Lateral chromatography test strip for confirming sample sufficiency, preparation device, diagnostic system and detection method

Abstract

The invention discloses a lateral chromatography detection test strip based on a depletion-capture structure, a sample preparation device, a diagnosis system and a detection method. The near end of the detection strip is provided with a gasket for sample application, the gasket comprises a perforated filter membrane and a red blood cell agglutination reagent, and the filter membrane can realize efficient plasma release and transmission; and the detection line, the albumin depletion line and the contrast line are sequentially arranged on the downstream membrane strip, and are provided with a double-gradient internal standard nano-probe area for signal calibration. The sample preparation device can independently and quickly obtain plasma from whole blood or filter oral liquid, and the diagnostic system works cooperatively to confirm sufficiency of the sample. According to the method, a detection line and a test line are taken as reference thresholds, and a calibration curve is combined to carry out quantitative analysis on a target. According to the invention, the accuracy and the stability of complex sample detection are improved, and the kit is suitable for in-vitro immunoassay in a POCT (Point-of-Care Testing) environment.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a lateral chromatography test strip, sample preparation device, diagnostic system and detection method based on red blood cell depletion and plasma fractionation filtration. It is specifically applied to the rapid qualitative or quantitative detection of target substances in oral swabs, whole blood or plasma samples, and belongs to the technical fields of in vitro diagnostics (IVD), point-of-care testing (POCT) and immunochromatographic analysis. Background Technology

[0002] Point-of-care (POC) testing technologies have their own strengths and weaknesses in the fields of nucleic acid and antigen / antibody detection: PCR and isothermal amplification can amplify pathogen DNA / RNA signals billions of times and are considered the gold standard, but they are very picky about the operating environment and purification steps. In particular, hemoglobin in whole blood has a strong inhibitory effect on the amplification reaction, forcing POC nucleic acid detection to rely on expensive instruments and complex pretreatment. In contrast, lateral flow immunochromatography (LFA) has a simple structure and intuitive readings, and has been widely used to detect serum antibodies, viral antigens or host protein biomarkers. However, when the sample is switched from high-concentration whole blood to low-concentration oral fluid with huge differences, the sensitivity and quantitative consistency drop rapidly.

[0003] In today's world, where clinical and home testing scenarios are constantly shifting, users often desire a single reagent that can produce high concentrations of antibodies / antigens using 5–100 μL of finger-prick blood, while also allowing for painless testing using approximately 150 μL of oral swab extract. However, the abundance of target analytes differs by one to two orders of magnitude between these two types of bodily fluids, and the concentration in oral samples fluctuates significantly, making direct comparison with blood sample volumes impossible. The lack of a reliable sample volume reference mechanism forces developers to create separate tests for blood and oral fluid, preventing the sharing of a single platform and reading threshold. The two samples require entirely different preparation processes: whole blood must be filtered through a small-pore membrane to remove red blood cells, resulting in slow flow rates and limited processing volume; while oral fluid contains almost no red blood cells but includes mucus and large particles, requiring a large-pore filter layer for faster flow rates but larger volume. This contradiction between filter size and throughput makes it difficult for existing POCLFA (Polymer-Assisted Clinical Trial) strips to handle both in a single test strip. The industry currently lacks a universal strategy for integrated processing of blood and oral fluid, limiting the development of truly user-convenient, multi-scenario point-of-care diagnostic products.

[0004] The prior art disclosed in this application mainly focuses on three approaches: ① paper-based μPADs utilizing red blood cell agglutination; ② single-layer depth filtration fiber pads (… Passive mechanical screening is performed (e.g.); ③ An albumin sufficiency line is added to the detection membrane to determine the sufficiency of the sample.

[0005] Existing technology 1: Paper-based RBC agglomeration μPAD

[0006] In 2012, Yang et al. reported a device that applied antibodies onto wax printing paper, utilizing the agglutination of red blood cells to retain them in situ and exudate plasma. The separation area and the colorimetric reaction area were integrated onto a single μPAD, enabling a proof-of-concept demonstration of finger-prick blood testing and reading. ([RSC Publication][1], [ResearchGate][2])

[0007] Prior art 2: Single-layer depth filter pad

[0008] of The HV series provides commercially available plasma separation media for lateral flow kits. The product uses a single-layer depth filtration structure formed by random stacking of natural / synthetic fibers, physically separating whole blood through a pore size gradient. Its selling points are rapid blood absorption and plasma filtration within a single layer, making it widely integrated into disposable cartridges for pregnancy testing, inflammatory marker detection, etc. ([Ahlstrom][3],[pdf.medicalexpo.com][4])

[0009] Existing technology 3: Patent for a detection strip with a sufficient sample volume line

[0010] Several companies have recently proposed printing a human albumin reaction line onto a nitrocellulose membrane for use as a Sample Adequacy Line (SAL). If albumin staining is insufficient, it alerts the operator that the sample volume is too small or the sample processing has failed; if staining is sufficient, the target line can be read. This concept was published in a patent family in 2023 and has been extended to alternatives such as lactoferrin for adequate labeling. ([Google Patents][5], [Google Patents][6])

[0011] Three major technical problems that still need to be solved by existing technologies:

[0012] (i) Passive agglomeration μPAD is prone to clogging and flow rate is difficult to control.

[0013] Paper-based methods rely entirely on capillary actuation: once red blood cells form large clumps, they clog the channels, leading to highly volatile plasma yield and detection time; this is especially true for clinically hyperlipidemic or viscous samples. ([PMC][7], [Cytiva][8]) These devices also lack any rinsing or pulsed flow-assisted mechanisms, and residual red blood cell fragments and free hemoglobin easily interfere with subsequent colorimetric analysis, limiting quantitative accuracy. Quantitative accuracy during the reaction process is limited. Using an open-weave filter membrane combined with red blood cell agglutination technology, along with a buffer rinsing option, can significantly reduce clogging and red blood cell fragmentation, fundamentally solving problems related to clogging, flow control, and sample yield.

[0014] (ii) Single-layer mechanical filter pads have poor compatibility and significant protein loss.

[0015] The pad needs to compromise between pore size retention and protein permeability, resulting in 10–30% irreversible adsorption of low or medium molecular weight proteins; when using high-viscosity oral extracts, the flow rate decreases significantly or even stops. ([PubMed][9],[MDPI]

[10] ,[Cytiva][8]) The open-woven filter membrane significantly reduces non-specific protein adsorption through a dual mechanism of antibody-induced low-shear agglutination and hydrophilic nanofiber traction of plasma, and allows the same strip to be used for both whole blood and oral fluids.

[0016] (iii) Existing SAL only detects sample volume and cannot correct for optical bias or abnormal monitoring process.

[0017] The albumin sufficiency line in the patent only provides a single threshold judgment of sufficiency or insufficiency, which cannot offset backlight noise caused by different mobile phone cameras, ambient light, and batch differences; it is also unable to identify operational faults such as flow interruption and side leakage. ([Google Patent][5], [MDPI]

[11] , [PMC]

[12] ) The new method combines a dual-gradient internal standard nanoprobe with an appropriate line, and provides two known optical density references in the same region, thereby significantly improving the reliability of process anomaly alarms and on-site quantitative analysis.

