A whole blood heparin binding protein fluorescence immunochromatography test strip based on hematocrit correction and detection system

Abstract

The application provides a whole blood heparin binding protein fluorescence immunoassay chromatographic test strip based on hematocrit correction and a detection system, relates to the technical field of in vitro diagnosis, and the whole blood heparin binding protein fluorescence immunoassay chromatographic test strip based on hematocrit correction provided by the application is characterized in that a GPA internal reference area and an HBP detection area are arranged on the same nitrocellulose membrane, long-life fluorescent probe-GPA antibodies marked on a combination pad and short-life fluorescent probe-HBP antibodies are utilized, and the independent collection of hematocrit internal reference signals and target analyte detection signals is realized simultaneously in a single chromatographic reaction; wherein the GPA is an anchoring protein of the red blood cell membrane transmembrane, is still anchored to the membrane fragments after the red blood cell hemolysis and rupture, and is not released freely, so that the internal reference signal is not interfered by free hemoglobin in the hemolytic sample, the matrix effect deviation caused by the difference in hematocrit among different individuals is eliminated, and the accurate quantitative detection of heparin binding protein in the whole blood sample is realized.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and in particular to a whole blood heparin-binding protein fluorescent immunochromatographic test strip and detection system based on hematocrit correction. Background Technology

[0002] Heparin-binding protein (HBP) is a cationic antimicrobial protein released by azurophilic granules of neutrophils, possessing key pathophysiological functions such as inducing increased vascular endothelial permeability and activating inflammatory cascade responses. Numerous clinical studies have confirmed that HBP plasma concentrations significantly increase in the early stages of sepsis (2-4 hours post-infection), and its levels are strongly correlated with disease severity, organ failure progression, and 28-day mortality, making it a highly sensitive and timely biomarker for early warning of sepsis. Therefore, achieving rapid, accurate, and centrifugation-free whole blood HBP detection in primary hospitals, emergency departments, and ICUs has significant clinical value for the early diagnosis and treatment of sepsis.

[0003] Currently, HBP detection mainly relies on enzyme-linked immunosorbent assays (ELISA) on laboratory platforms. This requires centrifugation of whole blood samples to separate plasma, a time-consuming procedure dependent on central laboratory equipment, and cannot meet the needs of point-of-care diagnostics. To overcome this bottleneck, recent studies have attempted to migrate HBP detection to immunochromatographic fluorescent test strip platforms. However, such direct whole blood testing methods face a fundamental technical obstacle that has not yet been systematically resolved—matrix effect bias caused by hematocrit (HCT). Since the proportion of red blood cells in whole blood directly affects sample viscosity, antibody diffusion rate, steric hindrance of antigen-antibody binding, and fluorescence signal quenching efficiency, the same HBP concentration can produce detection biases as high as ±37% in different HCT samples, far exceeding the clinically acceptable error limit. This bias is not random noise but is highly linearly negatively correlated with HCT, indicating that it is essentially a systematic matrix interference that must be eliminated through HCT correction.

[0004] In existing technologies, HCT correction strategies mainly fall into three categories: (1) External blood routine equipment method: After measuring HCT with an independent blood cell analyzer, the reading is manually entered into the reader for calibration. Although this method is accurate, it violates the core design principle of POCT of "single device, one-stop, no external equipment", increases the complexity of operation, detection cost and time delay, and is difficult to implement in primary care and pre-hospital emergency scenarios; (2) Hemoglobin (Hb) internal control method: Anti-Hb antibody is coated on the test strip as the HCT internal control, and the concentration of free Hb is used to indirectly reflect the amount of red blood cells. However, the incidence of hemolysis in clinical whole blood samples is as high as 10%–20%. The free Hb released by hemolysis will specifically bind to the internal control antibody, resulting in a false increase in the internal control signal, which in turn leads to overcorrection, causing the HBP detection deviation to deteriorate sharply and completely lose its correction function; (3) Electrochemical / Optical HCT Sensing Method: Microelectrodes or reflective light sensors are integrated on the test strip, and HCT is calculated by measuring blood conductivity or transmittance. This type of solution requires additional circuit design and signal processing modules, which significantly increases the complexity of the test strip manufacturing process and the hardware cost of the reader. It is also susceptible to interference from substances such as lipemia and jaundice, resulting in poor stability. No mature products have yet been launched on the market.

[0005] In summary, existing technologies fail to simultaneously meet the three essential requirements of clinical applicability, robustness against interference, and calibration accuracy. Therefore, there is an urgent need for a whole blood HBP fluorescence immunoassay method based on a novel internal control principle, requiring no external equipment, and effectively overcoming common interferences such as hemolysis.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a whole blood heparin-binding protein fluorescent immunochromatographic test strip based on hematocrit correction, so as to at least solve one of the technical problems existing in the prior art.

[0008] The second objective of this invention is to provide a whole blood heparin-binding protein fluorescence immunoassay system.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: This invention provides a fluorescent immunochromatographic test strip for whole blood heparin-binding protein based on hematocrit correction, comprising: The base plate, and the sample pad, conjugation pad, nitrocellulose membrane and absorbent pad that are sequentially overlapped and fixed on the base plate along the chromatography direction; The conjugate pad contains an anti-human blood group glycoprotein A monoclonal antibody labeled with a first fluorescent probe and an anti-human heparin-binding protein monoclonal antibody labeled with a second fluorescent probe. Wherein, the first fluorescent probe is a long-lived fluorescent probe, and the second fluorescent probe is a short-lived fluorescent probe; The nitrocellulose membrane is provided with an internal reference zone and a detection zone sequentially along the chromatography direction; The internal reference region is coated with a monoclonal antibody against human blood group glycoprotein A, and the detection region is coated with a monoclonal antibody against human heparin-binding protein.

[0010] Furthermore, the nitrocellulose membrane is also provided with a quality control area; The internal reference zone, the detection zone, and the quality control zone are arranged sequentially along the chromatography direction of the nitrocellulose membrane.

[0011] Furthermore, the first fluorescent probe is a lanthanide fluorescent probe; and / or, The second fluorescent probe is a quantum dot fluorescent probe.

[0012] Furthermore, the excitation wavelength range of the lanthanide fluorescent probe is 300 nm to 400 nm, and the emission wavelength range is 600 nm to 630 nm; and / or, The excitation wavelength range of the quantum dot fluorescent probe is 600nm~650nm, and the emission wavelength range is 630nm~680nm. Preferably, the distance between the emission center wavelength of the lanthanide fluorescent probe and the emission center wavelength of the quantum dot fluorescent probe is greater than or equal to 30 nm.

