Kit for joint inspection of seven cardiopulmonary functions

Abstract

The invention provides a joint detection kit for seven cardiopulmonary functions, and belongs to the technical field of immunochemical detection. The invention provides a joint detection kit for seven items of cardiopulmonary functions. The joint detection kit consists of a micro-fluidic chip, a quantitative blood collector, a disposable peripheral blood taking needle and a diluent, the micro-fluidic chip is composed of a PMMA base material chip, a blood filtering film and a cover plate. The micro-fluidic chip comprises a sample adding area, a marking reaction area, a time control valve and a micro-channel detection area. A synchronous detection scheme of seven indexes of the cardiopulmonary function is integrated, seven results can be output through one-time detection, high-sensitivity detection of seven targets is achieved, and the performance indexes such as accuracy, linearity and repeatability are excellent.

Description

Technical Field

[0001] This invention relates to the field of immunochemical detection technology, and in particular to a seven-item combined test kit for cardiopulmonary function. Background Technology

[0002] As a core indicator for assessing bodily function, cardiorespiratory fitness directly reflects the coordinated working ability of the circulatory and respiratory systems. However, under the influence of multiple factors such as high-altitude environments, occupational exposure, and special physiological conditions, cardiovascular and respiratory diseases often exhibit synergistic damage. This combined pathology is characterized by its insidious nature and rapid progression, severely impacting bodily function.

[0003] Given the complexity and rapid progression of combined cardiopulmonary lesions, traditional single-detection methods are insufficient to meet clinical needs. Therefore, establishing a multi-dimensional assessment system is particularly important. Common markers for early myocardial injury and pulmonary infection include CK-MB, Myo, cTnI, NT-ProBNP, CRP, DDimer, and PCT. Since a single biomarker cannot meet all these requirements, multiple biomarkers can be combined for clinical diagnosis. Currently, the main detection methods for these markers are chemiluminescence, first-generation colloidal gold, or second-generation immunofluorescence. Chemiluminescence has advantages such as high sensitivity and wide detection range, but its equipment is expensive, large, requires high laboratory standards, and demands specialized operation, making it unsuitable for point-of-care testing scenarios such as field testing. Colloidal gold or immunofluorescence based on chromatography platforms can detect biomarkers in point-of-care testing (POCT) scenarios, but colloidal gold can only achieve qualitative detection of the target, and while immunofluorescence can quantify, its accuracy is lower. In addition, due to the inherent physical properties of the chromatography membrane, such as the inconsistent pore size and the inability to guarantee the consistency of liquid flow, the chromatography platform cannot achieve the joint detection of multiple markers.

[0004] Current technologies can detect a maximum of five indicators. Expanding the range of detection targets is crucial for the clinical diagnosis of complex, multifactorial diseases. However, as those skilled in the art know, achieving the technological leap from a five-in-one to a seven-in-one detection system is essentially a challenge stemming from the exponentially increasing complexity of the detection system. First, with the increase in detection indicators, antibody cross-reactivity within the microfluidic system increases significantly, leading to a decrease in detection specificity. This places higher demands on antibody selection and optimization, requiring high affinity and low cross-reactivity. Second, simultaneous detection of seven indicators requires more complex reagent ratios and labeling conditions. The optimal working concentrations of different antibodies may conflict, and balancing the reaction conditions among the indicators is a key challenge. Finally, the repeatability, sensitivity, and precision of the detection system are more difficult to guarantee in a multi-indicator environment, requiring sophisticated design and rigorous validation. These challenges make the development of seven-in-one detection technology a comprehensive leap in systems engineering capabilities. Summary of the Invention

[0005] The purpose of this invention is to provide a seven-item combined test kit for cardiopulmonary function, which simultaneously detects seven biomarkers with complementary diagnostic value to meet the needs of complex diagnosis of multifactorial pathogenesis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a seven-item cardiopulmonary function test kit, which consists of a microfluidic chip, a quantitative blood collection device, a disposable capillary blood collection needle, and a diluent;

[0008] The microfluidic chip consists of a PMMA substrate chip, a blood filtration membrane, and a cover plate.

[0009] The microfluidic chip includes: a sample dispensing area, a labeled reaction area, a time-controlled valve, and a microchannel detection area.

[0010] Preferably, it can simultaneously detect seven indicators: PCT, D-Dimer, CKMB, NT-proBNP, cTnI, CRP, and Myo.

[0011] Preferably, the blood filtration membrane is disposed between the sample application area and the labeling reaction area.

[0012] Preferably, the labeled reaction zone has micropillars with conical hemispherical tops arranged in a cross pattern, and the micropillars are coated with fluorescent antibodies for seven indicators.

[0013] Preferably, the time-controlled valve has a regular, continuous mountain-shaped structure and is coated with a rate-limiting agent.

[0014] Preferably, the microchannel detection area has micropillars with conical hemispherical tops arranged in a cross pattern, and the micropillars are coated with capture antibodies for seven indicators.

[0015] Preferably, the seven capture antibodies are arranged in the following order from the sample application end: PCT, D-Dimer, CKMB, NT-proBNP, CRP, cTnI, and Myo.

