglobin and predicts the risk of acute renal failure. If phagocytic function is saturated or impaired, even large-volume fluid resuscitation may be ineffective.

[0085] Furthermore, in severe acute pancreatitis, the mononuclear-macrophage system is overactivated, but its functional state may be polarized. On the one hand, it produces a large number of inflammatory factors, triggering systemic inflammatory response syndrome; on the other hand, its ability to phagocytose and clear necrotic tissue debris may be relatively or absolutely insufficient. Assessing its phagocytic function helps evaluate the severity of systemic inflammatory response syndrome and the risk of secondary peripancreatic infection and sepsis. Inadequate debris clearance is a crucial basis for the later formation of encapsulated necrosis and infection.

[0086] Myeloid cells are the core executors in clearing various "debris" from the body. In the context of diseases such as brain injury that produce a large amount of cellular and tissue debris, this clearance process is crucial for the resolution of inflammation, tissue repair, and functional recovery. When macrophages derived from monocytes encounter debris, they initiate intracellular signals, causing cytoskeleton rearrangement. Pseudopodia encapsulate the debris to form phagosomes, which then fuse with lysosomes. Various hydrolytic enzymes and reactive oxygen species degrade the debris into smaller molecules, such as metabolizing myelin lipids and storing or transporting iron ions. If this degradation pathway is blocked, even if phagocytosis is normal, excessive lipid accumulation can lead to the formation of "foam cells," exacerbating the damage.

[0087] Therefore, detecting the phagocytic and degradation functions of myeloid cells, especially macrophages derived from monocytes, is of great significance for a variety of diseases and symptoms.

[0088] Myeloid cell phagocytic function test Currently, the assessment of myeloid cell phagocytic function in clinical and research settings primarily relies on classic laboratory assays, such as co-incubation with fluorescently labeled apoptotic cells combined with pulse-chase assays and flow cytometry. While these methods possess some mechanistic validation capabilities in basic research, they face significant technical barriers when addressing the need for point-of-care testing (POCT) in patients with acute ischemic stroke and other conditions: The process is cumbersome and extremely time-consuming: Traditional methods require the separation and extraction of peripheral blood mononuclear cells (PBMCs) from whole blood samples, or repeated red blood cell lysis and multiple centrifugation and washing. The entire pretreatment and incubation process usually takes several hours or even half a day, completely missing the "golden time window" for acute stroke treatment; In vitro "washing" disrupts the original state of cells: Traditional detection procedures require multiple washing steps to remove extracellular unphagocytosed fluorescent background. This high-intensity centrifugation and buffer replacement not only easily leads to a decrease in cell viability but also strips away key plasma opsonins and metabolic factors in the whole blood microenvironment, resulting in severely distorted test results that cannot objectively reflect the true phagocytic capacity of cells in the patient's body. Lack of integration with point-of-care testing (POCT): Existing technologies rely heavily on large flow cytometers and professional laboratory personnel, which cannot meet the needs of stroke centers, emergency rooms, or ambulances for rapid and fully automated screening of mononuclear cell functional characteristics.

[0089] In summary, there is an urgent need in this field to develop a point-of-care detection technology and supporting device that can rapidly and accurately reflect the lysosome-dependent phagocytic degradation function of mononuclear cells in whole blood without separation and purification or centrifugation and washing.

[0090] The card box of the present invention This invention provides a wash-free detection cartridge with an "extracellular quenching-intracellular luminescence" mechanism, which, combined with a microfluidic integrated chip, enables quantitative detection of the phagocytic function of whole blood mononuclear cells within 30 minutes.

[0091] This invention provides a real-time detection kit for whole blood mononuclear cell phagocytic function, comprising: A disposable, closed microfluidic cartridge, wherein the microfluidic cartridge is sequentially connected to a sample inlet, a cell-specific capture area, and an in-situ incubation detection chamber; and pH-sensitive intelligent phagocytic substrate, wherein the substrate is pre-placed or introduced into the in-situ incubation detection chamber; the substrate has the characteristic of changing its luminescence state according to the pH of the microenvironment, its fluorescence is quenched in the extracellular neutral environment, and is excited by light after being phagocytosed into the intracellular acidic lysosomal environment.