[0013] Furthermore, the excitation wavelength of the lanthanide fluorescent probe is 365 nm, and the emission wavelength is 615 nm ± 10 nm; and / or, The quantum dot fluorescent probe has an excitation wavelength of 620 nm and an emission wavelength of 650 nm ± 10 nm.

[0014] Furthermore, the distance between the internal reference detection line corresponding to the internal reference region and the target analyte detection line corresponding to the detection region is 3mm~8mm.

[0015] The present invention also provides a whole blood heparin-binding protein fluorescence immunoassay system, comprising the above-mentioned whole blood heparin-binding protein fluorescence immunochromatographic test strip; and, A fluorescence reader, comprising dual excitation light sources, a single optical detection channel, and a data processing module; The dual excitation light source is configured to output a first excitation light adapted to the first fluorescent probe and a second excitation light adapted to the second fluorescent probe in a time-division manner; The single optical detection channel is configured to acquire a first emitted light signal when irradiated by the first excitation light, and to acquire a second emitted light signal when irradiated by the second excitation light; The data processing module has a built-in preset hematocrit prediction model and concentration correction model, configured to determine the predicted hematocrit value based on the first emitted light signal and the hematocrit prediction model, and to determine the corrected target analyte concentration based on the predicted hematocrit value, the concentration correction model and the second emitted light signal.

[0016] Furthermore, the single optical detection channel is equipped with a switchable narrowband bandpass filter assembly; Preferably, the fluorescence reader further includes a control module, which is configured to: drive the narrowband bandpass filter assembly to switch into a first filter adapted to the first fluorescence probe when acquiring the first emitted light signal; and drive the narrowband bandpass filter assembly to switch into a second filter adapted to the second fluorescence probe when acquiring the second emitted light signal.

[0017] Furthermore, the data processing module is configured as follows: Based on the peak integral area of ​​the first emitted light signal, the predicted value of hematocrit is calculated using the hematocrit prediction model. Based on the predicted hematocrit value, the correction coefficient is calculated using the concentration correction model; The original target analyte concentration is determined based on the peak integral area of ​​the second emitted optical signal, and the original target analyte concentration is corrected using the correction coefficient to obtain the corrected target analyte concentration.

[0018] Furthermore, in the data processing module: The hematocrit prediction model is a linear regression model obtained by fitting the peak integral area of ​​the first emitted light signal with the standard value of hematocrit. The predicted value of hematocrit is a×F_GPA+b, where F_GPA is the peak integral area of ​​the first emitted light signal, and a and b are constants determined by fitting with standard samples. The concentration correction model is a correction coefficient calculation model based on fitting the predicted hematocrit value with the original detection deviation. The correction coefficient K = cd × HCT predicted value, where the HCT predicted value is the predicted hematocrit value, and c and d are constants determined by fitting with standard samples. The corrected target analyte concentration Ccorrected = Coriginal / K, where Coriginal is the original target analyte concentration determined based on the second emitted light signal.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The heparin-binding protein (HBP) fluorescence immunochromatographic test strip based on hematocrit correction provided by this invention sets up a GPA internal control area and an HBP detection area on the same nitrocellulose membrane. Utilizing a long-lived fluorescent probe—GPA antibody—and a short-lived fluorescent probe—HBP antibody labeled on the binding pad, it simultaneously achieves independent acquisition of the hematocrit internal control signal and the target analyte detection signal in a single chromatographic reaction. GPA, as a transmembrane anchoring protein of the erythrocyte membrane, remains anchored to membrane fragments after hemolysis and rupture of erythrocytes without being released, ensuring that the internal control signal is not interfered with by free hemoglobin in the hemolyzed sample. Therefore, without the need for centrifugation pretreatment of the whole blood sample, the internal control signal corrects the HBP detection signal for hematocrit, eliminating matrix effect bias caused by differences in hematocrit between individuals, and achieving accurate quantitative detection of heparin-binding protein in whole blood samples.

[0020] The whole blood heparin-binding protein fluorescence immunoassay system provided by this invention integrates a whole blood heparin-binding protein fluorescence immunochromatographic test strip with a fluorescence reader. It utilizes dual excitation light sources to output excitation light adapted to two types of fluorescent probes in a time-division manner, and a single optical detection channel to acquire two emission light signals in a time-division manner. This allows the same optical channel to accommodate the acquisition of probe signals with different fluorescence mechanisms, such as long-lived and short-lived probes, eliminating the need for a dual-optical-path detection system. Simultaneously, the data processing module incorporates a hematocrit prediction model and a concentration correction model. Based on the first emission light signal, it automatically calculates the predicted hematocrit value and corrects the original target analyte concentration determined by the second emission light signal accordingly. Thus, in whole blood sample centrifugation-free detection scenarios, the test strip completes the dual-probe immunochromatographic reaction, and the reader automatically completes dual signal acquisition and algorithm correction, achieving integrated detection processes and automated result output. Detailed Implementation

[0021] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0022] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] According to one aspect of the present invention, a fluorescent immunochromatographic test strip for whole blood heparin-binding protein based on hematocrit correction is provided, characterized in that it comprises: The base plate, and the sample pad, conjugation pad, nitrocellulose membrane and absorbent pad that are sequentially overlapped and fixed on the base plate along the chromatography direction; The conjugate pad contains an anti-human blood group glycoprotein A monoclonal antibody labeled with a first fluorescent probe and an anti-human heparin-binding protein monoclonal antibody labeled with a second fluorescent probe. Wherein, the first fluorescent probe is a long-lived fluorescent probe, and the second fluorescent probe is a short-lived fluorescent probe; The nitrocellulose membrane is provided with an internal reference zone and a detection zone sequentially along the chromatography direction; The internal reference region is coated with a monoclonal antibody against human blood group glycoprotein A, and the detection region is coated with a monoclonal antibody against human heparin-binding protein.

[0025] GPA is a highly abundant protein specific to the surface of erythrocytes, and its content is positively correlated with the number of erythrocytes. As a transmembrane anchoring protein of erythrocytes, it remains anchored to membrane fragments after erythrocyte hemolysis and rupture, and is not released into the plasma. Therefore, even if the sample undergoes hemolysis of varying degrees, the internal control signal still stably corresponds to the original number of erythrocytes in the sample, and there is no signal drift due to the release of free hemoglobin, ensuring the accuracy of hematocrit correction in hemolyzed samples.

[0026] The internal control region is coated with anti-human blood group glycoprotein A monoclonal antibody, and the binding pad contains anti-human blood group glycoprotein A monoclonal antibody labeled with the first fluorescent probe, forming a double antibody sandwich structure to detect GPA in the sample. Therefore, the internal control signal can directly quantitatively characterize the number of red blood cells in the sample, providing a stable detection basis for hematocrit prediction.

