Method for removing interferent in biological sample, detection method of target protein, detection mechanism for target protein in biological sample and preparation method

Abstract

The invention discloses a method for removing interferents in a biological sample, a detection method of a target protein, a detection mechanism for the target protein in the biological sample and a preparation method. According to the method for removing the interferents in the biological sample, the isoelectric point of a target object to be detected is larger than a first value and smaller than a second value, and the method comprises the following steps that pH of the biological sample is adjusted to the first value; flowing the biological sample through a first ion exchange membrane to intercept a first impurity group; adjusting the pH value of the biological sample to a second value, wherein the second value is greater than the first value and the difference between the two is not less than 2 and not greater than 3; flowing the biological sample through a second ion exchange membrane to intercept a second impurity group, the isoelectric point of the second impurity group being greater than a second value when the isoelectric point of the first impurity group is less than the first value; when the isoelectric point of the first impurity group is larger than the second value, the isoelectric point of the second impurity group is smaller than the first value. The content of interferents can be effectively reduced, and interference during detection is reduced to the minimum.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a method for removing interfering substances from biological samples, a method for detecting target proteins, a detection mechanism for target proteins in biological samples, and a preparation method thereof. Background Technology

[0002] The advantages of saliva as an in vitro diagnostic sample are that it is non-invasive, convenient, painless, easy to collect and store, and rich in components (DNA, RNA, protein, hormones, etc.) that can reflect the overall health status. It can be used for early screening, monitoring and diagnosis of diseases (such as viruses, oral diseases, and certain systemic diseases), and is particularly suitable for home self-testing, large-scale epidemiological studies and point-of-care testing (POCT), reducing patient anxiety and medical costs.

[0003] However, saliva, as a biological sample, possesses unique complexity that directly affects the detection of low-concentration biomarkers. Saliva protein composition is complex, containing over 1000 proteins. High-abundance / structural proteins in saliva are the main sources of interference, and their main components are shown in Table 1.

[0004] Table 1

[0005]

[0006] Table 2 shows common diagnostic-related protein / hormone biomarkers in biological samples.

[0007] Table 2

[0008] The principle of ion exchange has been applied in protein chromatography for decades, with significant advantages, mainly in the following aspects: high selectivity: based on the difference in protein isoelectric point (pI), separation of <0.5 pI units can be achieved; high capacity: low concentrations of targets can be enriched from large-volume samples (pre-concentration factor 5-40 times); mild reaction conditions: carried out in buffer solution, without damaging protein biological activity; low cost: commercially available ion exchange fibers or membranes are very mature.

[0009] Currently, the following methods are mainly used to reduce non-specific binding of proteins and background interference: ① Optimizing lateral flow immunochromatographic materials and chemical conditions to reduce nonspecificity. This approach is the most common and mature type of commercially available lateral flow immunochromatographic test strips, and mainly includes: introducing blocking agents (such as BSA, casein, gelatin, etc.) into the sample pad, conjugate pad, or NC membrane; using nonionic or weakly ionic surfactants (such as Tween-20, Triton X-100) to reduce nonspecific adsorption; and weakening electrostatic interactions between proteins by adjusting the ionic strength and pH of the sample buffer or pretreatment solution.

[0010] ② Perform external sample purification or enrichment before lateral flow immunochromatography. This type of protocol mostly adopts the following methods: remove macromolecules or enrich targets by centrifugation, filtration, column chromatography, etc.; use ion exchange columns, affinity columns or magnetic beads to complete sample purification in vitro; dilute, lyse and adjust the ions of biological samples before loading and detection.

[0011] The research also includes applications combining ion exchange technology with lateral flow immunochromatography, proposing the integration of ion-selective membranes with electrodes to create an ion concentration polarization (ICP) region under an electric field, enriching the target analyte before immunoassay. Other applications incorporate other types of functional membranes into test strips, such as filtration membranes, molecular sieve membranes, and ultrafiltration membranes for physical retention of macromolecules; highly absorbent materials for altering flow rate or sample volume; and delay pads or hydrophobic barrier layers for timing control.