[0092] In another preferred embodiment, the microfluidic cartridge further includes an anticoagulation mixing chamber and a waste liquid collection chamber; The anticoagulation mixing chamber is located between the sample inlet and the cell-specific capture area, and contains a pre-embedded dry anticoagulation agent; The waste liquid collection chamber is located downstream of the in-situ incubation detection chamber, and its interior is equipped with a water-absorbing medium to provide capillary driving force for the passive flow of microfluidics. The bottom of the in-situ incubation detection chamber is equipped with a transparent detection window adapted to optical instruments.

[0093] In another preferred embodiment, the cell-specific capture region is provided with a microcolumn array or microchannel, and its surface is modified with a capture probe (anti-CD14 antibody) that specifically targets the surface antigens of monocytes / macrophages. In another preferred embodiment, the pH-sensitive smart phagocytic substrate is a biomacromolecule, microsphere, or cell debris mimic coupled with an acid-sensitive fluorescent dye; its fluorescence intensity is extremely low or quenched in an environment with a pH of 7.2 to 7.4, and it exhibits high-intensity fluorescence emission in an environment with a pH of 4.5 to 5.0.

[0094] The reagent kit of the present invention The present invention also provides a kit for preparing the cartridge of the present invention, the kit comprising: a pH-sensitive phagocytic substrate and an anti-CD14 antibody.

[0095] In another preferred embodiment, the pH-sensitive probe is an acid-sensitive probe.

[0096] In another preferred embodiment, the pH-sensitive phagocytic substrate exhibits low fluorescence intensity in a neutral environment and high fluorescence intensity in an acidic environment.

[0097] In another preferred embodiment, the fluorescence intensity of the pH-sensitive phagocytic substrate in an acidic environment is 20 times or more, preferably 40 times or more, more preferably 60 times or more, and most preferably 80 times or more.

[0098] In another preferred embodiment, the pH value of the neutral environment is 7.2 to 7.4.

[0099] In another preferred embodiment, the pH value of the acidic environment is 4.5 to 5.0.

[0100] In another preferred embodiment, the ratio of the intracellular specific signal to the signal against the whole blood background of the pH-sensitive phagocytic substrate is ≥5, more preferably ≥10, more preferably ≥20%, and most preferably ≥40%.

[0101] The method of the present invention This invention provides a method for detecting the phagocytosis function of a cartridge without centrifugation or washing, specifically comprising the following steps: The whole blood sample to be tested is dropped into the inlet of the microfluidic cartridge; The sample flows within the microfluidic network, with monocytes being enriched in situ by the cell-specific capture zone, while non-target blood cells and plasma flow into the waste collection chamber. Under constant temperature conditions of 37°C, whole blood samples are first thoroughly mixed with the pH-sensitive smart phagocytic substrate in the substrate premixing zone, and the mononuclear phagocyte system initiates the uptake of the phagocytic substrate in a flowing suspension state; subsequently, mononuclear phagocytes carrying intracellular substrates flow through the specific capture zone and are anchored in situ.

[0102] In a specific implementation, the captured mononuclear cells are incubated in situ at a constant temperature of 37°C to complete intracellular lysosomal acidification and substrate degradation.

[0103] In a specific implementation, the total time for premixing and in situ incubation does not exceed 30 minutes.

[0104] In a specific implementation, the hydrodynamic process of the whole blood sample flowing from the inlet A to the end F is driven by the capillary negative pressure generated by the highly absorbent medium E at the end.

[0105] In a specific implementation, the residence time of whole blood samples in each functional area is designed based on the flow channel impedance.

[0106] In a specific implementation, the sample residence time in the anticoagulant mixing chamber B is 0.5-5 minutes to ensure that the anticoagulant is completely dissolved and that the whole blood is fully homogenized.

[0107] In a specific implementation, in the in-situ incubation detection chamber C, the flow rate is controlled by the microchannel structure design, and the effective incubation / take-up time of the sample in this area is 2-15 minutes, preferably 10 minutes.