[0027] The GPA signal collected from the internal control area is used to quantitatively characterize the number of red blood cells. Based on this, the HBP signal collected from the detection area is corrected for hematocrit, eliminating the matrix effect bias caused by the difference in hematocrit between different individuals (normal range is about 15%-75%), making the HBP test results in whole blood samples closer to the gold standard value of plasma.

[0028] In some preferred embodiments, the nitrocellulose membrane is further provided with a quality control area; The internal reference zone, the detection zone, and the quality control zone are arranged sequentially along the chromatography direction of the nitrocellulose membrane.

[0029] In some preferred embodiments, the first fluorescent probe is a lanthanide fluorescent probe; and / or, The second fluorescent probe is a quantum dot fluorescent probe.

[0030] By defining the first fluorescent probe as a lanthanide fluorescent probe and the second fluorescent probe as a quantum dot fluorescent probe, and utilizing the significant differences between the two in their inherent optical properties such as fluorescence lifetime, emission peak narrowness, and Stokes shift, a necessary probe-level prerequisite is provided for the subsequent physical separation of the two fluorescent signals through time-resolved detection or time-division excitation / filter acquisition methods. This supports highly specific identification and low crosstalk reading of the intrinsic reference signal and the detection signal under the condition of sharing the same optical channel.

[0031] In some preferred embodiments, the excitation wavelength range of the lanthanide fluorescent probe is 300 nm to 400 nm, for example, but not limited to 300 nm, 320 nm, 350 nm, 380 nm, or 400 nm, preferably 365 nm; the emission wavelength range is 600 nm to 630 nm, for example, but not limited to 600 nm, 610 nm, 620 nm, or 630 nm, preferably 615 nm ± 10 nm; and / or, The excitation wavelength range of the quantum dot fluorescent probe is 600nm~650nm, for example, but not limited to 600 nm, 610 nm, 620 nm, 630 nm, 640 nm or 650nm, preferably 620nm; the emission wavelength range is 630nm~680nm, for example, but not limited to 630 nm, 640 nm, 650 nm, 660 nm, 670nm or 680nm, preferably 650nm±10nm.

[0032] In some preferred embodiments, the distance between the emission center wavelength of the lanthanide fluorescent probe and the emission center wavelength of the quantum dot fluorescent probe is greater than or equal to 30 nm.

[0033] By limiting the distance between the emission center wavelength of the lanthanide fluorescent probe and the emission center wavelength of the quantum dot fluorescent probe to be greater than or equal to 30 nm, sufficient wavelength separation is maintained between the emission spectra of the two fluorescent probes. Thus, when the two emission signals are acquired in a time-division manner using the same optical detection channel, a narrow-band bandpass filter can be used to effectively distinguish the emission signals of the two probes, reduce signal crosstalk caused by spectral overlap, and ensure the independence and accuracy of the acquisition of the GPA internal reference signal and the HBP detection signal.

[0034] In some preferred embodiments, the spacing between the internal reference detection line corresponding to the internal reference region and the target analyte detection line corresponding to the detection region is 3mm to 8mm.

[0035] This layout ensures that the two detection lines are in a distinguishable and stable capillary flow region during chromatography, avoiding both overlapping of the chromatography fronts and mutual interference of signals due to excessive spacing, and preventing increased flow rate differences and decreased quantitative consistency in different areas of the same test strip due to excessive spacing.

[0036] According to a second aspect of the present invention, a whole blood heparin-binding protein fluorescence immunoassay system is provided, comprising the above-described whole blood heparin-binding protein fluorescence immunochromatographic test strip; and, A fluorescence reader, comprising dual excitation light sources, a single optical detection channel, and a data processing module; The dual excitation light source is configured to output a first excitation light adapted to the first fluorescent probe and a second excitation light adapted to the second fluorescent probe in a time-division manner; The single optical detection channel is configured to acquire a first emitted light signal when irradiated by the first excitation light, and to acquire a second emitted light signal when irradiated by the second excitation light; The data processing module has a built-in preset hematocrit prediction model and concentration correction model, configured to determine the predicted hematocrit value based on the first emitted light signal and the hematocrit prediction model, and to determine the corrected target analyte concentration based on the predicted hematocrit value, the concentration correction model and the second emitted light signal.

[0037] The system combines whole blood heparin-binding protein fluorescence immunochromatographic test strips with a fluorescence reader. After the test strip completes the dual-probe immunochromatographic reaction of GPA in the sample that participates in the HBP target, the reader automatically completes signal acquisition and data processing, forming a complete detection closed loop from sample reaction to result output, without the need for external equipment or manual calculation.

[0038] The dual excitation light source is configured to output the first excitation light adapted to the first fluorescent probe and the second excitation light adapted to the second fluorescent probe in a time-division manner. This allows the two probes with different fluorescence mechanisms to be fully excited at their respective optimal excitation wavelengths, ensuring the fluorescence signal intensity of the two probes and avoiding the decrease in excitation efficiency caused by the mismatch of excitation wavelengths.

[0039] The single optical detection channel is configured to acquire the first emitted light signal when the first excitation light is irradiated and the second emitted light signal when the second excitation light is irradiated. By switching between time and time, the same detection channel can acquire both emitted light signals compatiblely. There is no need to set up a dual-optical-path detection system, which reduces the number of optical components and lowers the size, cost and debugging complexity of the instrument.

[0040] In some preferred embodiments, the single optical detection channel is equipped with a switchable narrowband bandpass filter assembly. By mechanically driving the switching of bandpass filters with different center wavelengths into the optical path, the same optical detection channel allows only fluorescence signals of specific emission wavelengths to pass through at different times. This enables time-division acquisition of two different emission wavelength fluorescence signals with a single photodetector, eliminating the need for an independent optical detection channel for each fluorescent probe, reducing the number of optical components and the complexity of optical path debugging, and lowering the instrument size and manufacturing cost.

[0041] In some preferred embodiments, the fluorescence reader further includes a control module configured to: drive the narrowband bandpass filter assembly to switch to a first filter adapted to the first fluorescent probe when acquiring the first emitted light signal; and drive the narrowband bandpass filter assembly to switch to a second filter adapted to the second fluorescent probe when acquiring the second emitted light signal, so that the filter switching action is automatically linked with the time-division output timing of the dual excitation light sources, ensuring that only the filter adapted to the emission wavelength of the current fluorescent probe is in the optical path during each acquisition period, thereby avoiding filter mismatch during the acquisition of the two emitted light signals and ensuring the accuracy and reliability of time-division acquisition.