[0016] This invention provides a seven-item cardiopulmonary function test kit. Based on a microfluidic chip, this kit can simultaneously detect seven key indicators—CK-MB, Myo, cTnI, NT-proBNP, CRP, D-Dimer, and PCT—on a 3cm × 7cm chip. Through system optimization, this invention improves detection sensitivity to cTnI 0.1 ng / ml, PCT 100 pg / ml, CRP 0.5 mg / L, NT 30 pg / ml, Myo 10 ng / ml, CKMB 1 ng / ml, and D-Dimer 100 ng / ml, while significantly shortening the testing time to less than 15 minutes. It also meets the on-site requirements of various POCT scenarios and fills the technological gap in multi-item simultaneous testing equipment. Attached Figure Description

[0017] Figure 1 This is a graph showing the initial fluorescence intensity results in Example 3;

[0018] Figure 2 This is a graph showing the fluorescence intensity results after secondary optimization in Example 3;

[0019] Figure 3 This is a graph showing the fluorescence intensity results after three optimizations in Example 3;

[0020] Figure 4 This is a graph showing the fluorescence intensity results when the ratio of each antibody in Example 4 is 1:1:1:1:1:1:1;

[0021] Figure 5 This is a graph showing the fluorescence intensity results after the ratio of each antibody was adjusted once in Example 4;

[0022] Figure 6 This is a graph showing the fluorescence intensity results after the secondary adjustment of the antibody ratios in Example 4;

[0023] Figure 7 This is a graph showing the fluorescence intensity results after three adjustments to the proportions of each antibody in Example 4;

[0024] Figure 8 This is a graph showing the fluorescence intensity results after four adjustments of the antibody ratios in Example 4;

[0025] Figure 9 This is a linear regression analysis graph of the Myo marker in Example 9;

[0026] Figure 10 This is a linear regression analysis graph of the CKMB biomarker in Example 9;

[0027] Figure 11 This is a linear regression analysis graph of the cTnI biomarker in Example 9;

[0028] Figure 12This is a linear regression analysis graph of the PCT biomarker in Example 9;

[0029] Figure 13 This is a linear regression analysis graph of the D-Dimer biomarker in Example 9;

[0030] Figure 14 This is a linear regression analysis graph of the NT-proBNP biomarker in Example 9;

[0031] Figure 15 This is a linear regression analysis graph of CRP markers in Example 9;

[0032] Figure 16 This is a comparison chart of the test results of Myo and the control product in Example 10;

[0033] Figure 17 This is a comparison chart of the test results of CKMB and the control product in Example 10;

[0034] Figure 18 This is a comparison chart of the measurement results of cTnI and the control product in Example 10;

[0035] Figure 19 This is a comparison chart of the PCT test results and the control product in Example 10;

[0036] Figure 20 This is a comparison chart of the test results of D-Dimer and the control product in Example 10;

[0037] Figure 21 This is a comparison chart of the test results of NT-proBNP and the control product in Example 10;

[0038] Figure 22 This is a comparison chart of the CRP test results and the control product in Example 10;

[0039] Figure 23 This is a schematic diagram of the microfluidic chip structure in Example 11 (the left image is the chip top cover, and the right image is the chip bottom plate);

[0040] Figure 24 This is a schematic diagram of the structure of the four key functional units in Example 11;

[0041] Figure 25 This is a schematic diagram of the sample application area structure in Example 11;

[0042] Figure 26 This is a schematic diagram of the labeled reaction zone structure in Example 11;

[0043] Figure 27 This is a schematic diagram of the time-controlled valve structure in Example 11;

[0044] Figure 28This is a schematic diagram of the microchannel detection area structure in Example 11;

[0045] Figure 29 This is a schematic diagram of the detection point structure in the microchannel detection area of ​​Example 11. Detailed Implementation

[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1: Preparation method of a seven-item cardiopulmonary function test kit

[0048] (1) Fluorescent antibody labeling

[0049] 1.1 Preparation of EDC and NHS solutions:

[0050] Weigh 5 mg of EDC and dissolve it in 0.5 mL of LME buffer. Mix well with a shaker for 1 min to prepare a 10 mg / mL solution. Prepare and use immediately.

[0051] Weigh 5 mg of NHS and dissolve it in 0.5 mL of LME buffer. Mix well with a shaker for 1 min to prepare a 10 mg / mL solution. Prepare and use immediately.

[0052] 1.2 Activating the carboxyl groups of fluorescent microspheres:

[0053] Add 130 μL of MES buffer to 65 μL of fluorescent microsphere stock solution (2%) and sonicate for 30 s.

[0054] Add 1.8 μL of NHS solution and mix with a shaker for 30 s. Then add 2.4 μL of EDC solution and sonicate for 30 s.

[0055] Place on a 37℃ constant temperature shaker and react for 25 min. Then centrifuge in a refrigerated centrifuge (4℃, 14000 rpm, 15 min) and discard the supernatant.

[0056] Add 120 μL of HEPES buffer to the fluorescent microsphere solution and sonicate to resuspend the fluorescent microspheres for 1 min before use.

[0057] 1.3 Preparation of fluorescent antibody complexes:

[0058] Take 105 μg of Ab (all seven fluorescent antibodies used were mouse antibodies) and add it to a certain volume of HEPES buffer solution. Shake and mix for 1 min to make the total volume 210 μL.

[0059] Take 100 μL of the fluorescent microspheres after sonication and quickly add them to the centrifuge tube containing the antibody solution and sonicate for 1 min.