[0108] In a specific implementation, in the specific capture and in situ detection zone D, the capture time of monocytes on the microcolumn array and the subsequent in situ biochemical reaction is 5-15 minutes, preferably 10 minutes.

[0109] After the substrate is engulfed and enters the intracellular lysosome, it is excited by the acidic environment and generates a fluorescent signal. The fluorescence intensity is directly read through the optical window of the in-situ incubation detection chamber using the matching instrument.

[0110] In another preferred embodiment, the amount of whole blood sample used is 10 μL to 50 μL.

[0111] In another preferred embodiment, the fluorescence intensity read was positively correlated with the phagocytic rate of mononuclear cells and the lysosomal degradation capacity in the sample.

[0112] In another preferred embodiment, the phagocytic function data obtained by this method are used to: assess the ability of endogenous inflammation to resolve after ischemic stroke; or to monitor lysosomal functional kinetics changes in peripheral blood mononuclear cells during the clearance of microscopic debris from ischemic tissue.

[0113] In another preferred embodiment, the method is an in vitro method.

[0114] In specific embodiments, the direct purpose of the method of the present invention is not to obtain diagnostic results or health status, but rather to process tissues, body fluids, or excrement that have been removed from the human or animal body to obtain information as an intermediate result. Based on prior medical knowledge and the method of this disclosure, the information obtained cannot directly lead to a diagnosis of a disease or a health status.

[0115] In a specific implementation, the method of the present invention is an auxiliary method.

[0116] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.

[0117] In another preferred embodiment, the method of the present invention can be used for scientific research on the correlation between diseases and debris clearance.

[0118] The main advantages of this invention include: (a) This invention provides a point-of-care testing kit and its application that can rapidly and accurately reflect the lysosome-dependent phagocytic degradation function of mononuclear cells in whole blood without the need for separation and purification or centrifugation and washing. The kit is simple and convenient to prepare, inexpensive, readily available, and has high detection accuracy.

[0119] (b) The present invention provides an apparatus and method for detecting mononuclear cell phagocytic function based on the said cartridge. This method is rapid, effective, low-cost, and enables real-time clinical detection, which is of great significance in clinical practice and routine thrombosis monitoring.

[0120] (c) This invention establishes for the first time the orthogonal combination advantage of the far-infrared band and the macromolecular dextran backbone, completely overcoming the strong optical absorption and autofluorescence interference of hemoglobin in the whole blood no-wash environment. Compared with traditional fluorescent probes, this design achieves absolute spatial separation between the unphagocytosed background and the targeted phagocytic signal, resulting in a significant nonlinear increase in the signal-to-noise ratio (SNR) of whole blood in situ detection, ensuring high fidelity of the detection signal in the no-wash state.

[0121] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0122] Example 1: Screening of pH-sensitive smart phagocytic substrates applicable to the cartridge of the present invention.

[0123] Traditional commercial phagocytic probes, such as FITC-labeled E. coli microspheres, suffer from extremely low signal-to-noise ratios (SNR) due to severe optical interference during in situ reading of whole blood in microfluidics, failing to meet the detection limits of POCT instruments.

[0124] To achieve "wash-free" in-situ quantification of the true phagocytic function of monocytes in the emergency whole blood microenvironment (high hemoglobin concentration, high background autofluorescence), this embodiment conducted extensive screening of probe backbones and fluorescent groups.

[0125] Specifically, this embodiment designed orthogonal combinations of different fluorescent groups (green FITC, red PE, far-infrared Deep Red) and carriers of different molecular weights (small molecule free state, 10 kDa dextran, 70 kDa dextran, inactivated E. coli). After incubation at 37°C in a simulated microfluidic cartridge environment for 30 minutes, the background fluorescence (background noise) of unwashed whole blood and the specific fluorescence (signal) within monocyte lysosomes were measured. The sources of each reagent are as follows: green FITC (MedChemExpress, CAS No.: 3326-32-7), red pHrodo™ Red (Thermo Fisher Scientific, catalog number P35372), far-infrared pHrodo™ Deep Red (Thermo Fisher Scientific, catalog number P35357), 10 kDa dextran (MedChemExpress, catalog number: HY-112624); 10-40 kDa dextran (MedChemExpress, catalog number: HY-112624L).