[0012] Traditional nitrocellulose membrane-based lateral flow immunochromatographic strips have inherent defects, such as non-specific adsorption (NC membranes have affinity for all types of proteins and lack selectivity), background protein competition (interfering proteins compete with target proteins for limited antibody binding sites), and insufficient sensitivity (the sensitivity of conventional colloidal gold lateral flow immunochromatographic strips is approximately at the μg / mL level). While existing technologies have attempted to improve the performance of lateral flow immunochromatography by addressing issues such as blocking and chemical inhibition, external sample purification, electrokinetic enrichment, physical filtration, and flow rate control, they all suffer from one or more of the following shortcomings: users need to undergo complex procedures to significantly reduce salivary background, thus increasing the complexity and number of steps involved; they fail to selectively process highly charged components in saliva; they are difficult to integrate with existing colloidal gold / latex lateral flow immunochromatographic platforms; or they rely on external equipment, making them unsuitable for ordinary users.

[0013] While traditional colloidal gold / latex lateral flow (LFA) test strips are simple, fast, and intuitive to use, their detection limits are relatively high compared to laboratory methods (such as ELISA). This is because the concentration of the target analyte in saliva is extremely low, and the presence of complex matrices such as mucins easily generates non-specific background signals. Low-concentration targets pose a challenge in saliva lateral flow immunochromatographic detection. Conventional test strip structures lack enrichment steps for trace analytes. Typically, to improve sensitivity, both sample pretreatment and signal enhancement are required, but this makes it difficult to ensure a seamless detection process, inevitably increasing the number of steps for the user, and the results are still not ideal. Summary of the Invention

[0014] To overcome the above-mentioned drawbacks, one of the objectives of this invention is to provide a method for removing interfering substances from biological samples, wherein the isoelectric point of the target analyte is greater than a first value and less than a second value, comprising the following steps: STEP 01: Adjust the pH of the biological sample to the first value; STEP 02: Pass the biological sample from step STEP 01 through the first ion exchange membrane to retain the first group of impurities; STEP 03: Adjust the pH of the biological sample from step STEP02 to a second value, where the second value is greater than the first value and the difference between the two is not less than 2 and not greater than 3. STEP 04: Pass the biological sample from step STEP 03 through the second ion exchange membrane to retain the second group of impurities. When the isoelectric point of the first impurity group is less than the first value, the isoelectric point of the second impurity group is greater than the second value; When the isoelectric point of the first impurity group is greater than the second value, the isoelectric point of the second impurity group is less than the first value.

[0015] Furthermore, the first value is 3.0 ± 0.5, and the second value is 5 ± 0.5.

[0016] Furthermore, the first value is 3.0 ± 0.2, and the second value is 5 ± 0.2.

[0017] Furthermore, the first value is 3.0, and the second value is 5. For example, the first impurity group consists of substances with an isoelectric point less than 3, and the second impurity group consists of substances with a pI greater than 5.

[0018] Furthermore, the first impurity group is mucin, and the second impurity group is at least one of amylase and IgA.

[0019] The second objective of this invention is to provide a method for detecting a target protein in a biological sample, which utilizes the aforementioned method to remove interfering substances from the biological sample before detecting the target protein.

[0020] Furthermore, the target protein is HCG. HCG has an isoelectric point slightly below 5. At pH 3, HCG is positively charged and will not be adsorbed by the anion exchange membrane; while at pH 5, HCG has become negatively charged and therefore will not be adsorbed by the cation exchange membrane, allowing it to pass through smoothly. Thus, the anion and cation exchange membranes respectively "deplete" the non-target proteins that are negatively charged at the first pH and positively charged at the second pH, allowing target molecules such as HCG to remain in a free state as much as possible throughout the process. This greatly improves the purity of the target HCG in the sample and significantly reduces interference noise during detection.

[0021] A third objective of this invention is to provide a detection mechanism for target proteins in biological samples, which enables the aforementioned method for detecting target proteins.

[0022] Furthermore, the detection mechanism can be used for at least one of a test kit, a test strip card, or a test strip. The detection mechanism includes a sample injection section and a detection section. The sample injection section includes an interference retention area, which includes the following layers through which the sample to be tested passes sequentially: The first functional layer is a glass fiber membrane layer with a pH value of 1. First ion exchange membrane; The second functional layer is a glass fiber membrane layer with a pH value of 2. The second ion exchange membrane is an ion exchange membrane with opposite charges to the first ion exchange membrane. The isoelectric point of the target analyte is between the first and second values. The lengths of the first functional layer, the first ion exchange membrane, the second functional layer, and the second ion exchange membrane decrease sequentially.