[0060] Place on a 37°C constant temperature shaker and react for 120 min. Then add 20 μL of blocking solution, sonicate for 1 min, and continue reacting for 120 min.

[0061] Centrifuge in a refrigerated centrifuge (4℃, 14000rpm, 15min) and discard the supernatant.

[0062] Add 200 μL of HEPES buffer and sonicate for 1 min. Centrifuge (4℃, 14000 rpm, 15 min), discard the supernatant, and repeat twice.

[0063] Finally, add 200 μL of fluorescent antibody protection solution and sonicate to resuspend for 1 min.

[0064] (2) Labeling of capture antibodies:

[0065] 1.1 Preparation of EDC and NHS solutions (same as 1.1 in (1)):

[0066] 1.2 Activating the carboxyl groups of latex microspheres:

[0067] Add 180 μL of LME ES buffer to 20 μL of latex microsphere solution (10%) and mix with a shaker for 1 min.

[0068] Add 4 μL of NHS solution and mix with a shaker for 1 min. Then add 5 μL of EDC solution and mix with an ultrasonicator for 1 min.

[0069] Place on a 37℃ constant temperature shaker and react for 25 min. Then centrifuge in a refrigerated centrifuge (4℃, 14000 rpm, 20 min) and discard the supernatant.

[0070] Add 190 μL of HEPES buffer and sonicate to resuspend the latex microspheres for 1 min.

[0071] 1.3 Preparation of capture antibody complexes:

[0072] Take 105 μg of antibody (all seven capture antibodies used were mouse antibodies) and add it to a certain volume of HEPES buffer solution. Shake and mix for 1 min to make the total volume 210 μL.

[0073] Take 100 μL of the ultrasonically resuspended latex microspheres and add them to the above antibody solution. Sonicate for 1 min.

[0074] Place on a 37°C constant temperature shaker and react for 120 min. Then add 20 μL of blocking solution, sonicate for 1 min, and continue reacting for 120 min.

[0075] Centrifuge in a refrigerated centrifuge (4℃, 14000rpm, 20min) and discard the supernatant.

[0076] Add 200 μL of HEPES buffer and sonicate for 1 min. Centrifuge (4℃, 14000 rpm, 20 min), discard the supernatant, and repeat once.

[0077] Finally, add 330 μL of HEPES buffer and sonicate for 1 min to resuspend.

[0078] (4) Pretreatment of blood filtration membrane

[0079] Weigh out 0.9% NaCl (inorganic salt), 3% BSA (inert protein), 1% trehalose (carbohydrate), 0.1% polyvinyl alcohol (macromolecule), 0.3% S11 (surfactant), 0.3% Tween 20, and 0.1% PC300. Dissolve them in 100mM Tris (buffer solution) to prepare the blood filtration membrane. Stir well and soak the blood filtration membrane thoroughly in the prepared blood filtration membrane pretreatment solution for 1 minute. Discard the excess liquid and lay the membrane flat on a plate. Place it in a constant temperature and humidity incubator at 37°C and 30% humidity overnight to dry. The next day, remove and collect the membrane, place it in a desiccant, seal it, and store it for later use.

[0080] (5) Chip substrate preprocessing

[0081] After pretreatment by irradiation activation, the chip substrate is ready for use.

[0082] Use an inkjet printer (or scribing machine) to add hydrophobic lines to the chip channels to prevent sample flow along the edges.

[0083] (6) Antibody fixation

[0084] After thoroughly mixing the seven fluorescent antibodies, spot them onto the chip mixing area manually or using a micro-spotting device, 2 μL / chip, and allow to air dry at room temperature. Then, spot the capture antibodies and quality control antibodies for each of the seven items sequentially onto the flow channels, 0.6 μL / spot, and allow to air dry at room temperature.

[0085] (7) Test card packaging

[0086] Place the blood filtration membrane into the groove of the blood filtration membrane on the test card after it has been dotted with fluorescent and capture antibodies and air-dried. Then, cover the test card with the top cover and run the ultrasonic welding program (welding distance 0.070-0.095mm). The test card is now sealed.

[0087] (8) Detection

[0088] Preparation of diluent: 3.5% sucrose, 2% glucose, 3% trehalose, 2.5% BSA, 1.2% NaCl, 0.4% HIER-R-012, 5% 2-methacryloyloxyethyl phosphorylcholine, 0.4% PEG6000, 9.5% hydroxypropyl-β-cyclodextrin, 0.08% preservative ProClin300, 0.35% Tween-20 and 0.2% S11, diluted with HEPES solution.

[0089] Collect 50 μL of fingertip blood using a disposable capillary lancet and a 50 μL blood collection device. Insert the collected blood collection device into a diluent tube containing 200 μL of diluent and shake to mix for about 1 minute. Lay the test card flat and squeeze the wall of the diluent tube to add all the mixture from the diluent tube to the test card. After waiting for 15 minutes, place the test card into the instrument for testing, and the test results will be available immediately.

[0090] Example 2: Preparation of diluent

[0091] The diluent formulation is based on HEPES buffer and contains the following components by weight percentage: 3.5% sucrose, 2% glucose, 3% trehalose, 2.5% BSA, 1.2% NaCl, 0.4% HIER-R-012, 5% 2-methacryloyloxyethyl phosphorylcholine, 0.4% PEG6000, 9.5% hydroxypropyl-β-cyclodextrin, 0.08% preservative ProClin300, 0.35% Tween-20, and 0.2% S11.