[0018] [1]:ttps: / / pubs.rsc.org / en / content / articlelanding / 2012 / lc / c1lc20803a? utm_source=chatgpt.com"Integrated separation of blood plasma from whole...-RSC Publishing"

[0019] [2]:https: / / www.researchgate.net / publication / 51806943_Integrated_separation_of_blood_plasma_from_whole_blood_for_microfluidic_paper-based_analytical_devices? utm_source=chatgpt.com"(PDF)Integrated separation ofblood plasma from whole blood for..."

[0020] [3]:https: / / www.ahlstrom.com / products / medical-life-sciences-and-laboratory / lateral-flow-test-pads / plasma-separation-pads / ?utm_source=chatgpt.com"Lateral flow plasma separation pads-Ahlstrom"

[0021] [4]:https: / / pdf.medicalexpo.com / pdf / ahlstrom-munksjoe / cytosep-plasma-separation-media / 69479-223998.html?utm_source=chatgpt.com" PlasmaSeparation Media- "

[0022] [5]:https: / / patents.google.com / patent / KR20230145131A / en?utm_source=chatgpt.com"KR20230145131A-Lateral flow assay with sample adequacy line..."

[0023] [6]:https: / / patents.google.com / patent / AU2020285366A1 / en?utm_source=chatgpt.com"Lateralflow test strips with competitive assay control-GooglePatents"

[0024] [7]:https: / / pmc.ncbi.nlm.nih.gov / articles / PMC11068687 / ?utm_source=chatgpt.com

[0025] "Sample-to-answer lateral flow assay with integrated plasma..."

[0026] [8]:https: / / www.cytivalifesciences.com / en / us / knowledge-center / diagnostic-assays-blood-separ ation-using-depth-filters?srsltid=AfmBOoqcOyE51WgS25Q3h4YPeztL5TqIheCpra0NEjO8Czoxfo PSm9KP&utm_source=chatgpt.com"Diagnostic assays:How depth filters work in blood separation-Cytiva"

[0027] [9]:https: / / pubmed.ncbi.nlm.nih.gov / 34677311 / ?utm_source=chatgpt.com"High-Performance Passive Plasma Separation on OSTE Pillar Forest"

[0028]

[10] :https: / / www.mdpi.com / 2079-6374 / 11 / 10 / 355?utm_source=chatgpt.com

[0029] "High-Performance Passive Plasma Separation on OSTE Pillar Forest"

[0030]

[11] :https: / / www.mdpi.com / 2079-6374 / 13 / 6 / 623?utm_source=chatgpt.com"Proof-of-Concept:Smartphone-and Cloud-Based Artificial...-MDPI"

[0031]

[12] :https: / / pmc.ncbi.nlm.nih.gov / articles / PMC11763061 / ?utm_source=chatgpt.com"Machine Learning-Based Quantification of Lateral Flow AssayUsing..." Summary of the Invention

[0032] In response to the urgent need for rapid quantitative analysis of whole blood and oral swab samples in point-of-care testing (POCT), existing paper-based microfluidic devices (μPADs) or single-layer mechanical filter pads are insufficient to simultaneously achieve complete removal of red blood cells, intact preservation of proteins, and stable flow rate control. This results in large fluctuations and poor repeatability of test results, limiting their large-scale promotion in primary healthcare and home monitoring scenarios.

[0033] The core technical problems to be solved by this invention include: (1) passive agglutination μPAD is easily blocked by red blood cell aggregation, and the flow rate is uncontrollable; (2) monolayer The filter pad is difficult to balance between retaining red blood cells and passing through target proteins, which can easily cause 10-30% irreversible protein adsorption and poor compatibility with high-viscosity oral extracts; (3) The existing sample sufficient line only gives a single threshold judgment, which cannot correct for different light sources, cameras and batch differences, and cannot detect abnormal operation such as flow interruption or side leakage in real time.

[0034] To overcome the above-mentioned defects, this invention provides a series of synergistic technical solutions: First, the lateral chromatography test strip introduces a two-layer hierarchical depletion-capture structure of open-woven filter membrane + erythrocyte agglutination reagent at the first end, and attaches a hydrophilic nanofiber layer to the back of the filter membrane; Second, test lines, albumin depletion lines and dual-gradient internal standard nanoprobe regions are arranged on the membrane strip. At the same time, corresponding sample preparation devices, diagnostic systems and methods for confirming sample adequacy are also proposed, forming a complete closed loop from sample processing to result reading.

[0035] The open-structured woven filter membrane retains only large aggregates of erythrocytes under low-shear induction by erythrocyte agglutinin; the hydrophilic nanofiber layer reduces local viscous resistance through capillary traction; and the optional added washing buffer can stably deliver quantitative plasma components to downstream systems. For the first time, the albumin depletion line is coupled to two levels of optical density internal standards within the membrane strip, allowing for the simultaneous output of sample volume thresholds and calibration curve references in the same region.

[0036] The beneficial effects achieved by this invention are mainly reflected in the following aspects: ① The triple synergistic mechanism of agglutination-filtration-rinsing effectively avoids red blood cell blockage and flow control loss; ② It is compatible with whole blood and high-viscosity oral fluids in a single operation, significantly reducing non-specific protein adsorption and maintaining a high recovery rate; ③ The dual-gradient internal standard and albumin depletion line work together to automatically determine sample sufficiency, generate a quantitative calibration curve within 30 seconds, and monitor process abnormalities such as flow interruption and side leakage in real time, making the bedside testing results more robust and traceable.

[0037] This invention, through its innovative design integrating depletion-capture sample preparation structure, optional rinsing, and dynamic quality control, successfully overcomes the bottlenecks in the entire process from rapid plasma separation to accurate result interpretation. It is applicable to diverse POCT scenarios such as infectious diseases, inflammatory markers, and multi-target combined screening, and has outstanding comprehensive advantages such as simple operation, low cost, batch manufacturing friendliness, and strong scalability.

[0038] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0039] First, the present invention provides a diagnostic device suitable for oral samples and blood or plasma samples, which is suitable for point-of-care (POC) testing of both oral and blood samples, and has an internal control device to ensure that the oral sample collection method can optimize the test results.

[0040] The advantages and benefits of this invention lie in the design of a portable testing device (POC) for alternating use of two main clinical sample types (antibodies and antigens against blood biomarkers) – whole blood / plasma or oral fluids (such as saliva or intergingival fluid), while incorporating procedural controls to ensure sufficient protein equivalent to plasma when using the more variable oral fluid sample type, thereby ensuring the validity of the test results. This is particularly useful for point-of-care testing for home use or by undertrained healthcare workers, as some individuals may prefer oral fluid samples over finger-prick blood or venous whole blood samples.

[0041] For point-of-care testing (POC) of any specific biomarker found in plasma (such as hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B core antibody, HIV antibody, and hepatitis C antibody), whole blood / plasma samples may be more sensitive than oral fluid samples. However, it is generally believed that even with lower POC sensitivity, the wider acceptance and use of oral fluid home testing leads to more successful diagnostic tests (total diagnostics). In contrast, even with higher POC sensitivity, whole blood testing has a narrower range of applications. By having a single test that can be used with both whole blood and oral fluid, end-users can always choose the most appropriate test for their specific circumstances, thereby maximizing overall diagnostics.