[0023] Furthermore, the preparation method of the test strip includes the following steps: In the region corresponding to the sample injection area of ​​the nitrocellulose membrane with the gold-labeled pad, the second ion exchange membrane, the second functional layer, the first ion exchange membrane, and the first functional layer are sequentially arranged in a direction away from the nitrocellulose membrane (NC membrane). The second ion exchange membrane, the conjugate pad, the NC membrane, and the absorbent pad overlap by approximately 1-2 mm, and the lengths of the first functional layer, the first ion exchange membrane, the second functional layer, and the second ion exchange membrane decrease sequentially. After the biological sample to be tested is added to the sample injection section, it flows sequentially through the first functional layer, the first ion exchange membrane, the second functional layer, and the second ion exchange membrane. Most of the interfering substances are retained in the sample injection section, while the target analyte flows smoothly to the detection section.

[0024] The present invention has the following beneficial effects: ① This application utilizes a first functional layer with a pH value of 1, causing some interfering substances in the sample to become charged as they pass through the first functional layer and be retained by the first ion exchange membrane. When the sample continues to pass through the second functional layer, another portion of the interfering substances in the sample becomes charged, with a different charge than the interfering substances retained by the first ion exchange membrane, and is adsorbed and retained by the second ion exchange membrane. Since the second and first ion exchange membranes are ion exchange membranes with opposite charges, this allows for the retention of as many different types of interfering substances as possible in the biological sample. This ensures that the analytes in the biological sample can pass smoothly through both the first and second ion exchange membranes without being retained. This application does not significantly increase processing steps or costs, cleverly utilizing two types of charged ion exchange membranes to retain most impurities in the sample, thereby minimizing interference during subsequent detection of the analytes in the biological sample.

[0025] ② In contrast to the conventional "binding / elution" mode, this scheme belongs to the "flow-depletion" mode: the ion exchange membrane is not used to enrich the target, but rather acts as a "trap" to capture charged impurities during sample flow, allowing the target to pass freely. In this way, it is equivalent to integrating a one-step sample purification function inside the test strip, eliminating the need for additional purification steps and processes for biological samples, thus greatly improving the detection efficiency for biological samples.

[0026] ③ This invention utilizes the basic structure of existing test strips or test cards, but adjusts the functional and structural layers of the sample introduction section. Therefore, by introducing an ion exchange membrane based on charge selection inside the lateral flow immunochromatographic test strip, and through a synergistic composite design with the lateral flow immunochromatographic membrane in terms of structure and microenvironment, it achieves effective depletion of the complex background of saliva without the need for external equipment or additional operational steps. This significantly reduces non-specific binding and improves the capture ability and detection sensitivity of the target. No additional impurity removal steps are required during the entire detection process. By cleverly utilizing two oppositely charged ion exchange membranes, most impurities in the sample are retained, thus greatly improving the detection sensitivity of the analyte. Attached Figure Description

[0027] Figure 1 This is a three-dimensional structural diagram of a detection mechanism for target proteins in biological samples according to an embodiment of the present invention. Figure 1 ; Figure 2 This is a three-dimensional structural diagram of a detection mechanism for target proteins in biological samples according to an embodiment of the present invention. Figure 2 ; Figure 3 for Figure 2 A magnified view of a portion of point A in the middle; Figure 4 This is a schematic diagram of an ion exchange lateral flow immunochromatographic coupling test strip according to an embodiment of the present invention; Figure 5 To improve the detection performance of the ion exchange and lateral flow coupling test strip of the present invention; Figure 6 To utilize the detection effect of the test strips in the comparative example.

[0028] In the picture: 1. Sample inlet; 2. Detection section; 11. First functional layer; 12. First ion exchange membrane; 13. Second functional layer; 14. Second ion exchange membrane; 21. Binding pad; 22. NC membrane; 23. Absorbent pad; 24. Liquid inlet hole; 25. Detection window; 3. Backplate. Detailed Implementation