[0092] Example 3 investigated the order in which the seven target antibodies were arranged.

[0093] 1. Initial Test

[0094] First, the antibody sequence for the seven assays was initially set on the chip, starting from the loading end: Myo, CRP, DD, NT-proBNP, PCT, cTnI, and CKMB. Then, zero-value samples were used for testing to assess baseline stability under this sequence, with three replicates (Figures 1, 2, and 3). Results are shown below. Figure 1 (Myo: 100-160, CRP: 170-240, DD: 260-320, NT-proBNP: 340-400, PCT: 410-490, cTnI: 510-580, CKMB: 590-670) as shown.

[0095] Experimental results show that the baseline fluctuates significantly under the initial arrangement, especially in the Myo, CRP, and DD regions, where it is disordered. Significant false signals also appear in the PCT, cTnI, and CKMB segments, indicating that this arrangement has obvious cross-interference problems.

[0096] 2. Secondary optimization

[0097] Based on the initial experimental results, the order of the capture antibodies was adjusted. The new order is: PCT, CKMB, Myo, CRP, DD, cTnI, NT-proBNP. The zero-value sample test was repeated three times on the new chip (Figures 1, 2, and 3). The results are as follows... Figure 2 (PCT: 100-160, CKMB: 170-240, Myo: 260-320, CRP: 340-400, DD: 410-490, cTnI: 510-580, NT-proBNP: 590-670) as shown

[0098] Experimental results show that, compared with the initial sorting, the baseline of the first segment has improved and the overall false signal level has decreased, indicating that this sorting has alleviated the cross-reactivity problem to a certain extent and improved the detection stability.

[0099] 3. Three optimizations

[0100] To further optimize system performance, the antibody arrangement order was readjusted: starting from the sample loading end, the order was PCT, DD, CKMB, NT-proBNP, cTnI, CRP, and Myo. Zero-value samples were tested on the updated chip, with three replicates (Figures 1, 2, and 3). The results are as follows... Figure 3 (PCT: 100-160, D-Dimer: 170-240, CKMB: 260-320, NT-proBNP: 340-400, CRP: 410-490, cTnI: 510-580, Myo: 590-670) as shown.

[0101] The results showed that the overall baseline was more stable, and there were no obvious fluctuations or false signals at each detection site, indicating that the arrangement optimization was effective, effectively reducing cross interference and improving the reliability and accuracy of the system.

[0102] Example 4: Fluorescent Antibody Ratio Optimization Experiment

[0103] 1. Preparation of premixed antibody solution

[0104] Seven fluorescent antibodies were prepared for each of the seven items, with a final concentration of 0.5 mg / ml. Equal volumes of the seven labeled fluorescent antibodies were added to centrifuge tubes and vortexed for 1 minute to ensure thorough mixing, resulting in a mixed antibody solution with an initial ratio of 1:1:1:1:1:1:1. This solution was then stored at 4°C in the dark for later use.

[0105] 2. Initial Test

[0106] A standard mixture containing clinically positive cutoff values ​​for each detection target (cTnI 0.32 ng / ml, PCT 500 pg / ml, CRP 10 mg / L, NT-proBNP 300 pg / ml, Myo 59.6 ng / ml, CK-MB 5.4 ng / ml, D-Dimer 500 ng / ml) was prepared. A detection chip was prepared using a fluorescent antibody solution with an initial ratio (components in a 1:1:1:1:1:1:1 ratio). After adding the standards, the chip was incubated for 15 minutes before detection. The results are shown in Table 1 below. Figure 4 (PCT: 100-160, D-Dimer: 170-240, CKMB: 260-320, NT-proBNP: 340-400, CRP: 410-490, cTnI: 510-580, Myo: 590-670) as shown.

[0107] Table 1. Fluorescence values ​​and detection limits of seven labeled fluorescent antibodies.

[0108]

[0109] The results showed that the Myo signal intensity was relatively appropriate, while the CRP signal was too strong, and the DD (D-Dimer), CKMB, cTnI, PCT and NT (NT-BNPpro) signals were too weak, suggesting that the antibody ratio needs to be further adjusted.

[0110] 3. Iterative optimization of antibody ratio

[0111] Based on the signal intensity analysis results, the proportions of each antibody were gradually adjusted:

[0112] One adjustment: The proportions of DD, CKMB, cTnI, PCT, and NT were increased from 1 to 3, while the others remained unchanged. The results are shown in Table 2. Figure 5 (PCT: 100-160, D-Dimer: 170-240, CKMB: 260-320, NT-proBNP: 340-400, CRP: 410-490, cTnI: 510-580, Myo: 590-670) as shown.

[0113] Table 2. Fluorescence values ​​and detection limits of seven labeled fluorescent antibodies.

[0114]

[0115] The results show that the test results indicate that these five signals are still too low.

[0116] Second adjustment: The proportions of the above five items were further increased to 4, while CRP remained at 1. The results are shown in Table 3 and... Figure 6(PCT: 100-160, D-Dimer: 170-240, CKMB: 260-320, NT-proBNP: 340-400, CRP: 410-490, cTnI: 510-580, Myo: 590-670) as shown.

[0117] Table 3. Fluorescence values ​​and detection limits of seven labeled fluorescent antibodies.