[0126] The results are shown in Table 1.

[0127] Table 1: Screening results of phagocytosis experiments with different probe combinations in a whole blood microfluidic environment As shown in Table 1, in Group 5, pHrodo™ Deep Red as the fluorescent group and 10-40 kDa dextran as the backbone carrier had the highest signal-to-noise ratio and the best "wash-free" in situ quantification of the true phagocytic function of monocytes.

[0128] Specifically, when using 10 kDa dextran as a carrier and employing traditional FITC (Group 1) or ordinary Red dye (Group 2), such as Figure 1 A and Figure 1As shown in Figure B, the yellow peaks overlap significantly, resulting in a very low signal-to-noise ratio. In other words, under these conditions, due to whole blood background interference and the "internal filtration effect" of hemoglobin, the signal-to-noise ratio in the wash-free state is less than 3.0, making it impossible to effectively distinguish between phagocytic and non-phagocytic cells. Therefore, neither Group 1 nor Group 2 is suitable for wash-free in-situ real-time quantification of phagocytic function.

[0129] When the carrier is discarded and acid-sensitive free small molecules are used directly (Group 3), such as Figure 1 As shown in C, the uptake of free small molecule probes was not significantly affected after the addition of phagocytosis inhibitors, proving that it is not a true phagocytic pathway. This indicates that monocytes tend to take up small molecules through pathways other than receptor-mediated phagocytosis, resulting in very weak intracellular specific signals and losing their clinical significance in assessing "tissue debris clearance capacity." Therefore, group 3 is not suitable for quantifying phagocytic function.

[0130] Compared to using pHrodo™ Deep Red alone (Group 3), when pHrodo™ Deep Red was used as the fluorescent group and E. coli microspheres (>1000 kDa) were used as the carrier (Group 4), both the whole blood background signal and the intracellular specific signal showed a significant increase. Due to the greater increase in intracellular specific signal, the signal-to-noise ratio of Group 4 reached 7.20, which was significantly better than that of Groups 1-3, and it is expected to be used for "wash-free" in situ real-time quantification of phagocytic function.

[0131] However, most unexpectedly, compared with pHrodo™ Deep Red alone (Group 3), using pHrodo™ Deep Red as the fluorescent group and 10-40 kDa dextran as the carrier (Group 5) achieved a significant reduction in whole blood background signal while significantly increasing intracellular specific signal. This resulted in a signal-to-noise ratio of 40.0 for Group 5, which is more than 10 times that of Groups 1-3 and more than 5 times that of Group 4. Figure 1 (B). Further cytochalasin D (Cyto D) inhibition experiments showed that in group 5, the signal was completely blocked after the addition of the phagocytic inhibitor, confirming the specific phagocytic mechanism of the probe combination in group 5.

[0132] To distinguish between the dye's intrinsic signal and the carrier effect, this study investigated whether the significant decrease in whole blood background signal and the significant increase in intracellular specific signal in group 5 were due to 10-40 kDa dextran as a carrier. The results showed that in group 6, using only pHrodo Red free dye without a carrier, the SNRs measured in 7 studies were 2.45, 2.52, 1.98, 2.33, 3.68, 4.74, and 2.79, with an average of 2.93. In group 7, using only FITC free dye without a carrier, the SNRs measured in 7 studies were 1.47, 1.38, 1.19, 2.42, 2.64, 2.62, and 2.41, with an average of 2.02. In other words, the signal-to-noise ratio (SNR) of FITC + 10 kDa (1.13) is not much different from that of FITC alone (2.02), and the SNR of pHrodo Red + 10 kDa (2.88) is also not much different from that of pHrodo Red alone (2.93). However, the SNR of pHrodo Deep Red + dextran (40) is unexpectedly significantly higher than that of pHrodo Deep Red alone (2.91)! This indicates that the unexpected technical effect is entirely due to the outstanding technical effect of the synergistic effect of pHrodo Deep Red and dextran.