[0029] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0030] In some embodiments, the present invention provides a method for removing interfering substances from biological samples. This invention utilizes the difference in isoelectric point between the target analyte and most other interfering substances to remove most of the interfering substances from the biological sample. The isoelectric point of the target analyte is greater than a first value and less than a second value. Specifically, the method includes the following steps: STEP 01: Adjust the pH of the biological sample to the first value. The first and second values ​​can be a single point value or a range value. Please note that the first and second values ​​here do not strictly refer to the lower and upper limits of the isoelectric point of the analyte. Rather, the first value is less than or equal to the lower limit of the isoelectric point of the analyte; the second value is greater than or equal to the upper limit of the isoelectric point of the analyte. For example, if the analyte is HCG, and its isoelectric point is 3.5-4.5, then the first value can be 3.5, or it can be 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, or other values; then the second value can be 5.0, or it can be 5.1, 5.3, 5.4, 5.5, or other values. Correspondingly, if the analyte is salivary mucin, and its isoelectric point is 3.0-4.0, then the first value can be set to 2.3, 2.5, 2.7, etc., and the second value can be set to 4.5, 5.0, etc.

[0031] STEP 02: Pass the biological sample from step STEP 01 through the first ion exchange membrane 12 to retain the first group of impurities; STEP 03: Adjust the pH of the biological sample from step STEP 02 to a second value. The second value is greater than the first value, and the difference between the two is not less than 2 and not greater than 3. The second value can be a single point value or a range value.

[0032] STEP 04: Pass the biological sample from step STEP 03 through the second ion exchange membrane 14. When the isoelectric point of the first impurity group is lower than the first value, the isoelectric point of the second impurity group is higher than the second value. Therefore, the first impurity group will be negatively charged at the first pH value. At this time, the first ion exchange membrane 12 is set as an anion exchange membrane, so the first impurity group can be captured and retained by the cations of the anion exchange membrane. For example, the isoelectric point of the first impurity group is 2-3, and the first value is 3. The first impurity group can be a single substance or a mixture of substances. Correspondingly, the second value is set to 5, so the second impurity group is the impurity with an isoelectric point greater than 5. The second impurity group is positively charged at this time. At this time, the second ion exchange membrane 14 is set as a cation exchange membrane, so the second impurity group can be adsorbed onto the second ion exchange membrane 14 and retained.

[0033] Accordingly, when the isoelectric point of the first impurity group is greater than the second value, the isoelectric point of the second impurity group is less than the first value. In this case, the first ion exchange membrane 12 is a cation exchange membrane, and the second ion exchange membrane 14 is an anion exchange membrane.

[0034] In some embodiments, the target protein in the biological sample is detected after the interfering substances in the biological sample are removed using the method of the present invention.

[0035] In some embodiments, the biological sample testing apparatus of the present invention can be obtained by improving existing test strips or test cards. The testing apparatus can be a test card, a test strip, or a testing device.

[0036] In some embodiments, the detection mechanism is a test strip or test card. The ion exchange lateral flow coupling test strip of the present invention can be obtained by utilizing the basic structure of existing test strips or test cards, preparing and adjusting each functional layer during the preparation process, and assembling them.

[0037] In some embodiments, the ion exchange lateral flow coupling test strip of the present invention includes a sample injection section 1 and a detection section 2. The sample injection section 1 includes an interference retention area, which includes a first functional layer 11, a first ion exchange membrane 12, a second functional layer 13, a second ion exchange membrane 14, a conjugate pad 21, an NC membrane 22, and an absorbent pad 23 through which the sample to be tested passes in sequence.

[0038] In some embodiments, the first functional layer 11 is a glass fiber membrane layer with a pH of a first value (or a membrane layer of other materials such as a polyester fiber membrane layer). The first functional layer 11 can be a glass fiber membrane layer treated with a first sample processing solution with a pH of a first value, or it can be a glass fiber membrane layer with a pH of exactly the first value that is used in the prior art (meaning that the pH of the raw material system used in the glass fiber membrane layer processing is exactly close to the first value, thus making the pH of the processed glass fiber membrane layer the first value). The first functional layer 11 is located at the very front of the test strip and is used to carry and preliminarily process the biological sample to be tested.

[0039] In some embodiments, the first ion exchange membrane 12 can be obtained by treating a first sample treatment solution with a pH of a first value, or it can be obtained by directly using an ion exchange membrane in the prior art, and ensuring that the processing raw materials during the processing are near the first value, so that the pH of the prepared first ion exchange membrane 12 is the first value.