[0118]

[0119] The results show that the PCT, DD, and NT signals are still relatively weak.

[0120] Three adjustments: A more refined ratio was attempted, set to NT(10):DD(4):PCT(4):Myo(2):CKMB(2):cTnI(2):CRP(1), the results are shown in Table 4 and... Figure 7 (PCT: 100-160, D-Dimer: 170-240, CKMB: 260-320, NT-proBNP: 340-400, CRP: 410-490, cTnI: 510-580, Myo: 590-670) as shown.

[0121] Table 4. Fluorescence values ​​and detection limits of seven labeled fluorescent antibodies.

[0122]

[0123] The results show that the signal values ​​have improved, but the PCT, DD, and NT signals are still insufficient, and the CRP signal is too high.

[0124] Four adjustments were made, and the final optimized ratio was determined to be DD(4):CKMB(4):cTnI(4):PCT(3):NT(3):Myo(2):CRP(0.5). The results are shown in Table 5 and Figure 8 (PCT: 100-160, D-Dimer: 170-240, CKMB: 260-320, NT-proBNP: 340-400, CRP: 410-490, cTnI: 510-580, Myo: 590-670) as shown.

[0125] Table 5. Fluorescence values ​​and detection limits of seven labeled fluorescent antibodies.

[0126]

[0127] At this ratio, the signals of various indicators are evenly distributed, and the overall performance reaches the best balance.

[0128] Example 5 Accuracy Measurement

[0129] 1. Preparation

[0130] Three batches of chips were prepared, and the testing equipment was calibrated and in stable operation. A mixed reference standard with two concentration levels (low and high) was selected as the test sample: the low concentration level group (level 1) included D-Dimer 300 ng / ml, CKMB 3 ng / ml, cTnI 0.2 ng / ml, PCT 200 pg / ml, NT-proBNP 100 pg / ml, Myo 30 ng / ml, and CRP 2 mg / L; the high concentration level group (level 2) included D-Dimer 4000 ng / ml, CKMB 60 ng / ml, cTnI 20 ng / ml, PCT 60000 pg / ml, NT-proBNP 20000 pg / ml, Myo 300 ng / ml, and CRP 80 mg / L.

[0131] 2. Testing

[0132] The mixed reference samples at both low and high levels were added to three different batches of microfluidic chips. After incubation, they were tested sequentially on the same detection device. Each batch of chips performed three replicate tests on each concentration sample, for a total of six tests per batch (three for high and three for low), and a total of 18 tests across the three batches. After each test, the fluorescence signal values ​​corresponding to each marker and the converted concentration results were recorded to ensure data completeness and accuracy.

[0133] 3. Calculation

[0134] After all tests were completed for each batch of chips, the obtained data were organized and statistically analyzed. Each level was measured three times, and the test results were recorded as (X...). i ), calculate the relative deviation (B) according to formula (1) respectively. i ).

[0135] B i =(X i -T) / T×100% Formula (1)

[0136] In the formula: B i —Relative deviation; X i —Test value; T—Reference material label value.

[0137] 4. Data Analysis and Evaluation

[0138] Based on the experimental results, in three replicates at two levels across three batches, the relative deviation (B) of the seven markers was [missing information]. i All values ​​were ≤10%. The results are shown in Table 6 below.

[0139] Table 6. Accuracy test results of seven markers.

[0140]

[0141] Example 6 Repeatability Test

[0142] 1. Preparation

[0143] Three batches of chips were prepared, and the testing equipment was calibrated and in stable operation. Two mixed reference standards at low and high concentrations were selected as test samples: the low concentration group included D-Dimer 300 ng / ml, CKMB 3 ng / ml, cTnI 0.2 ng / ml, PCT 200 pg / ml, NT-proBNP 100 pg / ml, Myo 30 ng / ml, and CRP 2 mg / L; the high concentration group included D-Dimer 4000 ng / ml, CKMB 60 ng / ml, cTnI 20 ng / ml, PCT 60000 pg / ml, NT-proBNP 20000 pg / ml, Myo 300 ng / ml, and CRP 80 mg / L.

[0144] 2. Testing

[0145] The mixed reference samples at both low and high levels were added to three different batches of microfluidic chips. After incubation, they were tested sequentially on the same detection device. Each batch of chips performed 10 replicate tests on each concentration sample, for a total of 20 tests per batch (10 for high and 10 for low), and 60 tests in total for the three batches. After each test, the fluorescence signal values ​​and converted concentration results for each marker were recorded to ensure data completeness and accuracy.

[0146] 3. Calculation

[0147] After all tests were completed for each batch of chips, the obtained data were organized and statistically analyzed. For each detection marker, the mean (M), standard deviation (SD), and coefficient of variation (CV) of 10 repeated tests were calculated at both high and low concentration levels. The coefficient of variation was calculated according to formula (2). Finally, the results of the three batches of chips were summarized to evaluate the repeatability of the present invention across different batches.

[0148] CV=SD / M×100% Formula (2)

[0149] Where: CV—coefficient of variation; SD—standard deviation of 10 measurements; M—mean of 10 measurements.

[0150] 4. Data Analysis and Evaluation

[0151] The experimental results show that the coefficient of variation (CV) for repeatability at both levels across the three batches of each biomarker is ≤10%. The results are shown in Table 7 below.