[0042] Second, many point-of-care (POC) diagnostic tests are currently available to detect disease-related molecules commonly found in blood, such as antibodies against HIV or hepatitis C virus, or hepatitis B surface antigen. Consumers tend to prefer POC tests using samples such as saliva or oral swabs rather than blood samples, but current tests designed for blood samples cannot be used with oral samples, and vice versa. The technical solution of this invention allows for testing using both blood and oral / swab samples, while also controlling the presence of sufficient blood / plasma equivalents in the oral or swab sample, thereby expanding the commercial market size and improving diagnostic efficacy due to the wider acceptance of oral / swab sample testing in the general population. Further application of this solution ensures the quality of swab samples used to diagnose infections such as SARS-CoV-2 and influenza by controlling the presence of sufficient respiratory epithelial cell equivalents in the oral or swab sample, thus gaining a competitive advantage and enhancing the confidence of customers and healthcare institutions.

[0043] China is a world leader in point-of-care (POC) device manufacturing, possessing a mature supply chain covering all components, including plastics, membranes, and chemical and biological reagents (such as antibodies and recombinant proteins). However, there is a lack of coordination between POC products for blood samples and those for oral or swab samples, leading to inefficiencies in test development, manufacturing, validation, marketing, and implementation. The technical solution of this invention achieves coordination and unification between blood testing and oral / swab testing by ensuring that oral / swab samples can function correctly as substitutes or equivalents for blood samples. This makes all steps in the development, manufacturing, and validation of POC tests more efficient, providing a competitive advantage for domestic and international manufacturers adopting this technical solution.

[0044] Blood-based point-of-care (POC) tests have been used to detect HIV, hepatitis B, hepatitis C, and syphilis (to name just these four) for 20 to 30 years. These tests are widely used in screening to help prevent mother-to-child transmission of HIV, hepatitis B, and syphilis (a triple elimination target of the World Health Organization). However, blood samples are less convenient than oral or swab samples, so these POC tests are not widely accepted by patients and healthcare professionals (unlike swab tests for COVID-19). Only in recent years have oral sample POC tests for HIV and hepatitis C been developed and approved, while oral sample POC tests for hepatitis B and syphilis are not yet available. This is mainly because oral / swab sample tests have lower clinical sensitivity than blood sample tests, as the molecules being tested (antibodies or antigens) are usually present in plasma, and the levels of these antibodies / antigens in saliva or oral swab samples largely reflect plasma seepage from the gingival sulcus. Proper oral swab collection techniques can yield sufficient plasma from the site, but current point-of-care (POC) test kits cannot control for proper collection. Incorrect swab collection techniques can significantly reduce the clinical sensitivity of POC tests, even if oral swab tests and blood tests have the same analytical sensitivity (antibody / antigen detection limits). This invention addresses this long-standing problem by providing a method to identify invalid tests with insufficient plasma equivalent content (positive results are still considered valid). Invalid tests identified in this way need to be repeated without affecting the clinical sensitivity of the test. Current oral swab tests lack validity controls based on plasma equivalent content, leading to false negatives and reduced clinical sensitivity. Using this method, for the first time, oral swab-based testing for various diseases such as hepatitis B and syphilis can be performed, while also improving the detection of HIV and hepatitis C. The same method will improve the performance of blood-based POC tests (ensuring sufficient blood sample is added) and oral / swab tests for diseases such as COVID-19 and influenza (ensuring sufficient respiratory epithelial cells are added).

[0045] For point-of-care (POC) testing technologies based on oral swabs, the reliability of detecting blood biomarkers such as HIV, HCV, HBV, and syphilis is generally questioned due to operator skill levels and significant variations in the equivalent plasma volume used for testing. In the United States, oral HIV testing products like OraSure, while demonstrating high sensitivity in controlled studies, have failed to reproduce this result in real-world applications. Despite this limitation and lower sensitivity, oral HV testing products have been approved for sale and use in many countries to leverage their wider acceptance, increased testing capacity, and greater case diagnostics, despite the lower sensitivity of a single test. This invention overcomes this technical bias by determining whether a sufficient quantity of sample was collected (and used for testing). Similarly, when used for point-of-care blood testing or oral / swab testing for respiratory infections such as SARS-CoV-2 or influenza, this invention overcomes the technical bias regarding the reliability of these tests, particularly for home use or use by non-professionals. For example, when point-of-care testing (POC) for SARS-CoV-2 is performed by an individual for work or visitation, there is no guarantee that the testing procedure will be performed correctly to obtain a valid negative result. That is, an individual may intentionally collect insufficient samples to avoid the consequences of a positive result. This invention avoids this situation because individuals must collect sufficient blood (for hand-based blood testing), an equivalent amount of plasma (for hand-based oral / swab testing, equivalent to hand blood testing), or respiratory epithelial cells (for oral / swab-based respiratory disease testing) for a negative test result to be considered valid. This improves the overall reliability and usability of point-of-care testing. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the operation process of the filtration and drying plasma device provided in the embodiment of the present invention;

[0047] Figure 2 This is a schematic diagram of the first control test line method provided in the embodiment of the present invention;

[0048] Figure 3 This is a schematic diagram showing representative results of the first control test line method provided in the embodiments of the present invention;

[0049] Figure 4 This is a schematic diagram of the second control test line method provided in the embodiment of the present invention;

[0050] Figure 5 This is a schematic diagram showing representative results of the second control test line method provided in the embodiments of the present invention;

[0051] Figure 6 This is a schematic diagram showing representative results of the third control test line method provided in the embodiments of the present invention;

[0052] Figure 7 This is a schematic diagram illustrating representative results of the preferred test method for detecting HIV antibodies provided in the embodiments of the present invention;

[0053] Figure 8 This is a schematic diagram of the C-type dual-pass detection provided in an embodiment of the present invention;

[0054] Figure 9 This is a schematic diagram of the bidirectional HIV / syphilis antibody (left) and hepatitis B surface antigen (HBsAg) detection (right) provided in an embodiment of the present invention;

[0055] Figure 10 This is a representative schematic diagram of the results provided in this embodiment of the invention, which uses a third control test line method to detect the nucleocapsid antigen of the novel coronavirus in order to diagnose pneumonia caused by the novel coronavirus, targeting the intracellular antigen (glyceraldehyde-3-phosphate dehydrogenase).

[0056] Figure 11 This is a schematic diagram of the three-dimensional structure of the equipment test strip made of nitrocellulose membrane or similar material provided in the embodiments of the present invention;

[0057] Figure 12 This is a schematic diagram of a diagnostic device for detecting target substances in oral samples and blood or plasma samples, provided by an embodiment of the present invention;

[0058] Figure 13 This is a schematic diagram of a three-dimensional structure of a test strip made of nitrocellulose membrane or similar material, provided in an embodiment of the present invention.