[0042] In some preferred embodiments, the data processing module is configured as follows: Based on the peak integral area of ​​the first emitted light signal, the predicted value of hematocrit is calculated using the hematocrit prediction model. Based on the predicted hematocrit value, the correction coefficient is calculated using the concentration correction model; The original target analyte concentration is determined based on the peak integral area of ​​the second emitted optical signal, and the original target analyte concentration is corrected using the correction coefficient to obtain the corrected target analyte concentration.

[0043] This configuration enables the data processing module to automatically complete the entire process from raw signal acquisition to final corrected concentration output in a fixed logical order, without the need for manual intervention in intermediate steps. This avoids random errors introduced by manual table lookup or calculation, and improves the consistency and repeatability of the detection results.

[0044] In some preferred embodiments, the data processing module includes: The hematocrit prediction model is a linear regression model obtained by fitting the peak integral area of ​​the first emitted light signal with the standard value of hematocrit. The predicted value of hematocrit is a×F_GPA+b, where F_GPA is the peak integral area of ​​the first emitted light signal, and a and b are constants determined by fitting with standard samples. The concentration correction model is a correction coefficient calculation model based on fitting the predicted hematocrit value with the original detection deviation. The correction coefficient K = cd × HCT predicted value, where the HCT predicted value is the predicted hematocrit value, and c and d are constants determined by fitting with standard samples. The corrected target analyte concentration Ccorrected = Coriginal / K, where Coriginal is the original target analyte concentration determined based on the second emitted light signal.

[0045] The hematocrit prediction model employs a linear regression model (HCT predicted value = a × F_GPA + b) based on fitting the peak integrated area of ​​the first emitted light signal with the standard value of hematocrit. The concentration correction model employs a correction coefficient calculation model (K = cd × HCT predicted value, C correction = C original / K) based on fitting the predicted value of hematocrit with the original detection deviation. Both models use measured data from standard samples to determine constants, establishing a deterministic mathematical mapping relationship from the integrated area of ​​the fluorescence signal to the predicted value of hematocrit and then to the correction coefficient. This quantifies the detection deviation caused by the difference in hematocrit in whole blood samples into a calculable correction coefficient. Through division operations, the concentration of the original target analyte is systematically corrected, achieving quantitative compensation for the detection deviation.

[0046] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0047] Example 1: Preparation of test strips This embodiment provides a whole blood heparin-binding protein fluorescent immunochromatographic test strip based on hematocrit correction, including a base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad that are sequentially overlapped and fixed on the base plate along the chromatography direction.

[0048] The nitrocellulose membrane has an internal control zone (near the sample pad end) and a detection zone (near the absorbent pad end) arranged sequentially along the chromatography direction, with a 5mm spacing between the internal control zone and the detection zone. Specific coating process parameters are as follows:

[0049] I. Preparation of Fluorescently Labeled Antibodies 1. Eu 3+ Labeling with anti-GPA antibody (first fluorescent probe labeling) Principle: Using lanthanide chelates (such as Eu) 3+ -NTA or Eu 3+ -DTPA) uses an amino-coupled method to label anti-GPA monoclonal antibodies, and utilizes long-lifetime fluorescence properties to achieve time-resolved detection.

[0050] step: Antibody pretreatment: Take anti-human GPA monoclonal antibody (concentration 1.0 mg / mL, mouse anti-human CD235a, clone number HIR2), dialyze overnight with 0.05M MES buffer (pH 6.0) to remove free amino and salt ions.

[0051] Eu 3+ Chelate activation: Take Eu 3+ -NHS ester (Sigma-Aldrich, catalog number E1207), according to antibody: Eu 3+ Dissolve in anhydrous DMF at a molar ratio of 1:10 and activate at room temperature for 30 minutes.

[0052] Coupling reaction: Activated Eu 3+ The chelate was added dropwise to the antibody solution, and the mixture was stirred at 4°C in the dark for 2 hours.

[0053] Purification: Labeled antibodies were separated using a Sephadex G-25 chromatography column (GE Healthcare) with PBS containing 0.1% BSA (pH 7.4) as the elution buffer, and the first elution peak was collected.

[0054] Labeling efficiency determination: Labeling efficiency was measured at 280 nm (antibody) and 340 nm (Eu) using a UV spectrophotometer.3+ Absorbance, calculate labeling rate (target value: 5-8 Eu) 3+ (Molecules / antibodies).

[0055] Storage: After aliquoting, store at -20℃ protected from light. Dilute with PBS containing 5% sucrose to 0.5 mg / mL before use.

[0056] 2. Quantum dot-labeled anti-HBP antibody (second fluorescent probe labeling) Principle: Carboxylated quantum dots (such as CdSe / ZnS, emission wavelength 650nm) are used to conjugate anti-HBP monoclonal antibodies via EDC / NHS cross-linking.

[0057] step: Quantum dot pretreatment: Take carboxylated quantum dot solution (concentration 1 μM, emission wavelength 650nm, Invitrogen, catalog number Q21311MP), centrifuge with 0.01M PBS (pH 7.2) (12000×g, 10 min), wash twice, and resuspend to 1 mL.

[0058] Antibody activation: Take anti-human HBP monoclonal antibody (concentration 1.5 mg / mL, rabbit anti-human), add EDC (final concentration 5 mM) and NHS (final concentration 10 mM), and activate at room temperature for 15 minutes.

[0059] Coupling reaction: The activated antibody was added to the quantum dot solution at a molar ratio of antibody to quantum dots of 5:1. The mixture was stirred at 37°C in the dark for 1 hour.

[0060] Blocking and purification: Add 10% BSA solution to block unreacted sites, let stand at 4°C for 30 minutes; filter with a 0.22 μm filter membrane to remove aggregates, and store at 4°C.

[0061] Labeling efficiency verification: The emission intensity of quantum dots (excitation 620 nm, emission 650 nm) was measured by a fluorescence spectrophotometer to ensure that the fluorescence quantum yield of the labeled quantum dots was retained at ≥80%.

[0062] II. Preparation method of binding pad Principle: The two labeled antibodies are mixed and sprayed onto a glass fiber conjugate pad. The antibodies are then fixed by drying to ensure uniform release of antibodies during chromatography.

[0063] step: Pretreatment of the conjugate pad: Take a glass fiber membrane (such as Ahlstrom 8964), soak it in PBS (pH 7.4) containing 0.5% Triton X-100 and 2% BSA for 30 minutes, dry it at 37°C for 2 hours, and set it aside.

[0064] Antibody mixture preparation: Eu 3+Labeled anti-GPA antibody (0.5 mg / mL) and quantum dot-labeled anti-HBP antibody (0.8 mg / mL) were mixed at a volume ratio of 1:1, and 5% sucrose and 0.1% PVP were added as a protective agent. The mixture was then vortexed to mix.