[0152] Table 7. Results of repeatability tests for seven biomarkers

[0153]

[0154] Example 7: Inter-batch precision determination

[0155] 1. Preparation

[0156] Three batches of chips were prepared, and the testing equipment was calibrated and in stable operation. A mixed reference standard with two concentration levels (low and high) was selected as the test sample: the low concentration level group included D-Dimer 300 ng / ml, CKMB 3 ng / ml, cTnI 0.2 ng / ml, PCT 200 pg / ml, NT-proBNP 100 pg / ml, Myo 30 ng / ml, and CRP 2 mg / L; the high concentration level group included D-Dimer 4000 ng / ml, CKMB 60 ng / ml, cTnI 20 ng / ml, PCT 60000 pg / ml, NT-proBNP 20000 pg / ml, Myo 300 ng / ml, and CRP 80 mg / L.

[0157] 2. Testing

[0158] The mixed reference samples at both low and high levels were added to three different batches of microfluidic chips. After incubation, they were tested sequentially on the same detection device. Each batch of chips performed 10 replicate tests on each concentration sample, for a total of 20 tests per batch (10 for high and 10 for low), and a total of 60 tests across the three batches (30 for high and 30 for low). After each test, the fluorescence signal values ​​and converted concentration results for each marker were recorded to ensure data completeness and accuracy.

[0159] 3. Calculation

[0160] After all tests were completed for each batch of chips, the obtained data were organized and statistically analyzed. The mean (M) and standard deviation (SD) of 30 measurements were calculated for both high and low concentration levels, and the coefficient of variation (CV) was obtained according to formula (2). Finally, the results of the three batches of chips were summarized to evaluate the precision performance of the present invention across different batches.

[0161] 4. Data Analysis and Evaluation

[0162] The results are shown in Table 8. According to the experimental results, the coefficient of variation (CV) of each marker at both levels is ≤10%, which meets the requirements.

[0163] Table 8 Results of inter-batch precision determination of seven biomarkers

[0164]

[0165] Example 8: Validation Experiment of Blank Limit (LoB) and Detection Limit (LoD)

[0166] 1. Preparation

[0167] To verify the LoB and LoD performance of this invention, three batches of chips were prepared, including two blank samples and two low-value samples (D-Dimer 85 and 90 ng / ml, CKMB 0.85 and 0.9 ng / ml, cTnI 0.085 and 0.09 ng / ml, PCT 85 and 90 pg / ml, NT-proBNP 20 and 25 pg / ml, Myo 8.5 and 9 ng / ml, CRP 0.35 and 0.4 mg / l). The blank samples were used to evaluate LoB, and the low-concentration samples were used to evaluate LoD. The detection equipment was calibrated and in stable operating condition. Repeated measurements were performed on the four samples for three consecutive testing days.

[0168] 2. Testing

[0169] The LoB claimed in this invention consists of: D-Dimer 10 ng / ml, CKMB 0.2 ng / ml, cTnI 0.02 ng / ml, PCT 50 pg / ml, NT-proBNP 10 pg / ml, Myo 1 ng / ml, and CRP 0.1 mg / l.

[0170] The LOD claimed in this invention is: D-Dimer ≤ 100 ng / ml, CKMB ≤ 1 ng / ml, cTnI ≤ 0.1 ng / ml, PCT ≤ 100 pg / ml, NT-proBNP ≤ 30 pg / ml, Myo ≤ 10 ng / ml, and CRP ≤ 0.5 mg / l.

[0171] On each test day, two blank samples and two low-concentration samples were added to three different batches of microfluidic chips. After incubation for 15 minutes, the samples were sequentially detected on the same device. Each sample was measured four times on each test day, and the testing was conducted continuously for three days, resulting in a total of 48 data sets for each batch (2 blanks × 4 times / day × 3 days + 2 low-concentration samples × 4 times / day × 3 days). The fluorescence signal values ​​of each marker obtained from each detection and their converted concentration results were recorded to ensure data completeness and accuracy.

[0172] 3. Calculation

[0173] The data recorded for each batch were sorted in descending order. The number of blank samples with test results less than or equal to the claimed LOB number was not less than 87%, and the number of low-concentration samples with test results greater than or equal to the claimed LOB number was not less than 87%. The results are shown in Tables 9 to 12.

[0174] Table 9. Blank sample detection results for Myo, CKMB, cTnI, and PCT

[0175]

[0176] Table 10 shows the detection results of blank samples for D-Dimer, NT-proBNP, and CRP.

[0177]

[0178] Table 11 Detection results of low-value samples of Myo, CKMB, cTnI, and PCT

[0179]

[0180] Table 12 shows the detection results of low-value samples of D-Dimer, NT-proBNP, and CRP.