[0059] Figure 14 This is a schematic diagram of another diagnostic device for detecting target substances in oral samples and blood or plasma samples provided in an embodiment of the present invention;

[0060] Figure 15 This is a schematic diagram of a diagnostic component for detecting target substances in oral samples and blood or plasma samples, provided by an embodiment of the present invention;

[0061] Figure 16 This is a schematic diagram of another diagnostic component for detecting target substances in oral samples and blood or plasma samples provided in an embodiment of the present invention;

[0062] In the diagram: 1. Plastic backing; 2. Sample application pad; 3. Reagent mixing pad; 4. Equipment test strip; 5. Absorbent pad; 6. Test line; 7. Control line; 8. Equipment box; 9. Observation window; 10. Sampling and buffer solution loading wells; 11. Spare sample and buffer solution loading wells; 12. Exhaustion control line; 13. Control test line; 14. Solid opaque structure; 15. Lower part of the equipment box. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0064] In routine screening using a combination of oral swabs and fingertip capillary blood samples, the clinical and in vitro diagnostic industries have long been hampered by significant differences in sample composition, lengthy processing procedures, and high reliance on operator skills. Particularly when the target is a low-abundance inflammatory factor or early screening protein marker, excessive albumin and erythrocyte lysis products can trigger a strong matrix effect in the lateral chromatography channel, leading to a sharp drop in signal-to-noise ratio and making on-site quantification difficult. These obstacles are further amplified in emerging application scenarios such as large-scale population cohorts, gene-environment interaction monitoring, and home testing, necessitating a fully automated, end-to-end system capable of freely switching between trace amounts of plasma and oral exudate and automatically completing matrix purification.

[0065] The integrated sampling-pretreatment device of this invention uses an open-pore high-density porous cellulose membrane as its core. It loads IgG or IgM antibodies that specifically recognize human erythrocyte glycoprotein A antigen via a non-covalent binding method (drying treatment). Utilizing the rehydration and multivalent properties of the antibodies, a self-aggregation reaction is initiated within seconds of the addition of the liquid sample. After being captured, erythrocytes form a through-cell microplastic, and the plasma they carry permeates through the membrane under lateral capillary force, minimizing cell membrane rupture and thus reducing downstream chromatographic channel blockage and background scattering signals from the source.

[0066] Plasma permeating through the filter membrane carries the target low-abundance protein and endogenous high-abundance albumin, then flows into a finely constructed three-line detection zone. The upstream section contains a small-molecule capture zone with an anti-human albumin affinity peptide, providing the first quality control zone for result interpretation. The subsequent test line is immobilized with a monoclonal capture antibody and a signal amplification complex (gold-labeled / fluorescent microspheres optional), enabling quantitative or qualitative identification of the target through immune sandwich or competitive modes.

[0067] To overcome the random influence of external temperature, humidity, and sample differences on chromatography speed, this system incorporates a dual-validation quality control line at the end of the detection zone. Firstly, the constant binding of the goat anti-mouse secondary antibody to the labeled probe reflects whether chromatography has proceeded throughout the entire process. Secondly, a residual albumin capture band is reserved downstream of the anti-albumin loss zone. When sample processing is insufficient or the protein concentration falls below the threshold, this band immediately develops color, alerting the operator to resample and preventing false negative results from entering the decision-making process.

[0068] The reading process employs an integrated miniature spectral sensor array, utilizing multi-point sampling and time-resolved algorithms to correct for membrane surface non-uniformity, outputting semi-quantitative concentration values ​​after R-logit conversion. The quantitative assessment module incorporates dynamic calibration curves based on multi-source data, automatically switching the judgment window according to the baseline levels of different populations, ensuring consistent and comparable results across diverse application scenarios such as vaccine response monitoring, chronic disease management, and drug efficacy tracking.

[0069] The entire system significantly shortens the manual intervention chain in sample pretreatment during industrial implementation, reduces reliance on dedicated centrifugation, cold chain, and high-precision pipetting equipment, and enables mixed oral and fingertip screening to be implemented in primary healthcare settings, remote mobile clinics, and even home environments. Its working mechanism balances the engineering feasibility and theoretical rigor of high-throughput testing, providing a replicable technological paradigm for the in vitro diagnostics industry under the trends of decentralization and multi-fluid fusion.

[0070] The first aspect of this invention is sample preparation using erythrocyte agglutination. It is known in the art that antibodies against glycoprotein A or other erythrocyte (RBC) surface antigens (such as type B / band 3 protein) can be used to agglutinate erythrocytes. If these antibodies are contained in an open-woven (macroporous) filter membrane (such as Ahlstrom 8951 or 8950), large volumes of whole blood can be processed rapidly without causing filter clogging (PMID: 30700508). The novelty of this invention lies in using the same sample pad material (an open-woven filter membrane with agglutinating antibodies, rapid flow rate) for oral fluid samples. For example, Ahlstrom 8950 and 8951 filters are recommended for oral fluid samples (separation of mucin), nasal samples, and serum / plasma, but not for whole blood; while Ahlstrom 142, 1668, 1660, 1662, and 1663 filters are recommended for whole blood, but not for oral fluids. By using filter membranes with agglutinating antibodies, such as the Ahlstrom 8950 or 8951, the same filter membrane (antibody-treated sample pad) can be used for whole blood and oral fluid samples, and of course, serum / plasma. However, for this method to be useful, the relative content of equivalent plasma proteins in blood and saliva samples must be controlled, which depends on the second aspect of the embodiments of the present invention.

[0071] A second aspect of this invention is a method for controlling the equivalent amount of plasma protein collected in an oral fluid sample. Albumin is a protein that is very abundant in human plasma, with a normal concentration of approximately 45 mg / mL. Therefore, a 10 μL whole blood sample contains approximately 5 μL of plasma or 22.5 μg of albumin. By incorporating a test line into a point-of-care testing device that indicates the presence of a similar total amount of albumin in a blood sample (10 μL containing approximately 22.5 μg of total albumin) or an oral fluid swab sample (e.g., 100 μL containing at least 2 μg of total albumin, more preferably at least 20 μg), point-of-care testing provides procedural control to ensure that there is sufficient plasma in the oral fluid sample to obtain valid results. The same procedural control also ensures that the blood or plasma sample has been correctly applied. This method can use any plasma protein that has a relatively stable value in the subject, such as albumin, transferrin, IgG or IgM antibodies, but plasma proteins with large variability (host reactive proteins), such as C-reactive protein and ferritin, should not be used, nor should plasma proteins with common genetic defects, such as IgA deficiency.

[0072] Using anti-erythrocyte antibodies for agglutination reactions is not a new method; the novelty of this embodiment lies in applying the same method to the preparation of a sample device that can be used for whole blood or oral fluid samples, and controlling the addition of the sample through the second aspect of this embodiment.

[0073] The same method can also be used in sample preparation devices for oral or whole blood samples for nucleic acid amplification detection, so that these tests can be optimized for blood or oral fluid tests according to end-user preferences.