[0065] Spraying process: Using a BioDot XYZ3050 sprayer, the antibody mixture was uniformly sprayed onto the pretreated conjugate pad at a speed of 1.0 μL / cm, with a spraying width of 0.8±0.1 mm.

[0066] Drying and storage: Dry at 37℃ and relative humidity <30% for 4 hours. After drying, immediately seal in an aluminum foil bag, add desiccant, and store at 4℃ for later use.

[0067] Performance verification: Take the prepared binding pad, add 100 μL of PBS, and observe the antibody release rate (target: complete release within 30 seconds with no residue).

[0068] The coated NC membrane, sample pad (Ahlstrom; model 8964; size 25cm*30cm) and absorbent pad (Wuhan Sirui; model SR-05; size 250mm*300mm) are sequentially overlapped on the base plate, cut into 4 mm wide test strips, placed in the cartridge, and sealed with desiccant for storage.

[0069] Example 2 Construction of a whole blood heparin-binding protein fluorescence immunoassay system This embodiment provides a whole blood heparin-binding protein fluorescence immunoassay system, including the test strip prepared in Example 1 and a fluorescence reader.

[0070] The fluorescence reader includes dual excitation light sources, a single optical detection channel, and a data processing module.

[0071] Dual excitation light sources include a 365nm LED light source (compatible with Eu). 3+ The system uses a 620nm LED light source (adapted to quantum dot excitation) and a 620nm LED light source (adapted to quantum dot excitation), with time-sharing output of the two light sources and a switching time of less than 50ms.

[0072] The single optical detection channel is equipped with a switchable narrowband bandpass filter assembly: the first filter is a 615±10nm bandpass filter (compatible with Eu). 3+ The first filter is a 650±10nm bandpass filter (adapted for quantum dot emission). The switching between the two filters is automatically controlled by the embedded chip of the reader, and the switching time is less than 50ms.

[0073] The scanning timing of the dual excitation light source and single optical detection channel is as follows: During the first scan, the 365nm excitation light source is turned on, and a 615±10nm bandpass filter (first filter) is inserted into the single optical detection channel. The intrinsic parameter region is scanned in 0.1mm increments to acquire Eu. 3+ The emission light signal emitted by the marker at 615nm was used to obtain the peak integrated area of ​​the first emission light signal F_GPA. During the second scan, the 365nm excitation source was turned off and the 620nm excitation source was turned on. Within a single optical detection channel, the control module switched to a 650±10nm bandpass filter (the second filter) to scan the detection area at the same time, acquiring the emission light signal of the quantum dot marker at 650nm, and obtaining the peak integrated area of ​​the second emission light signal F_HBP. The same photodetector was used for both scans, and time-division acquisition was achieved through the synchronous switching of the excitation source and the filter, with a total scan time of less than 2 seconds.

[0074] The data processing module has a built-in preset hematocrit prediction model and concentration correction model. The hematocrit prediction model is a linear regression model obtained by fitting the peak integral area of ​​the first emitted light signal (F_GPA) with the standard value of hematocrit: HCT predicted value = a × F_GPA + b. The concentration correction model is a correction coefficient calculation model obtained by fitting the predicted hematocrit value with the original detection deviation: correction coefficient K = cd × HCT predicted value, and the corrected target analyte concentration Ccorrected = Coriginal / K. Wherein, Coriginal is the original HBP concentration obtained by substituting the peak integral area of ​​the second emitted light signal (F_HBP) into the calibration curve of a conventional plasma sample. The constants a, b, c, and d are determined by fitting with standard samples, as detailed in Example 4.

[0075] Example 3: Spectral Selection Principle of 365nm / 620nm Dual Excitation and 615nm / 650nm Dual Emission This embodiment illustrates the selection principle and technical basis for the combination of dual excitation and dual emission spectra.

[0076] The first scan used 365nm excitation / 615nm emission acquisition GPA-Eu. 3+ Internal reference signal. Europium ion (Eu) 3+ As a lanthanide element, its fluorescence emission originates from within the 4f electron shell. 5 D0→ 7 F j (J=0-6) energy level transition, where 5 D0→ 7 The 615nm emission corresponding to the F2 transition is an ultrasensitive electric dipole transition, with the highest fluorescence intensity and an extremely sharp emission peak (approximately 10nm full width at half maximum). Eu 3+The maximum excitation wavelength is around 337nm, but this embodiment chooses 365nm as the excitation light, mainly based on the following considerations: ① 365nm is in the near-ultraviolet region, and LED light source technology at this wavelength is mature, low-cost, and has high output stability, making it suitable for integration into portable fluorescence readers; ② Eu 3+ It still has a high absorption efficiency at 365nm (about 60%-70% of the peak absorption), which can generate sufficient fluorescence signal intensity; ③ There is a Stokes shift of up to 250nm between the 365nm excitation light and the 615nm emission light, which is far greater than that of ordinary fluorescent dyes (usually 20-50nm), which can effectively avoid the interference of excitation light scattering on emission light collection, and a high signal-to-noise ratio can be obtained without a complex optical filtering system.

[0077] The second scan used 620nm excitation / 650nm emission to acquire HBP quantum dot detection signals. Quantum dots (QDs) are semiconductor nanocrystals with characteristics such as broad-band excitation, narrow-symmetry emission, high fluorescence quantum yield, and excellent photostability. This scheme selected quantum dots with an emission peak at 650nm to label the HBP detection antibody, based on the following criteria: ① Quantum dots have a broad excitation spectrum (continuously excitable from 300-700nm), and 620nm excitation light can effectively excite quantum dots to produce 650nm emission; ② The 650nm emission peak is similar to Eu... 3+ The 615nm emission peaks have a wavelength spacing of 35nm, and the half-maximum width of the quantum dot emission peak is about 30nm. 3+ The emission peak half-width is about 10nm, and the two can be completely separated in the spectrum; ③650nm is at the edge of the near-infrared region, and the autofluorescence background of blood samples in this band is low, which is beneficial to improving the detection signal-to-noise ratio.

[0078] The two scans share the same optical detection channel and are performed in a time-sharing manner. Each scan uses only a single excitation source, thus eliminating the problem of simultaneous crosstalk between excitation beams. However, since the two scans share the same optical detection channel, the following spectral compatibility must be ensured: ① The same filter system must be compatible with switching between 615nm and 650nm emission wavelengths. This scheme uses a switchable narrowband bandpass filter assembly in the detection optical path. The 615±10nm filter is used during the first scan, and the 650±10nm filter is used during the second scan. The filter switching is automatically controlled by the embedded chip of the reader, with a switching time of less than 50ms; ② The 365nm excitation beam does not interfere with the second scan (the 365nm source is turned off during the second scan); ③ The 620nm excitation beam does not interfere with the first scan (the 620nm source is turned off during the first scan); ④ The emission spectra of the two fluorescent probes do not overlap, Eu 3+The half-width at half-maximum (WHM) of the 615nm emission peak is only about 10nm (610-620nm), while the half-width at half-maximum (WHM) of the 650nm emission peak of the quantum dot is about 30nm (635-665nm). The peak spacing between the two is 35nm. With the use of narrow-band filters of 615±10nm and 650±10nm, it can be ensured that only the emission signal of the corresponding fluorescent probe is collected in each scan, with a crosstalk rate of less than 0.5%.