[0181]

[0182] 4. Data Analysis and Evaluation

[0183] Comparing the blank sample results with the claimed LOB (Level of Birth) shows that for Myo, all results in batches one and three are less than the claimed requirement, yielding a percentage of 24 / 24 = 100%; for batch two, 23 results are less than the claimed requirement, yielding a percentage of 23 / 24 = 95.83%, which is greater than the minimum percentage of 87% corresponding to a sample size of 24 in the consensus. For CKMB, batch one has 23 results less than the claimed requirement, yielding a percentage of 23 / 24 = 95.83%; all results in batches two and three are also less than the claimed requirement, yielding a percentage of 24 / 24 = 100%. For cTnI, both batches one and two have 23 results less than the claimed requirement, yielding a percentage of 23 / 24 = 95.83%; all results in batch three are also less than the claimed requirement, yielding a percentage of 24 / 24 = 100%. For PCT batch 1, 23 results were less than the declared requirements, yielding a 23 / 24 = 95.83% percentage; for batches 2 and 3, all results were less than the declared requirements, yielding a 24 / 24 = 100% percentage. For D-Dimer batch 1, 23 results were less than the declared requirements, yielding a 23 / 24 = 95.83% percentage; for batches 2 and 3, all results were less than the declared requirements, yielding a 24 / 24 = 100% percentage. For NT-proBNP batches 1 and 3, all results were less than the declared requirements, yielding a 24 / 24 = 100% percentage; for batch 2, 23 results were less than the declared requirements, yielding a 23 / 24 = 95.83% percentage. For CRP batch 1, 23 results were less than the declared requirements, yielding a 23 / 24 = 95.83% percentage; for batches 2 and 3, all results were less than the declared requirements, yielding a 24 / 24 = 100% percentage. Therefore, the claimed LoB verification is considered successful.

[0184] In the low-concentration sample test results, only one of the seven biomarkers in PCT batch 3 had a result lower than the claimed LoB, yielding a percentage of 23 / 24 = 95.83%. The results for the remaining six biomarkers in each batch were higher than the claimed LoB, with a percentage calculated as 24 / 24 = 100%. Since the test results for all seven biomarkers exceeded the minimum percentage of 87% corresponding to 24 sample sizes in the industry standard, the LoD claim verification of this invention was successful.

[0185] Example 9: Linear Range Determination

[0186] 1. Preparation

[0187] Three batches of chips were prepared. The linear range and measurement range declared in this invention are: D-Dimer [100, 5000] ng / ml, CKMB [1, 80] ng / ml, cTnI [0.1, 30] ng / ml, PCT [100, 80000] pg / ml, NT-proBNP [30, 30000] pg / ml, Myo [10, 400] ng / ml, and CRP [0.5, 100] mg / l. One real high-value clinical sample (H) and one real low-value clinical sample (L) were selected from near the upper and lower limits of this range. The high-concentration and low-concentration samples were mixed in the following proportions: (10H + 0L, 9H + 1L, 7.5H + 2.5L, 5H + 5L, 2.5H + 7.5L, 0H + 10L), with dilutions of 0, 0.1, 0.25, 0.5, 0.75, and 1, respectively. The sample was diluted to six concentrations, and each concentration was measured five times using chips from the same batch. The sample concentrations used in this experiment were: Myo 8 ng / ml, CKMB 0.9 ng / ml, cTnI 0.09 ng / ml, PCT 95 pg / ml, D-Dimer 95 ng / ml, NT-proBNP 28 pg / ml, and CRP 0.4 mg / L; the high concentrations were: Myo 410 ng / ml, CKMB 85 ng / ml, cTnI 32 ng / ml, PCT 81000 pg / ml, D-Dimer 5100 ng / ml, NT-proBNP 31000 pg / ml, and CRP 110 mg / L.

[0188] 2. Testing

[0189] Six prepared concentration samples were added to the chip for detection. After 15 minutes of incubation, they were detected sequentially on the same device. Each sample was measured four times, yielding 24 data sets for each biomarker, for a total of 168 data points for the seven biomarkers. The fluorescence signal values ​​of each biomarker and their converted concentrations were recorded for each test to ensure data completeness and accuracy.

[0190] 3. Calculation

[0191] Calculate the average value of the test results of each concentration of each marker, with X as the dilution and Y as the measured average value, plot a scatter plot and mark the regression equation, and calculate the correlation coefficient; if the linear correlation coefficient r>0.990, the linear range initially meets the requirements.

[0192] 4. Data Analysis and Evaluation

[0193] The linear correlation coefficient r for the three batches is greater than 0.990, and the linear range meets the requirements. The linear relationship representation graph for each marker is shown below. Figures 9-15 As shown

[0194] Example 10 Comparative Verification

[0195] 1. Sample preparation

[0196] This invention was compared and validated with control products (Beckman Coulter ACCESS2 for CKMB, cTnI, PCT, NT-proBNP, and Myo; Beckman Coulter AU680 fully automated biochemical analyzer for DD; and Mindray BC-5390 CRP blood analyzer for CRP). For each target, 30 representative samples were selected from clinical samples (a total of 210 samples), covering low, medium, and high concentration levels to ensure that the concentration gradient covered the linear detection range.

[0197] 2. Testing

[0198] Thirty prepared samples were added to the microfluidic chip of this invention and the sample processing system of the control product. For the product of this invention, after sample addition, incubation was performed for 15 minutes to allow the reaction to proceed fully, followed by instrument detection. Each sample was tested twice in the system of this invention. Simultaneously, the same samples were tested using the control product, strictly following the standard operating procedures of each instrument. Each sample was also tested twice, and the average value was taken as the control result. The detection of the four detection systems (this invention, Beckman, D-Dimer detector, and CRP detector) was carried out simultaneously to reduce potential errors. Data were recorded, with 30 samples for each target, each sample tested twice, and two platforms tested simultaneously, resulting in 840 data points (7 markers × 30 samples × 2 replicates × 2 platforms).