[0074] Figure 1 The Filter Drying Plasma Device (PMID: 30700508) illustrates a schematic diagram of the rapid separation of large volumes of whole blood using an open-cell fiber filter paper (Ahlstrom 8951) pad impregnated with anti-erythrocyte antibodies (such as anti-glycoprotein A antibodies). Figure 1 A: Device components. Figure 1 B, C: Add 100 μL of whole blood to the sample pad. Within 3 minutes at room temperature, red blood cells agglutinate, while plasma (and white blood cells) begin to migrate along the rest of the 8951 pad, into the nitrocellulose, and then into the Whatman 903 plasma collection filter paper. Figure 1 D). White blood cells are trapped at the interface between 8951 and nitrocellulose (nitrocellulose has a smaller pore size than white blood cells), but because their number is relatively small compared to red blood cells, they do not impede the flow of samples or other reagents. Figure 1 E: Additional buffer is added to drive away residual plasma from agglutinated red blood cells, thereby maximizing plasma collection on the Whatman 903 plasma collection filter membrane. Figure 1The amount of plasma recovered in F). For oral fluid samples, the same sample pad (based on the Ahlstrom 8951 or a similar open-woven fabric filter) can also be used. Anti-glycoprotein A antibodies do not function in this sample preparation but do not cause any harm; therefore, sample pads integrated with anti-erythrocyte antibodies can be used for both sample types, thus integrating oral fluid and whole blood sample testing. Note that the device shown here (filtered dried plasma, manufactured as a VL-Plasma device) is only used for plasma separation and drying, but this arrangement of the sample pad with the nitrocellulose membrane (containing test lines, control lines, and other lines on a longer strip) and the absorbent pad is typical of all lateral flow assays.

[0075] Figure 2 and Figure 3 An exemplary control line method is demonstrated for detecting the presence of a sufficient amount of equivalent plasma in an oral fluid sample, while also ensuring that such a sample has been added when the preferred sample type is blood or plasma. It is noteworthy that current blood-based detection methods lack this feature and cannot provide procedural controls to ensure that a sample has been added (i.e., the same procedural control result would be obtained even if water or buffer solution were added).

[0076] Figure 2 middle:

[0077] A: Sufficient equivalent plasma (albumin) in the sample = sample albumin competition, and weak / no control test line visible.

[0078] B: Low / no plasma equivalent (albumin) in the sample = reduced competition between sample albumin and the strong control test line.

[0079] Comparison of control test line intensity = approximate estimate of plasma equivalent volume

[0080] Figure 3 middle:

[0081] A: Schematic diagram of lateral flow test with test lines (XYZ) and control lines.

[0082] B: Example results of high, normal, low / absent plasma equivalent (albumin) in the sample.

[0083] Since the XYZ analytes were not present in the test sample, high and normal results were negative, but low results were invalid.

[0084] For control test lines, it is important to avoid potential cross-reactions between reagents used to detect plasma load and the target analyte. Figure 2 and Figure 3In the example shown, colloidal gold labeled with human serum albumin is mixed with the sample (e.g., when the sample is added to the sample pad). The control test line consists of an anti-human serum albumin antibody. In the presence of whole blood or plasma samples, or in oral fluid samples with sufficient plasma equivalent, albumin in the sample will compete with the gold-albumin conjugate, resulting in a weaker or absent control test line. Figure 2 A). If no sample is added (due to operational error), or if the plasma equivalent in the oral fluid sample is insufficient, the competition for the gold-albumin conjugate is reduced or absent, resulting in a stronger control test line. Figure 2 B), which would be considered an invalid test result. However, the drawback of this method is that a positive reaction (the appearance of a visible line on the control test line) indicates an invalid test result (insufficient sample size), which contradicts the usual understanding of procedural control.

[0085] exist Figure 4 and Figure 5 In the example shown, a solution to this problem or deficiency is presented: instead of using the anti-albumin test line as a control test line, it is used to consume the albumin-gold conjugate in the sample when there is insufficient competition from albumin (as previously described). The unbound albumin-gold conjugate is then captured on a second test line, which serves as the control test line. In this case, the control gold conjugate contains albumin and a second, unrelated antigen, such as chicken IgY (i.e., each gold particle is bound to a mixture of albumin and chicken IgY, in this case, albumin-IgY-gold). Figure 4 A: with Figure 2 Similarly, if a blood or plasma sample is present, or if the oral fluid sample contains sufficient plasma equivalent, the albumin-IgY-gold conjugate will be competitively eliminated by albumin in the sample and will not bind to the anti-albumin consumption line, but will continue to flow along the device. The second detection line (control detection line) contains anti-chicken IgY antibody. If the albumin-IgY-gold complex is competitively eliminated by albumin in the sample on the anti-albumin detection line, it will react on the anti-IgY detection line, thus producing a strong positive detection line signal. In this case, a valid result (sufficient plasma or plasma equivalent in the sample) will form a strong control detection line (…). Figure 4 A). If no sample is added, or the plasma equivalent in the oral fluid sample is insufficient, then the albumin-IgY-gold complex will not compete for binding with the anti-albumin test line, nor will it bind with the control test line. Figure 4 B). The advantage of this method is that a positive reaction (visible band on the control test line) indicates that the test result is valid (sufficient sample), which makes the understanding of the procedure control intuitive and clear.

[0086] Figure 4 middle:

[0087] A: Sufficient plasma equivalent (albumin) in the sample = strong control detection line, weak / lossless line

[0088] B: Low / absent plasma equivalent (albumin) in the sample = weak / no control detection line, strong exhaustion line

[0089] Comparison of the intensity of the depletion line with the control line = accurate estimation of plasma equivalent volume.

[0090] Figure 5 middle:

[0091] A: Schematic diagram of a transverse flow test with test leads (XYZ), loss lines, and control lines.

[0092] B: Example results of high, normal, low / absent plasma equivalent (albumin) in the sample.

[0093] Since the XYZ analytes were not present in the test sample, high and normal results were negative, but low results were invalid.

[0094] Samples with high, normal, or low plasma equivalent levels.

[0095] It is important to note that comparing the intensity of the exhaustion line with that of the control test line provides the most accurate estimate of the equivalent plasma volume. The relative intensities of the lines can be adjusted using methods well-known in the art (including, but not limited to, the amount of antibody in the test line, the amount of antigen on the gold marker, and the optical density of the gold marker added to the test) to aid visual interpretation. As a preferred example, when using oral fluid samples, if the intensities of the control and exhaustion lines are approximately equal, it indicates that a preferred or minimum equivalent plasma volume has been used (valid test). A stronger control line than the exhaustion line is acceptable (valid test) because it means that more than the preferred or minimum amount of equivalent plasma has been added to the test; a stronger exhaustion line than the control line is unacceptable (invalid test) because it means that less than the preferred or minimum amount of equivalent plasma has been added to the test. When using whole blood or plasma, a valid test result should always show the control line as darker than the exhaustion line. However, if no sample is added due to operational error, the exhaustion line may be darker than the control line, or the control line may be lighter or absent, indicating an invalid test result.

[0096] Optionally, the anti-albumin (depletion) test line can be placed at the beginning of the test reaction band, i.e., before the start of the observation window, or it can be obscured or hidden by setting a solid feature within the observation window, so that the operator cannot see the result of this test line and can only see the control test line. The advantage of this is that the operator will only see the control test line, and only a positive reaction at this line indicates a valid test, which is consistent with the conventional interpretation of point-of-care (POC) tests widely used for decades. An example of this format (control test line method) is shown in... Figure 6 The diagram is presented in the form of an illustration, in which the features of the reagent kit obscure the view of the depletion line.

[0097] Figure 6 middle:

[0098] A: Schematic diagram of a sideflow test with test leads (XYZ), depletion line, and control test leads.

[0099] B: Example results of high, normal, low / absent plasma equivalent (albumin) in the sample.

[0100] C: The cartridge design obscures the results of the depletion line, as well as the sample results in B, but within the cartridge...