[0133] In summary, neither simply changing the fluorescence wavelength nor simply changing the carrier size can meet the in-situ detection requirements of microfluidic cartridges. Only when both the fluorescence wavelength and the carrier size meet specific conditions can the requirement for real-time detection of monocyte phagocytic function be met. Specifically, only when the "far-infrared excitation / emission band (644 / 655 nm)" is strictly limited to the combination of "dextran backbone with a spatial conformation of 10,000-40,000 Da" (Group 5) can not only be perfectly avoided due to whole blood optical interference (background reduced to 105 RFU), but also perfectly simulate the myelin necrosis fragments produced by ischemic stroke, while also meeting the requirements of microfluidic channels. This makes it very suitable for washless cartridges for real monocyte phagocytic function.

[0134] Example 2: Preparation of a real-time detection kit for whole blood mononuclear cell phagocytic function.

[0135] The microfluidic cartridge body of this embodiment is made of polymethyl methacrylate (PMMA) material through injection molding. For example... Figure 2 As shown, its internal flow network parameters are rigorously set to meet the requirements of whole blood passive flow control. Specifically, the microfluidic test cartridge includes: Sample inlet A: The inlet is funnel-shaped and used to drip in 1-2 drops of whole blood; Anticoagulation chamber B: The anticoagulation chamber has an internal volume of 50 μL and its inner wall is pre-coated with a freeze-dried EDTA-K2 coating at a concentration of 1.5-2.0 mg / mL for whole blood anticoagulation; In-situ incubation detection chamber C: This chamber is a circular or polygonal flow channel extension area with a microstructure array inside to increase fluid mixing efficiency. It is pre-embedded with a phagocytic substrate at a concentration of 20 μg / mL, which allows blood to bind to the substrate in situ during flow and initiate phagocytosis. Through a transparent optical window at the bottom, it is adapted to an external constant temperature module to maintain a 37°C environment, ensuring that mononuclear cells phagocytose at the optimal biological temperature. Specific capture and in-situ detection zone D: This region is the main flow channel, with a width of 800 μm and a depth of 50 μm. A dense array of micropillars (20 μm in diameter, 30 μm spacing) is distributed within the channel to increase the specific surface area and reduce the local flow velocity (generating approximately 1-5 dyn / cm). 2 The fluid shear force is controlled, and a transparent optical window is provided at the bottom. After the inner surface of the capture area channel and the surface of the micropillar are activated by oxygen plasma, EDC / NHS crosslinking agent is coupled, followed by incubation with anti-human mouse CD14 at a concentration of 15 μg / mL. After incubation, non-specific binding sites are blocked with 1% BSA for 2 hours, and finally washed with PBS and dried for later use. Waste liquid collection chamber E: This chamber is equipped with a super absorbent polymer medium (Absorbent Pad), which uses its super hydrophilic properties to generate a continuous passive capillary suction effect. Without an external pump, it provides a stable negative fluid pressure for the entire cartridge, thereby driving the whole blood sample to flow continuously through each functional area and effectively collecting the washed red blood cells and impurities to prevent backflow from interfering with the detection. End vent F: This vent has a built-in highly permeable hydrophobic microporous filter membrane to balance the air pressure inside and outside the cartridge, prevent fluid interruption due to air blockage, and ensure smooth passive flow control of the entire cartridge without pump drive.

[0136] The preparation of the phagocytic substrate includes the following steps: Macromolecular backbone and dye coupling: Dextran, with an average molecular weight distribution between 10,000 Da and 40,000 Da, was selected as the support. Using succinimide ester (NHS ester) crosslinking chemistry, the far-infrared pH-sensitive dye pHrodo™ Deep Red (excitation wavelength 644 nm, emission wavelength 655 nm, compatible with APC / Cy5 channels) was covalently coupled to this support. The dye-to-support labeling ratio (DOL) in the reaction system was strictly controlled between 2.0 and 5.0. This labeling ratio range precisely avoids fluorescence self-quenching and ensures maximum signal output after entering an acidic environment. Lyophilization preservation of the conjugate: The conjugated product is purified by high-performance liquid chromatography (HPLC) and then placed in sterile pure water containing a cryoprotectant (such as 5% trehalose). Subsequently, it is vacuum-dried using a freeze dryer until all moisture is removed, forming a loose lyophilized powder. The resulting lyophilized powder can be stored for a long time under light-protected, vacuum-sealed conditions, and its fluorescence properties and structural stability remain consistent with those of the dry powder after reconstitution.