[0079] In addition, Eu 3+ The fluorescence lifetime of the α-dot is 100-1000 μs (milliseconds), while that of the quantum dot is 5-50 ns (nanoseconds), a difference of approximately four orders of magnitude. This scheme, in the first scan (GPA-Eu... 3+ Time-gated technology is introduced in the internal reference signal acquisition: signal acquisition begins 50μs after the 365nm excitation pulse ends. By this time, any short-lived background fluorescence in the sample (including nanosecond-level fluorescence of quantum dots, autofluorescence of NC films, etc.) has completely decayed, while Eu... 3+ The long-lived fluorescence maintains high intensity, effectively eliminating background fluorescence interference and improving the detection signal-to-noise ratio of the internal reference signal. The second scan (HBP quantum dot signal acquisition) does not require the use of time-resolved mode due to the high fluorescence brightness and good signal-to-noise ratio of the quantum dots.

[0080] Example 4: Determination of parameters for the hematocrit prediction model and concentration correction model In this embodiment, constants a, b, c, and d in the hematocrit prediction model and concentration correction model are determined by fitting standard samples.

[0081] Calibration samples were obtained from 10 healthy volunteers aged 18-50 years, with no history of infection in the past month, no hematological diseases, and a hematopoietic stem cell count (HCT) between 40% and 50%. Each volunteer had 5 mL of EDTA-K2 anticoagulated venous blood collected after fasting. All samples were thoroughly mixed and used for gradient preparation. The mixed whole blood was centrifuged at 4°C and 3000 rpm for 10 min to separate the upper plasma layer, middle white membrane layer, and lower red blood cell layer. Red blood cells and plasma were mixed at the corresponding volume ratios for 13 HCT gradients: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%. Three parallel samples, each at least 1 mL, were prepared for each gradient. After preparation, the actual HCT values ​​were measured using a fully automated hematology analyzer as the reference values.

[0082] Thirty-nine blank samples (unspecified HBP) ​​from the above 13 HCT gradients were collected and tested using the detection system described in Example 2. 100 μL of sample was added to each test strip, and the samples were incubated at 37°C for 10 min before acquiring the F_GPA signal. Using the actual true HCT value as the independent variable and the corresponding F_GPA peak area as the dependent variable, a linear fit was performed using the least squares method to obtain the hematocrit prediction model: HCT predicted value = 0.00197 × F_GPA + 5.18, with a goodness of fit R0. 2 =0.994, the 95% confidence interval slope is [0.00192, 0.00202] and the intercept is [4.92, 5.44], i.e., a=0.00197, b=5.18. No outliers were found by the Glubbdox test with α=0.05, and the HCT prediction bias is ≤1.2%.

[0083] For each HCT gradient, samples were spiked with recombinant HBP concentrate, with four spiking gradients: 0 ng / mL blank, 10 ng / mL low concentration, 50 ng / mL medium concentration, and 200 ng / mL high concentration. All samples were tested within 2 hours of preparation. True HBP values ​​were determined using an HBP ELISA kit (Beijing Bio-Lab Technology Co., Ltd., catalog number ZN2034). Before testing, plasma samples were centrifuged at 3000 rpm for 10 min. The F_HBP obtained from the test strip was substituted into the HBP calibration curve (established using a four-parameter logistic regression model) plotted from routine plasma samples to obtain the original HBP concentration Coriginal. The relative deviation was calculated as (Coriginal - true concentration) / true concentration × 100%.

[0084] Statistical analysis shows a strong linear negative correlation between the original bias and HCT. The fitted linear regression equation for the bias and HCT is: bias (%) = -1.187 × HCT + 52.1, R0. 2 =0.991. Based on the linear relationship between the deviation and HCT, the formula for the correction coefficient is derived: K = 1.521 - 0.01187 × HCT predicted value, i.e., c = 1.521, d = 0.01187. Therefore, the formula for the corrected HBP concentration is Ccorrected = Coriginal / K.

[0085] The 260 spiked samples were split into 182 training samples and 78 test samples in a 7:3 ratio. The training set was used to validate the performance of the test set. Results showed that the relative bias of all test set samples after correction ranged from -8.7% to +7.9%, with 100% sample bias ≤10%. The biases of extreme HCT samples (15% and 75%) after correction were +7.9% and -8.7%, respectively, representing a reduction of over 80% compared to the uncorrected bias. The linear regression equation between the corrected concentration and the ELISA gold standard was y = 0.989x + 0.72, R0. 2 =0.992, significantly better than the uncorrected R.2 =0.874.

[0086] Example 5: Validation of Spectroscopic Compatibility of Dual Fluorescent Probes To verify the lanthanide fluorescent probe (Eu) 3+ To assess spectral compatibility between quantum dot fluorescent probes and quantum dot fluorescent probes when acquired in the same optical detection channel at different times, the following crosstalk tests were conducted: (1) Only Eu sample added 3+ The antibody-labeled sample: When the first scan sequence (365nm excitation, 615±10nm filter) of Example 2 was used for acquisition, the F_GPA signal was normal; when the second scan sequence (620nm excitation, 650±10nm filter) was used for acquisition, the detected signal intensity was only 0.3% of the normal signal.

[0087] (2) Samples with only quantum dot labeled antibodies: When the second scan sequence (620nm excitation, 650±10nm filter) of Example 2 was used for acquisition, the F_HBP signal was normal; when the first scan sequence (365nm excitation, 615±10nm filter) was switched to acquisition, the detected signal intensity was only 0.4% of the normal signal.

[0088] (3) Samples with two labeled antibodies added simultaneously: The samples were collected according to the complete time-sharing scanning sequence of Example 2. The F_GPA and F_HBP signals were consistent with those when the samples were added separately, and the crosstalk rate was less than 0.5%.

[0089] The above results indicate that Eu 3+ The 35nm wavelength spacing between the 615nm emission peak (half-width of about 10nm) and the 650nm emission peak (half-width of about 30nm) of the quantum dot, combined with narrow bandpass filters of 615±10nm and 650±10nm (out-of-band cutoff depth OD≥4), can effectively achieve the separation and acquisition of the two emitted light signals, meeting the spectral compatibility requirements of dual-probe time-division detection.