[0199] 3. Calculation

[0200] After testing, all sample data were compiled. For each sample, the average of two repeated tests was calculated, forming 30 paired datasets of the present invention and control products. A scatter plot was plotted with the control product's test results on the X-axis and the present invention's test results on the Y-axis. Linear regression analysis was then performed to calculate the coefficient of determination R. 2 The regression equation is used to assess the correlation between the two.

[0201] In addition, the slope b value of each marker was calculated to determine whether it fell within the interval of 0.95 to 1.05, R0 2 Whether the R-value is not lower than 0.90 is an important indicator for evaluating the consistency between the two methods. If the R-value for all targets is... 2 If the regression slope b is ≥0.90 and within the preset range, then the present invention is considered to have good linear correlation and consistency with the control product. The results are as follows... Figures 16-22 As shown.

[0202] 4. Comparison of test results between the present invention and the control product

[0203] Analysis of detection data from 210 samples for seven biomarkers (Myo, CKMB, cTnI, PCT, D-Dimer, NT-proBNP, and CRP) showed that the detection results of the present invention and the control product were highly correlated on all targets, and the R values ​​of each target were significantly higher. 2 The values ​​all exceeded 0.90, and the slope b values ​​of the regression equation were all within the range of 0.95 to 1.05, which met the preset consistency standard.

[0204] Example 11

[0205] The reagent kits used in the above embodiments are composed of microfluidic chips ( Figure 23 As shown, the microfluidic chip of this invention consists of a quantitative blood collection device, a disposable capillary blood collection needle, and a special diluent. The chip mainly comprises a PMMA substrate chip, an internal filtration membrane, and a plastic cover. Compared to conventional chromatography test strips, this chip utilizes ultrasonic bonding technology to construct an airtight capillary system, containing four key functional units (…). Figure 24 (As shown): Sample loading area, labeled reaction area, time-controlled valve, and microfluidic detection area. The sample loading area ( Figure 25 A blood filtration membrane is placed between the labeled reaction area and the marked reaction area (as shown). Figure 26 As shown, several types of micropillars with conical hemispherical apexes are arranged in a cross pattern for pre-storing fluorescently labeled antibodies; time-controlled valves ( Figure 27 (As shown) is a regular, continuous mountain-shaped structure, coated with a rate-limiting agent to control the reaction time between the sample and the fluorescently labeled antibody; the microfluidic detection area ( Figure 28 As shown, several types of conical hemispherical micropillars are also arranged in a cross pattern to immobilize the capture antibodies for the seven items. The chip is encapsulated using ultrasonic bonding technology to form an airtight capillary detection system.

[0206] After precisely spotting seven capture antibodies onto the microfluidic detection area ( Figure 29 As shown, coating the entire flow channel with antibody protection solution (or only applying it to the sites) can prevent air bubbles from forming in the flow channel and prevent the captured antibodies from interacting with each other after being washed away by the fluid. This ensures the stability of the antibodies in the fluid environment and significantly improves the shelf life and detection stability of the kit.

[0207] This kit is based on the double-antibody sandwich method. A small sample (50 μL) is mixed with diluent and directly added. After blood cells are separated by a blood filtration membrane, fluorescently labeled antibodies are released and bind to their targets in the labeled reaction zone under rate-limited valve control. The antibodies then enter the microfluidic channel and specifically bind to immobilized capture antibodies. Quantitative detection results for seven biomarkers can be obtained within 15 minutes. This design enables simultaneous detection of multiple indicators while ensuring accuracy and repeatability, fully meeting the testing requirements of point-of-care testing (POCT). In the future, it can also realize convenient functions such as home testing and disease risk warning, enabling early detection, early warning, early intervention, and early treatment of diseases.

[0208] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reagent kit for a combined detection of seven cardiopulmonary functions, characterized in that, It consists of a microfluidic chip, a quantitative blood collection device, a disposable capillary blood collection needle, and a diluent; The microfluidic chip consists of a PMMA substrate chip, a blood filtration membrane, and a cover plate. The microfluidic chip includes: a sample dispensing area, a labeled reaction area, a time-controlled valve, and a microchannel detection area.

2. The cardiopulmonary function seven-item combined test kit as described in claim 1, characterized in that, It can simultaneously detect seven indicators: PCT, D-Dimer, CKMB, NT-proBNP, cTnI, CRP, and Myo.

3. The cardiopulmonary function seven-item combined test kit as described in claim 1, characterized in that, The blood filtration membrane is placed between the sample application area and the labeling reaction area.

4. The cardiopulmonary function seven-item combined test kit as described in claim 1, characterized in that, The labeled reaction zone has micropillars with conical hemispherical tops arranged in a cross pattern, and the micropillars are coated with fluorescent antibodies for seven indicators.

5. The cardiopulmonary function seven-item combined test kit as described in claim 1, characterized in that, The time-controlled valve has a regular, continuous mountain-shaped structure and is coated with a rate-limiting agent.

6. The cardiopulmonary function seven-item combined test kit as described in claim 1, characterized in that, The microfluidic detection area has micropillars with conical hemispherical tops arranged in a cross pattern, and the micropillars are coated with capture antibodies for seven indicators.

7. The cardiopulmonary function seven-item combined test kit as described in claim 6, characterized in that, The seven capture antibodies are arranged in the following order from the sample application end: PCT, D-Dimer, CKMB, NT-proBNP, CRP, cTnI, and Myo.

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