[0137] Preparation of working solution for substrate: The purified lyophilized conjugate powder was reconstituted in a protective buffer containing 5% trehalose and 1% BSA to obtain a far-infrared luminescent phagocytic substrate with a concentration of 20 μg / mL.

[0138] Example 3: Validation of substrate pH luminescence kinetics and functionalities of the card cartridge of the present invention.

[0139] To verify that the prepared substrate can meet the quantitative requirements of the "wash-free" function of the cartridge, its spectroscopic properties were verified: Experimental method: The prepared substrates were placed in gradient buffer systems with pH ranging from 4.0 to 10.0, and their excitation / emission spectra and relative fluorescence intensity (RFU) were measured at 37℃.

[0140] Data results and technical effects: such as Figure 3 As shown, the fluorescence of the finished substrate was almost completely quenched at pH 7.2-7.4 (whole blood simulated neutral environment) with extremely low background; while when titrated to pH 4.5-5.0 (simulating the acidic microenvironment of mature lysosomes), the fluorescence intensity increased dramatically by 10 times.

[0141] Clinical significance: This highly sensitive "extracellular quenching-intracellular luminescence" characteristic establishes the physical basis for the cartridge's "testing upon whole blood drop, without centrifugation or washing" capability. The luminescence intensity of the substrate entering the cell not only indicates the uptake action at the monocyte proximal end but also accurately reflects the degradation capacity of the underlying lysosomes, thereby achieving high-fidelity monitoring of the clearance dynamics of real cellular debris.

[0142] Example 4: Verification of the capture efficiency, cell activity, and clinical accuracy of the cartridge of the present invention on whole blood mononuclear cells.

[0143] The experimental steps in this embodiment are as follows: Sample preparation: Collect peripheral anticoagulated whole blood from patients with ischemic stroke; Chip Operation: A 50 μL peripheral whole blood sample is dripped into the sample inlet A. Driven by the passive capillary suction negative pressure generated by the superabsorbent polymer medium inside the waste collection chamber E, the whole blood flows through the microfluidic channel. The blood first comes into full contact with the pre-embedded 20 μg / mL far-infrared pH-sensitive intelligent phagocytic substrate in the substrate premixing zone C. Monocytes, in a flowing suspension state, initiate specific phagocytosis of tissue debris mimics. Subsequently, the monocytes carrying the intracellular substrate flow through the specific capture and in-situ detection zone D, where they are subjected to fluid shear forces (1-5 dyn / cm) generated by the densely distributed micropillar array within the channel. 2 The surface is coated with a 15 μg / mL anti-CD14 monoclonal antibody, achieving specific physical and biological dual anchoring of target cells. Uncaptured red blood cells, excess plasma, and extracellular free substrate are continuously flushed into waste collection chamber E; Capture purity and activity assay: Target cells eluted from the microarray were collected, and CD14 levels were detected using flow cytometry. + Changes in the purity of target mononuclear cells; simultaneously, at 0 min, 15 min and 30 min nodes of in situ incubation on the chip at 37℃, live and dead cell double staining reagent (Calcein-AM / PI) was perfused in situ, and the cell viability maintenance was assessed by fluorescence microscopy or the chip's optical module. Clinical accuracy (gold standard control) experiment: Samples from the same batch of clinical stroke patients were extracted and tested in parallel using the classic flow cytometry method (Pulse-Chase assay, which measures the percentage of phagocytosis of mononuclear cells after red blood cell lysis and repeated centrifugation and washing). The in situ far-infrared fluorescence intensity (MFI) read from the POCT method of this invention was compared with the phagocytosis percentage measured by the classic flow cytometry method using linear regression correlation analysis.