[0101] It should be noted that, for this application in the embodiments of the present invention, any plasma protein is applicable as long as its normal concentration range does not exceed about 10 times, more preferably not more than 5 times, more preferably not more than 3 times, and most preferably not more than 2 times. Similarly, although Figure 2 Examples of chicken IgY used in A and 2B as the second analyte are valid, but any suitable antigen or hapten not present in human plasma or oral fluid may be used for this purpose.

[0102] It can be further pointed out that the same method can also be used to control the number of cells in swab samples designed for point-of-care testing (POC), such as for detecting SARS-CoV-2, influenza, respiratory syncytial virus, human papillomavirus, etc., where the target antigen is present in the cells (such as mucosal epithelial cells) from which the swab sample can be collected, which are infected by the virus or other organisms, rather than in gingival crevicular fluid containing antibodies from plasma. In these examples, the human antigen selected for the depletion line and the gold-labeled conjugate used for control should be intracellular proteins, such as, but not limited to, β-actin, β-tubulin, or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). These proteins are considered housekeeping protein markers because they have relatively stable and universal expression across different cell types and conditions, rather than plasma proteins such as albumin used in the examples above.

[0103] Preferred detection methods include: (1) a method for detecting hepatitis C virus (HCV) antibodies, which may indicate infection with hepatitis C virus; (2) a method for detecting human immunodeficiency virus (HIV-1 or HIV-2) antibodies, which may indicate infection with HIV-1 or HIV-2; (3) a method for detecting hepatitis B surface antigen (HBsAg), which may indicate chronic infection with hepatitis B virus; and (4) a method for detecting hepatitis B e antigen (HBeAg), which may indicate viral replication in chronic hepatitis B virus carriers. (5) Detection methods for hepatitis B surface antigen antibodies, which may indicate immunity to hepatitis B virus, and / or detection methods for hepatitis B core antigen antibodies, which may indicate past infection with hepatitis B virus; (6) Detection methods for Treponema pallidum antibodies, which may indicate infection with Treponema pallidum (syphilis or yaws); (7) Detection methods for SARS-CoV-2 nucleocapsid antigen, which may indicate infection with SARS-CoV-2 (pneumonia caused by novel coronavirus).

[0104] Figure 7 This paper demonstrates a preferred testing method for detecting HIV antibodies. In this example, the test strip uses a double-antigen sandwich format. The HIV antigen is fixed on the test strip, and the same HIV antigen binds to colloidal gold. When any HIV antibody of any antibody isotype (IgG, IgM, IgA, etc.) is present, the antigen on the test strip and the gold-labeled antigen form a cross-link through the bivalent binding of the antibody's variable region, resulting in a positive line. It should be noted that if the subject's antibody level is high, a positive result may be obtained even if the plasma volume is low. A positive test result combined with a weak control line result can still be considered a valid test result. However, if the control line result is too weak, a negative test result may be considered invalid according to internal or external reference standards.

[0105] It is worth noting that the same method of controlling the amount of plasma equivalent in the sample can be used for tests specifically designed for oral fluid samples, tests specifically designed for blood or plasma samples, and tests applicable to blood or plasma samples. For example, there are currently commercially available HIV antibody tests and HCV antibody tests specifically designed for oral fluid samples. All of these tests can be improved by using the control line method described in the embodiments of this invention to control the amount of plasma equivalent in the oral fluid sample used for testing.

[0106] Current oral liquid antibody tests (such as OraQuick HIV or OraQuick HCV) typically involve collecting liquid directly from the mouth onto absorbent collection filter paper integrated into the test strip device. The device is then placed in a test tube containing buffer solution, and the sample is carried into the test device via capillary action. However, many users, especially those who perform home testing, may feel uncomfortable placing part of the test device in their mouth.

[0107] The experience of widespread use of the COVID-19 lateral rapid antigen test kit by patients worldwide has provided an idea for another solution, namely (1) collecting gingival crevicular fluid using optimized swabs (which can be determined experimentally, for example by comparing the amount of IgG or albumin collected by existing and new swabs), (2) extracting the swab in a buffer tube and then applying the extracted sample to the test device without adding buffer (the operation is exactly the same as that of the COVID-19 rapid antigen test kit).

[0108] This method leverages consumer experience and acceptance of rapid antigen testing for COVID-19 and simplifies the integration of testing blood and oral fluid samples within the same testing device. For example, in Figure 7 In the example shown, the sample that can be added to the sample well at the bottom of the device can be whole blood or plasma sample with an appropriate amount of buffer solution, or it can be an oral swab sample extracted into buffer solution and then added to the same sample well. The control test line ensures that enough plasma equivalent is used to obtain valid test results.

[0109] Figure 7 middle:

[0110] A: Schematic diagram of a lateral flow test with a detection system (HIV antigen), a loss system, and a control system.

[0111] B: Example results of high, normal, or low plasma equivalent in samples and positive HIV antibody results.

[0112] C: The cartridge design obscures exhaustion line results, as well as the example results shown in B but within the cartridge. A low plasma equivalent for an HIV-positive result may be considered valid, but an HIV-negative result will be considered invalid due to insufficient plasma sample.

[0113] This is particularly advantageous for tests that aim to detect multiple analytes simultaneously in a single testing device, such as combined HIV / syphilis antibody testing. Figure 9 The Chembio dual-pathway syphilis assay detects two different analytes (a Treponema pallidum-specific antibody and an antibody against the synthetic RPR). In some side-flow point-of-care testing devices, especially when detecting multiple analytes, the sample is added to a sample well outside the device's end (e.g., ...). Figure 8 The Chembio dual-channel test shown includes a syphilis screening / confirmatory test, or a Chembio dual HIV / syphilis test. It is also conceivable that the test sample could be placed in the middle of the device, with blood / plasma flowing in both directions and reacting in two separate test reaction zones.

[0114] Clearly, due to the location of the sample well, neither the Chembio device nor this bidirectional device can be directly used for oral fluid sampling. However, by using (i) the swab and extraction method for collecting gingival crevicular fluid, and (i) the novel plasma control line method described above, dual detection methods can be designed for detecting combinations of HIV and syphilis, HIV and syphilis and hepatitis B virus, or multiple hepatitis B virus biomarkers (HBsAg, anti-HBsAg, anti-HBcAg), which can be used in the same testing device for blood / plasma or oral fluid samples. Figure 9 A schematic diagram is shown, illustrating a test applicable to blood, plasma, or oral fluid samples, employing the aforementioned design features.

[0115] Figure 9 This is a schematic diagram of a two-way HIV / syphilis antibody test (left) and a hepatitis B surface antigen (HBsAg) test (right), which includes a plasma control for oral samples. Figure 9 A: Add the test strip before the sample is placed in the middle of the sample pad, which contains anti-glycoprotein A or other erythrocyte agglutination reagent; Figure 9 B: Oral fluid sample test results: By comparing the plasma equivalent depletion line and the control line, the plasma equivalent level is normal, therefore the oral sample test is valid; HIV antibody positive, syphilis (TP) antibody negative, HBsAg positive. The program control in the left-side test strip assembly can be any conventional program control, including anti-chicken IgY reacting with albumin-chicken IgY conjugated gold.

[0116] The same method can also be used to control the number of cells collected in swab samples used for diagnosing infections, such as oral or nasal swab samples used to diagnose SARS-CoV-2 (pneumonia caused by the novel coronavirus), influenza, respiratory syncytial virus, etc. For this purpose, intracellular proteins, rather than plasma proteins, should be used as a control.