[0090] Example 6: Clinical whole blood sample validation Thirty clinical whole blood samples were enrolled for validation, with HCT ranging from 17% to 72%, covering clinical scenarios such as sepsis, postoperative infection, and common fever. HBP concentration in each sample was simultaneously determined using the detection system described in Example 2 (direct detection of whole blood) and the plasma ELISA method (plasma was collected after centrifugation at 3000 r / min for 10 min).

[0091] The results showed that the kappa value of the consistency between the test strip values ​​and the plasma ELISA values ​​before correction was 0.76, indicating a significant deviation. After correction using the correction formula determined in Example 3, the kappa value of consistency increased to 0.94, reaching a level of almost perfect consistency. Among them, the deviation of the 5 hemolyzed samples (free hemoglobin concentration 1.2-3.8 g / L) after correction was ≤3.2%, proving that the GPA internal control can still effectively correct for HCT differences under hemolytic conditions.

[0092] Example 7: Comparative Experiment of GPA Internal Reference and Hemoglobin Internal Reference under Multiple Interference Conditions To fully verify the technical advantages of the GPA internal control compared to the hemoglobin (Hb) internal control, three test strips were prepared in parallel according to the preparation method in Example 1: GPA internal control test strip (the internal control area is coated with anti-human blood group glycoprotein A monoclonal antibody, and the binding pad contains Eu). 3+ Labeled anti-human blood group glycoprotein A monoclonal antibody), Hb internal control test strip (internal control area coated with anti-human hemoglobin monoclonal antibody, conjugate pad containing Eu) 3+ The labeling anti-human hemoglobin monoclonal antibody was prepared in the same manner as in Example 1, and the test strips without internal control were coated only with HBP detection antibody and had no internal control area.

[0093] Interference samples were prepared based on whole blood from 10 healthy volunteers (HCT adjusted to 45%). Recombinant HBP concentrate was added to each interference gradient sample, with three spiking gradients: 0 ng / mL, 50 ng / mL, and 200 ng / mL, and three replicates for each concentration. During detection, 100 μL of sample was added to each test strip, incubated at 37°C for 10 min, and then the signal was acquired according to the first / second scan sequence of Example 2. The stability of the internal control signal was evaluated using the coefficient of variation (CV%). The corrected HBP concentration was calculated according to the formula in Example 4. The relative deviation was calculated using the ELISA measurement as the gold standard. One-way ANOVA was used for inter-group comparisons, with P < 0.05 considered statistically significant.

[0094] 1. Comparison of internal control signal stability under hemolytic interference conditions Hemolytic interference samples were prepared by repeated freeze-thaw cycles to create hemolyzed whole blood, which was then mixed with baseline whole blood. Six gradients of free hemoglobin concentration were set: 0, 0.5, 1.0, 2.0, 3.0, and 5.0 g / L. The GPA internal control signal remained within the range of 18655–18821 AU (fluctuation <0.9%) within the free Hb concentration range of 0–5 g / L. There were no statistically significant differences in signal between each concentration gradient and the control group (P>0.05), with an overall CV ≤2.8%. The Hb internal control signal showed a significant positive bias with increasing free Hb concentration: the signal increased by 6.0% at a free Hb concentration of 0.5 g / L and by 42.5% at 5 g / L. The differences between each concentration gradient and the control group were statistically significant (P<0.01), with an overall CV of 18.6%. This is because the free hemoglobin released during hemolysis specifically cross-binds with the anti-Hb antibodies immobilized on the NC membrane, causing the signal intensity of the internal control detection line to continuously increase with increasing free Hb concentration, thus failing to accurately reflect the actual number of red blood cells in the sample. See the table below:

[0095] Taking a 50 ng / mL spiked sample as an example, the HBP detection deviation in the GPA internal control group remained within +2.9% at all hemolysis gradients, showing no significant difference from the non-hemolysis control. In the Hb internal control group, the deviation increased sharply after correction as the degree of hemolysis worsened, reaching +8.7% at 0.5 g / L free Hb and as high as +38.4% at 5 g / L. This is because the Hb internal control signal was falsely elevated due to interference from free Hb, resulting in overcorrection after being substituted into the correction formula, leading to a systematic underestimation of HBP concentration. In the non-internal control group, the deviation remained relatively stable at around +3.1% at all hemolysis gradients, but the deviation across the entire HCT range (15%-75%) reached ±37.2%, failing to meet clinical testing requirements. See the table below:

[0096] 2. Comparison of signal stability of internal control under lipemia interference conditions Lipid-affected samples were added to a fat emulsion injection solution, with six triglyceride concentration gradients: 0, 5, 10, 15, 20, and 25 mmol / L. The CV of the GPA internal control signal was ≤2.8% within the triglyceride range of 0–25 mmol / L, with no significant difference compared to the control group at each concentration (P>0.05). The CV of the Hb internal control signal increased slightly with increasing lipid concentration (maximum 7.7%), but the signal mean did not show a systematic shift, indicating that lipid concentration had a relatively small impact on both internal control signals. However, the stability of the GPA internal control was still significantly better than that of the Hb internal control (P<0.05). See the table below:

[0097] 3. Comparison of internal reference signal stability under jaundice interference conditions Bilirubin standard was added to jaundice interference samples, and six gradients of total bilirubin concentrations were set: 0, 100, 200, 300, 400, and 500 μmol / L. The CV of the GPA internal control signal was ≤2.8% within the bilirubin range of 0–500 μmol / L, and there was no significant difference between each concentration and the control group (P>0.05). The CV of the Hb internal control signal increased slightly with increasing bilirubin concentration (maximum 5.5%), possibly related to the weak absorption of bilirubin near 635 nm, which interferes with the excitation efficiency of Hb fluorescent markers. The stability of the GPA internal control was significantly better than that of the Hb internal control (P<0.01). See the table below:

[0098] 4. Comparison of internal control signal stability under varying white blood cell / platelet concentration conditions Leukocyte concentration gradient samples were separated from the white blood cell layer by centrifugation and then re-added proportionally, with a setting of 1×10⁻⁶. 9 5×10 9 10×10 9 20×10 9 30×10 9 There are 5 gradients per L; platelet concentration gradient samples are added proportionally after centrifugation to separate platelet-rich plasma, with a setting of 50 × 10⁶. 9 100×10 9 200×10 9 350×10 9 500×10 9 There are 5 gradients per L. White blood cell concentration 1×10⁹ / L 9 / L~30×10 9 Within the range of / L, the CV of the internal control for GPA is ≤2.9%, and the CV of the internal control for Hb is ≤3.2%; platelet concentration is 50×10 9 / L~500×10 9 Within the range of / L, the CV of the GPA internal control was ≤2.9%, and that of the Hb internal control was ≤3.2%. Both internal controls showed good stability under varying leukocyte and platelet concentrations, but the CV of the GPA internal control was lower than that of the Hb internal control at all gradients, and the difference was statistically significant (P<0.05). See the table below:

[0099]

[0100] The above comparative experiments under multiple interference conditions show that: ① Hemolysis interference is an inherent defect of the Hb internal reference system, and the Hb internal reference signal increases linearly with increasing free Hb concentration (R0). 2=0.987), with a signal deviation of +42.5% under 5g / L free Hb conditions, and a corrected HBP detection deviation of +38.4%, failing to meet clinical testing requirements; ② GPA internal control possesses natural hemolytic resistance. As a transmembrane protein of erythrocytes, GPA remains anchored to membrane fragments after hemolysis and is not released into the plasma. Within the free Hb concentration range of 0~5g / L, the signal CV of GPA internal control is ≤2.8%, and the corrected HBP detection deviation is ≤2.9%, showing no significant difference from the non-hemolytic control; ③ GPA internal control is also superior to Hb internal control under conditions of lipemia, jaundice, and changes in white blood cell / platelet concentration. The CV of GPA internal control under all interference conditions is ≤3.1%, significantly lower than the corresponding value of Hb internal control (maximum 18.6%). Therefore, this protocol selects GPA instead of Hb as the internal control marker for HCT, solving the long-standing technical problem of hemolytic sample correction failure in whole blood immune detection.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent immunochromatographic test strip for whole blood heparin-binding protein based on hematocrit correction, characterized in that, include: The base plate, and the sample pad, conjugation pad, nitrocellulose membrane and absorbent pad that are sequentially overlapped and fixed on the base plate along the chromatography direction; The conjugate pad contains an anti-human blood group glycoprotein A monoclonal antibody labeled with a first fluorescent probe and an anti-human heparin-binding protein monoclonal antibody labeled with a second fluorescent probe. Wherein, the first fluorescent probe is a long-lived fluorescent probe, and the second fluorescent probe is a short-lived fluorescent probe; The nitrocellulose membrane is provided with an internal reference zone and a detection zone sequentially along the chromatography direction; The internal reference region is coated with a monoclonal antibody against human blood group glycoprotein A, and the detection region is coated with a monoclonal antibody against human heparin-binding protein.

2. The whole blood heparin-binding protein fluorescent immunochromatographic test strip according to claim 1, characterized in that, The nitrocellulose membrane is also provided with a quality control area; The internal reference zone, the detection zone, and the quality control zone are arranged sequentially along the chromatography direction of the nitrocellulose membrane.

3. The whole blood heparin-binding protein fluorescent immunochromatographic test strip according to claim 1, characterized in that, The first fluorescent probe is a lanthanide fluorescent probe; and / or, The second fluorescent probe is a quantum dot fluorescent probe.

4. The whole blood heparin-binding protein fluorescent immunochromatographic test strip according to claim 3, characterized in that, The lanthanide fluorescent probe has an excitation wavelength range of 300 nm to 400 nm and an emission wavelength range of 600 nm to 630 nm; and / or, The excitation wavelength range of the quantum dot fluorescent probe is 600nm~650nm, and the emission wavelength range is 630nm~680nm. Preferably, the distance between the emission center wavelength of the lanthanide fluorescent probe and the emission center wavelength of the quantum dot fluorescent probe is greater than or equal to 30 nm.

5. The whole blood heparin-binding protein fluorescent immunochromatographic test strip according to claim 4, characterized in that, The lanthanide fluorescent probe has an excitation wavelength of 365 nm and an emission wavelength of 615 nm ± 10 nm; and / or, The quantum dot fluorescent probe has an excitation wavelength of 620 nm and an emission wavelength of 650 nm ± 10 nm.

6. The whole blood heparin-binding protein fluorescent immunochromatographic test strip according to claim 2, characterized in that, The distance between the internal reference detection line corresponding to the internal reference zone and the target analyte detection line corresponding to the detection zone is 3mm to 8mm.

7. A whole blood heparin-binding protein fluorescence immunoassay system, characterized in that, Including the whole blood heparin-binding protein fluorescent immunochromatographic test strip according to any one of claims 1 to 6; and, A fluorescence reader, comprising dual excitation light sources, a single optical detection channel, and a data processing module; The dual excitation light source is configured to output a first excitation light adapted to the first fluorescent probe and a second excitation light adapted to the second fluorescent probe in a time-division manner; The single optical detection channel is configured to acquire a first emitted light signal when irradiated by the first excitation light, and to acquire a second emitted light signal when irradiated by the second excitation light; The data processing module has a built-in preset hematocrit prediction model and concentration correction model, configured to determine the predicted hematocrit value based on the first emitted light signal and the hematocrit prediction model, and to determine the corrected target analyte concentration based on the predicted hematocrit value, the concentration correction model and the second emitted light signal.

8. The whole blood heparin-binding protein fluorescence immunoassay system according to claim 7, characterized in that, The single optical detection channel is equipped with a switchable narrowband bandpass filter assembly; Preferably, the fluorescence reader further includes a control module, which is configured to: drive the narrowband bandpass filter assembly to switch into a first filter adapted to the first fluorescence probe when acquiring the first emitted light signal; and drive the narrowband bandpass filter assembly to switch into a second filter adapted to the second fluorescence probe when acquiring the second emitted light signal.

9. The whole blood heparin-binding protein fluorescence immunoassay system according to claim 7, characterized in that, The data processing module is configured as follows: Based on the peak integral area of ​​the first emitted light signal, the predicted value of hematocrit is calculated using the hematocrit prediction model. Based on the predicted hematocrit value, the correction coefficient is calculated using the concentration correction model; The original target analyte concentration is determined based on the peak integral area of ​​the second emitted optical signal, and the original target analyte concentration is corrected using the correction coefficient to obtain the corrected target analyte concentration.

10. The whole blood heparin-binding protein fluorescence immunoassay system according to claim 9, characterized in that, In the data processing module: The hematocrit prediction model is a linear regression model obtained by fitting the peak integral area of ​​the first emitted light signal with the standard value of hematocrit. The predicted value of hematocrit is a×F_GPA+b, where F_GPA is the peak integral area of ​​the first emitted light signal, and a and b are constants determined by fitting with standard samples. The concentration correction model is a correction coefficient calculation model based on fitting the predicted hematocrit value with the original detection deviation. The correction coefficient K = cd × HCT predicted value, where the HCT predicted value is the predicted hematocrit value, and c and d are constants determined by fitting with standard samples. The corrected target analyte concentration Ccorrected = Coriginal / K, where Coriginal is the original target analyte concentration determined based on the second emitted light signal.

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