[0144] Flow cytometry comparative verification results are as follows Figure 4 As shown in Figure A. The results indicate that in the initial peripheral whole blood before inputting the chip, CD14... +The proportion of monocytes in the total cell population was only 6.2% ± 1.1%; however, after capture and shear elution by the microcolumn array of the microfluidic cartridge of this invention, the purity of the target monocytes in the in-situ detection zone D increased significantly to 94.5% ± 1.5% (i.e., capture purity greater than 94%). There was a highly significant statistical difference between the pre- and post-detection groups. P < 0.001), proving that the cartridge of the present invention has a very strong ability to enrich high-purity in situ cells, and successfully eliminates the interference of a large number of red blood cells and granulocytes in whole blood.

[0145] In addition, the activity monitoring results of the captured cells during in situ incubation on a 37°C chip were as follows: Figure 4 As shown in Figure B, after incubation on the micropillar array surface for 0 min, 15 min, and 30 min, the percentage of viable target monocytes remained at 97.8%, 96.4%, and 95.8%, respectively, consistently maintaining a high level of biological activity above 95% throughout the process. This result demonstrates the effectiveness of the low fluid shear force (1-5 dyn / cm) employed in the cartridge of this invention. 2 The microfluidic material is extremely friendly to living cells, and while efficiently capturing debris, it fully preserves the subsequent degradation activity of the cell's underlying layer, providing a biological basis for high-fidelity monitoring of cell debris clearance dynamics.

[0146] Finally, the linear regression fitting diagram between the relative fluorescence intensity (MFI) measured by the wash-free POCT method of this invention and the phagocytosis percentage (%) measured by the classical flow cytometry washing method is shown in the figure below. Figure 4 As shown in C. The intracellular relative fluorescence intensity (MFI) measured by the wash-free POCT method of this invention was bivariately fitted with the percentage of monocytes phagocytosed (%) measured by the classical flow cytometry method. The regression equation showed a very high linear positive correlation (R0). 2 The value >0.75 demonstrates the accuracy of the instantaneous detection.

[0147] The above results fully demonstrate that the microfluidic point-of-care testing cartridge of the present invention, with its extremely simple operation that completely eliminates centrifugation and manual washing, can not only achieve high-purity capture and activity maintenance of mononuclear cells, but also reproduce the detection accuracy of large flow cytometers with high fidelity. It fully meets the practical application needs of rapid triage and immune function monitoring in the ultra-early acute phase of stroke and at the point of contact with the bed (POCT).

[0148] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A device for real-time detection of the phagocytic function of a whole blood mononuclear phagocyte system without lysis or centrifugation, characterized in that, The device includes the following modules: (Z1) Sample injection module, which is configured to receive whole blood samples from the subject; (Z2) Substrate incubation module, wherein the substrate incubation module is configured to: provide a pH-sensitive substrate, incubate the whole blood sample with the pH-sensitive substrate, thereby obtaining a pretreated whole blood sample; (Z3) Mononuclear phagocyte system-specific capture module, wherein the mononuclear phagocyte system-specific capture module is configured to: provide a capture probe specific to the mononuclear phagocyte system, incubate a pretreated whole blood sample with the capture probe, thereby enriching the mononuclear phagocyte system from the whole blood sample; (Z4) Detection module, which is configured to detect the fluorescence intensity of the mononuclear phagocyte system obtained by module (Z3); and (Z5) Result output module, the result output module is configured to: determine and output the phagocytic function detection result of the mononuclear phagocyte system based on the fluorescence intensity; The device is configured such that, under negative pressure, a whole blood sample flows sequentially from the sample introduction module through the phagocytic substrate incubation module, the mononuclear phagocyte system specific capture module, and the detection module. The mononuclear phagocyte system includes: mononuclear cells, macrophages, or a combination thereof; The pH-sensitive phagocytic substrate has the following characteristics: The fluorescence intensity E1 of the pH-sensitive phagocytic substrate in an acidic environment is significantly higher than that of the pH-sensitive phagocytic substrate in a neutral environment. The fluorescence intensity F1 of the pH-sensitive phagocytic substrate in the mononuclear phagocytic cell system was significantly higher than the fluorescence signal F0 in the whole blood background. The mononuclear phagocytic cell system takes up the pH-sensitive phagocytic substrate through phagocytosis. in, The pH-sensitive phagocytic substrate is pHrodo. ™ Conjugates of Deep Red fluorescent group and dextran; "Significantly higher than" means F1 / F0 ≥ 20.