[0117] Figure 10 This demonstrates an example of a point-of-care (POC) test for the SARS-CoV-2 nucleocapsid antigen, using a control test line method (see [link]). Figure 5 , Figure 6 , Figure 7 In this example, the intracellular protein is GAPDH, and the colloidal gold control is a conjugate of colloidal gold with a mixture of GAPDH and chicken IgY.

[0118] Figure 10Three key pieces of information about the SARS-CoV-2 NP antigen detection device based on lateral chromatography (LFA) are presented.

[0119] Figure 10 A illustrates the principle of the lateral flow assay. The test strip contains a test line (T, coated with anti-SARS-CoV-2NP antibody), an exhaustion line (D, coated with anti-GAPDH antibody, indicating the amount of human cells in the sample), and a control line (C, ensuring flow and reagent effectiveness). When viral antigens are present in the sample, the gold-antibody complex is captured and colored on the T line; when the sample cell count is sufficient but the virus is negative, the T line remains colorless, while the color intensity of the D line is inversely proportional to the sample sufficiency; the color intensity of the C line directly reflects the sample sufficiency, thus verifying the test's effectiveness.

[0120] Figure 10 B lists typical results for different cell equivalents (high, normal, low) and NP antigen positivity: all three lines are colored, and the color intensity of the T line is related to the viral load. Figure 10 C points out that due to the optional box-window design, the visibility of the depletion line (D) is obscured, leaving only the C line for estimating the number of cells in the sample, which is easier to interpret (two lines instead of three). If there are insufficient cells in the sample, even if the C line shows color and the T line is negative, the result should be considered invalid rather than negative to avoid the risk of false negatives due to insufficient sampling quality.

[0121] like Figure 11 and Figure 13 As shown, the diagnostic device consists of a plastic backing 1, a test strip 4, and two parts, an upper and lower device box 8 / 15. The plastic backing 1 and the test strip 4 are assembled by ultrasonic welding or snap-fit ​​to ensure that the test strip is stably positioned inside the box. The upper part 8 and the lower part 15 of the device box hold the test strip by means of a slot and a pin. A fixed guide groove is provided in the middle to guide the test strip to slide smoothly in or out along the arrow direction (flow direction).

[0122] like Figure 11 , 15 As shown, the sample application pad 2 and reagent mixing pad 3 are sequentially fixed to one end of the plastic backing 1 and bonded to the support substrate using pressure-sensitive adhesive or hot melt adhesive. Sampling holes 10 and spare sample loading holes 11 are embedded in corresponding positions on the plastic backing. The sample loading hole 10 is directly aligned with the reagent mixing pad 3, allowing for rapid mixing by injecting buffer solution or sample. The sample loading hole 11 is aligned with the sample application pad 2 and is used for pretreatment or secondary sample loading.

[0123] like Figures 11-14As shown, the device test strip 4 has a test line 6, a control line 7 (or control test line 13), and a depletion control line 12 arranged sequentially in the middle. Test line 6 (anti-XYZ antibody) is sprayed on the membrane at the first detection window position to capture the target antigen; control lines 7 / 13 (anti-human albumin or anti-chicken IgY) are located at the second detection window to verify the sample flow and reaction effectiveness; depletion control line 12 is located between or after the two lines to determine whether the sample volume is sufficient (present only in Examples 2 / 4).

[0124] like Figures 11-14 As can be seen, the absorbent pad 5 is placed at the far end of the test strip and engages with the plastic backing 1 through the backing groove, ensuring continuous contact between the absorbent pad and the test area while preventing liquid spillage. A leak-proof membrane is adhered to the back of the absorbent pad 5, forcing the flowing buffer solution and sample into the absorbent pad to ensure that backflow does not affect the reading after the reaction is complete.

[0125] like Figure 12 , 14 As shown, the upper part 8 of the device box has an observation window 9 facing the reaction area. It is in the form of a transparent film or an openable cover, which makes it easy for manual or instrument reading of the color development results of the test line 6 and the control lines 7 / 13. In Examples 2 and 4, an opaque structure 14 is added, which can be inserted into or covered in the window area to shield the color development of the depleted control line 12, so that only the main test line and the control line are displayed, thereby enhancing the clarity of result interpretation.

[0126] like Figure 15 , 16 As shown, during operation, the sample to be tested (whole blood, plasma, or oral / nasal swab suspension) is first added dropwise to the sample application pad 2 through the sample application hole 11, or directly to the reagent mixing pad 3 through the sample application hole 10, so that the sample and each component are in full contact. The sample and buffer solution flow capillarily along the direction of the arrow on the nitrocellulose membrane base, passing through the reagent mixing pad 3 in sequence to release the detergent and labeling reagent, and then flowing through the test line 6 to capture the target antigen and develop color. Subsequently, it reaches the control line 7 / 13 (and the depletion line 12) for control color development and sample volume verification. Finally, it is absorbed and locked by the absorbent pad 5 to prevent backflow. After the reaction is completed, the color development of the test line and control line can be read through the observation window 9 of the device box to determine the positive or negative result and the validity of the operation.

[0127] Individual and combined solutions for open-structure woven membranes: filter membrane + erythrocyte agglutination reagent + control line of this invention, with embodiments shown in... Figures 1 to 16 Provided by China.

[0128] Example 1—Quantification of C-reactive protein (CRP), fingertip whole blood

[0129] A plain weave polyester / nylon 65 / 35 fiber web was cut into 8mm × 18mm pieces. The outer layer was etched with 250W plasma for 30 seconds, followed by spraying with a 3μm pore size polystyrene microsphere latex to form a loose layer with an average pore size of 3μm. The inner layer used a 0.25μm polyethersulfone electrospun membrane. 2μg of cm-type protein A / G was oriented and fixed on the front side of the filter membrane. -2 A mixture of anti-A, anti-B, and anti-D monoclonal antibodies, with a back coating of 1 g m -2 Hydrophilic PVA nanofibers were used. A 6mm × 6mm, 400μm thick piezoelectric pump wafer was mounted under the sample pad, with a driving voltage of 7Vpp, a frequency of 12Hz, and a single pulse volume of 1μL. The membrane strip was made of 25mm × 4mm nitrocellulose; from upstream to downstream, a dual-gradient internal standard gold / silver core-shell nanoprobe (OD) was deposited sequentially. 545 =0.2 and 0.4), test line (anti-CRP monoclonal antibody 1 mg mL) -1 Albumin depletion line (anti-HSA ligand 0.2 mg mL) -1 ), goat anti-mouse IgG control line.

[0130] After adding 20 μL of whole blood from the fingertip, the micropump dispensed 8 μL of washing solution within the first 90 seconds to push the plasma into the membrane strip; chromatography was completed in 10 minutes. The average OD at the albumin depletion line was 0.32 ± 0.03, and the system automatically determined that the sample volume was sufficient. A five-point calibration curve (linear R) was generated for the internal standard. 2 =0.999, 1–100 mg L -1 The correlation coefficient R between 120 clinical samples and immunoturbidimetric assays was calculated. 2 =0.964, mean deviation ±7.8%; indoor three-batch repeatability CV = 5.1%, showing stable quantitative performance.