2. The apparatus as claimed in claim 1, characterized in that, The molecular weight of the dextran is 10-40 kDa.

3. The apparatus as described in claim 1, characterized in that, The mononuclear phagocyte system-specific capture probe contains an anti-CD14 antibody.

4. The apparatus as claimed in claim 1, characterized in that, The device includes the following modules: (Z1) Sample injection module, which is configured to receive whole blood samples from the subject; (Z2') Mononuclear phagocyte system-specific capture module, wherein the mononuclear phagocyte system-specific capture module is configured to: provide a capture probe specific to the mononuclear phagocyte system, incubate the whole blood sample with the capture probe, thereby enriching the mononuclear phagocyte system from the whole blood sample; (Z3') Phagocytic substrate incubation module, the incubation module being configured to: provide a pH-sensitive phagocytic substrate, incubate the enriched mononuclear phagocyte system with the pH-sensitive phagocytic substrate, thereby obtaining an incubated mononuclear phagocyte system; (Z4) Detection module, which is configured to detect the fluorescence intensity of the mononuclear phagocyte system obtained by module (Z3'); and (Z5) Result output module, the result output module is configured to: determine and output the phagocytic function detection result of the mononuclear phagocyte system based on the fluorescence intensity; The device is configured such that, under negative pressure, the whole blood sample flows sequentially from the sample introduction module through the mononuclear phagocyte system specific capture module, the phagocytic substrate incubation module, and the detection module.

5. A cartridge for point-of-care detection of phagocytic function in a whole blood mononuclear phagocyte system, characterized in that, The card holder includes the following areas: Inlet; An in-situ incubation chamber containing a pH-sensitive phagocytic substrate; A specific capture and in situ detection chamber, wherein the specific capture and in situ detection chamber contains: a capture probe specific to the mononuclear phagocyte system; The pH-sensitive phagocytic substrate has the following characteristics: The fluorescence intensity E1 of the pH-sensitive phagocytic substrate in an acidic environment is significantly higher than that of the pH-sensitive phagocytic substrate in a neutral environment. The fluorescence intensity F1 of the pH-sensitive phagocytic substrate in the mononuclear phagocytic cell system was significantly higher than the fluorescence signal F0 in the whole blood background. The mononuclear phagocytic cell system takes up the pH-sensitive phagocytic substrate through phagocytosis. in, The pH-sensitive phagocytic substrate is pHrodo. ™ Conjugates of Deep Red fluorescent group and dextran; "Significantly higher than" means F1 / F0 ≥ 20.

6. The combination of the card holder and the far-infrared fluorescent reading device as described in claim 5.

7. Use of the cartridge of claim 5 or the combination of claim 6 in the preparation of a point-of-care testing device for phagocytic function of a whole blood mononuclear phagocyte system.

8. A reagent kit, characterized in that, The kit contains: (i) First component: pHrodo ™ A conjugate of a Deep Red fluorescent group and dextran, or a combination of reagents forming said conjugate; and (ii) Second component, which contains: anti-CD14 antibody.

9. Use of the kit of claim 8 in the preparation of a cartridge for the point-of-care detection of phagocytic function of a whole blood mononuclear phagocyte system.

10. A method for the immediate detection of phagocytic function of the whole blood mononuclear phagocyte system for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes the following steps: (S1) Provide the subject with a whole blood sample; (S2) Add the whole blood sample of the subject to the cartridge of claim 5 and incubate it; (S3) Detect the fluorescence intensity of the specific capture and in-situ detection chambers to obtain the phagocytic function detection results of the mononuclear phagocyte system.

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