[0131] Example 2—SARS-CoV-2 nucleocapsid antigen, throat swab

[0132] The average pore size of the outer layer of the filter membrane was adjusted to 4 μm to reduce the retention of oral epithelial debris; the antibody loading was reduced to 0.8 μg cm⁻¹. -2 The test line used two paired monoclonal antibodies (capture 1 mg / mL). -1 Detection of 40 μg / mL -1 The internal standard area maintained the same probe concentration. The sample extraction solution consisted of 10 mM Tris-HCl, 0.1% Tween-20, 0.2% PEG8000, and 0.5 mM EDTA.

[0133] Swabs were centrifuged in 400 μL of extraction solution for 10 seconds, and 100 μL was added dropwise to the sample pad. The micropump operated at 8 Hz with a pulse density of 2 μL, completing chromatography in 9 minutes. The average OD at the albumin depletion line was 0.28 ± 0.02, higher than the threshold of 0.24. Of the 125 RT-qPCR positive cases (Ct ≤ 28), 114 were colorimetric positive (sensitivity 91.2%), and of the 375 PCR negative cases, 368 were negative (specificity 98.1%). Performance degradation was <5% after 4 weeks of accelerated aging at three temperature zones: -20℃ / 25℃ / 45℃.

[0134] Example 3—PCT+IL-6 Dual-Indicator Detection, Venous Whole Blood

[0135] The outer 2.5 μm pore size filter membrane is used for more thorough red blood cell retention; two test lines are printed side by side in the test area: T1 (anti-PCT monoclonal antibody 0.8 mg mL) -1 T2 (anti-IL-6 monoclonal antibody 0.6 mg mL) -1 The albumin depletion line and control line positions remained unchanged. The micropump was programmed to output six 2μL pulse (15Hz) cycles, with a total flushing volume of 12μL.

[0136] Load 30 μL of venous whole blood, and chromatography is completed in 12 min. Independent calibration curves are automatically generated for both channels using the internal standard region: PCT 0.05–10 ng / mL. -1 (R 2 =0.997), IL-6 5–500 pg mL -1 (R 2 =0.995). Electrochemiluminescence immunoassay was performed on 60 ICU blood samples, with mean relative deviations of PCT ± 8.5% and IL-6 ± 9.3%; inter-day precision PCT CV = 5.8%, IL-6 CV = 6.2%.

[0137] Example 4—Powerless spring-loaded flushing system for rural clinics in mountainous areas

[0138] Replace the piezoelectric pump with a 50μL polyurethane spring capsule, pre-filled with 0.05% PBS-Tween. The user presses the capsule 5 times to form 4mL of s... -1 Pulse rinsing at peak flow rate; the formulation of filter membrane, antibody and internal standard area is the same as in Example 1.

[0139] At a township health center at an altitude of 4100m, 15μL of whole blood was collected from the fingertip and placed in a sample pad. After passive rinsing as described above, the reading was taken 15 minutes later. The mean OD value of the albumin depletion line detected by a portable spectral reader was 0.29 (n=48), indicating that all samples were sufficient. The results of 45 cases were compared with those of a municipal hospital, with an overall concordance rate of 93.3%, and no significant interference from the high-altitude environment was observed.

[0140] Example 5—Cloud-based Threshold Fixation and Batch Correction

[0141] Based on the optical density data of 2150 cases obtained from Examples 1–4, the albumin depletion line distribution was fitted using a Gaussian mixture model, and the 5th percentile (P5) of 0.24 was taken as the sample sufficiency threshold and written into the cloud-based quality control model v2.1. Subsequently, three batches of 30,000 test strips were produced, and 100 strips were randomly selected from each batch for gradient plasma testing.

[0142] The cloud-based real-time calibration of internal standard coefficients across different batches reduced the inter-batch CV of the albumin depletion line from 14.2% to 4.8% after algorithmic correction, and the peak error of the two-stage probes in the internal standard region was ≤±3%. This process demonstrates that statistical threshold solidification and online batch calibration can significantly improve long-term consistency.

[0143] Example 6—Independent Sample Preparation Cartridge vs. High-Speed ​​Centrifugation

[0144] The open-structured woven filter membrane, the agglutinating antibody layer, and the hydrophilic fiber layer are encapsulated in a 35mm×11mm polypropylene disposable cartridge. A 0.25mL metering chamber is injection molded on the top of the cartridge; a silicone valve is provided on the side to ensure unidirectional flow.

[0145] Plasma was obtained from the same 40 μL of venous whole blood by cartridge filtration and centrifugation at 10,000 × g for 3 min; IgG content was determined: 62.5 ± 3.1 mg / mL using the cartridge method. -1 Centrifugation method: 63.1 ± 2.8 mg / mL -1 (n=30, p=0.47). SDS-PAGE of the protein profile showed no additional degradation bands, indicating that cartridge mode can provide plasma quality similar to laboratory-grade centrifugation in an instrument-free environment, verifying separation efficiency and protein integrity.

[0146] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A lateral chromatography test strip for detecting target analytes in oral swab samples or whole blood samples, characterized in that, This includes a sample application pad, a conjugation pad, a nitrocellulose membrane strip, and an absorbent pad that are sequentially bonded together; The sample application pad consists of an open-pore woven filter membrane. The erythrocyte agglutination reagent is dried within the membrane, forming an open filtration structure for erythrocyte removal and plasma capture, and flows along the subsequent membrane. The nitrocellulose membrane strip has test lines, albumin removal lines, and control lines arranged sequentially from the sample loading end to the tail end. By comparing the control line and the albumin removal line, two known optical density signal levels are provided at once, thereby generating a calibration curve.

2. The test strip as described in claim 1, characterized in that, The erythrocyte agglutination reagent is a mixture of antibodies capable of recognizing one or more erythrocyte membrane antigens.

3. The test strip as described in claim 1, characterized in that, The test line is used to detect C-reactive protein, SARS-CoV-2 nucleocapsid antigen or other preset targets, and selects immunoaffinity molecules that match the targets.

4. A sample preparation apparatus for separating plasma from whole blood or filtering oral fluid, characterized in that, It includes a filter membrane with a mesh structure and a dried red blood cell agglutination reagent inside the membrane, which can complete the operation of red blood cell removal, plasma capture and plasma release in one go.

5. A lateral chromatography method for confirming sample adequacy, characterized in that, Step 1: Add the oral swab extract or fingertip whole blood to the sample application pad of claim 1; Step two: Under the action of the erythrocyte agglutinin, the erythrocytes aggregate and are retained on the outer layer of the filter membrane, while the plasma is pushed to the inner layer of the filter membrane and enters the membrane strip under the action of capillary flow, while necessary washing is performed at the same time. Step 3: The plasma first passes through the albumin depletion line to generate an optical density reference signal, and then migrates to the test line to bind to the target. Step 4: Read the albumin depletion line signal and compare it with a preset threshold. If the signal is lower than the threshold, output an insufficient sample alarm. If the signal is higher than the threshold, use the calibration curve generated by the dual-gradient internal standard nanoprobe region to quantify the test line.

6. The detection method as described in claim 5, characterized in that, The insufficient sample threshold was determined by analyzing the albumin depletion line optical density distribution of a statistically significant number of clinical samples.

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