[0040] In some embodiments, the second functional layer 13 can be a glass fiber membrane (or a membrane of other materials such as a polyester fiber membrane) treated with a second sample processing solution with a pH value of the second value, or it can directly utilize a glass fiber membrane with a pH value of the second value in the prior art (meaning that the pH of the raw material system used in the glass fiber membrane processing is close to the second value, so that the pH of the processed glass fiber membrane is the second value).

[0041] In some embodiments, the second ion exchange membrane 14 can be treated with a second sample treatment solution with a second pH value, or it can directly utilize an ion exchange membrane in the prior art, and ensure that the processing raw materials during the processing are near the first value, so that the pH of the prepared first ion exchange membrane 12 is the first value.

[0042] In some embodiments, the first ion exchange membrane 12 is an anion exchange membrane, and the second ion exchange membrane 14 is a cation exchange membrane. The second value is greater than the first value, and the difference between the two is not less than 2 and not greater than 3. Accordingly, the first sample processing solution may use the following components: 7.5 mM sodium citrate-citric acid buffer (pH 3.0), 100 mM NaCl, 1% Triton X-100, 0.5% sodium caseinate, and 0.5% N-acetyl-L-cysteine; the second sample processing solution may use 25 mM acetate-sodium acetate buffer (pH 5.0).

[0043] In some embodiments, the anion exchange membrane may be a thin film with quaternary ammonium salt groups immobilized on its surface (a strong anion exchange membrane) as this medium. This membrane layer overlaps with the first functional layer 11 to ensure continuous flow. When a sample solution with a pH of approximately the first value flows through this membrane, the positively charged groups on the membrane selectively bind negatively charged protein molecules. Since most non-target impurities (especially acidic components such as mucins and bacterial metabolites) still carry a negative charge at such a low pH, they are firmly adsorbed onto the anion exchange membrane. The anion exchange membrane material should have sufficient porosity and binding capacity to avoid excessive retention causing a decrease in flow rate or rapid saturation. In some embodiments, a thin membrane material with high exchange capacity (such as a functionalized nonwoven fiber membrane) is used to achieve rapid and sufficient anion exchange. The size and shape of the membrane can be comparable to the width of the test strip and fit well with the upstream and downstream materials.

[0044] In some embodiments, the cation exchange membrane, immediately following the second functional layer 13, is a membrane layer with negatively charged groups on its surface (such as a strong cation exchange membrane with immobilized sulfonic acid or carboxylic acid groups). This membrane is used for selective adsorption of cationic proteins. When a sample solution with a pH of approximately the second value enters the cation exchange membrane region, all proteins in the solution that are currently positively charged will be captured and bound by the negatively charged groups on the membrane. Through the cation exchange membrane, most of the remaining cationic interferences (including a large number of enzymes and immunoglobulins) are further removed. The cation exchange membrane also needs to possess the characteristics of rapid binding and permeability. In some embodiments, the cation exchange membrane uses a membrane material with a high surface area and a suitable pore size to ensure both efficient adsorption and continuity of liquid flow.

[0045] In some embodiments, the anion exchange membrane may be an AXP-D quaternary ammonium modified anion exchange membrane purchased from ASTOM; the cation exchange membrane may be a CXP-S sulfonic acid modified cation exchange membrane purchased from ASTOM.

[0046] In some embodiments, the first value can be 3, and the second value can be 5. The first functional layer 11 is made of a hydrophilic material (e.g., glass fiber or non-woven cellulose membrane), and is pre-treated with a strongly acidic treatment solution with a pH of the first value (e.g., a treatment solution prepared using a 5-10 mM citric acid-sodium citrate buffer system or using such a buffer system) and other biological sample lysis and dispersion components. Taking a first value of 3 as an example, the function of this strongly acidic treatment solution is to acidify the entire biological sample to pH≈3 instantly after the sample is added, and to reduce the sample viscosity. The first functional layer 11 also simultaneously performs the filtering function of the test strip sample pad in the prior art, trapping large particulate impurities (such as food residue) in saliva to ensure smooth subsequent flow. First, the pH of the entire sample system is significantly acidified by the first functional layer 11, causing strongly acidic, negatively charged impurities such as mucin to fully expose their charge and be captured and retained by the anion exchange membrane. Then, as the sample passes through the second functional layer 13, which has been pre-soaked in a second sample processing solution with a pH of 2, the sample is slightly neutralized to a weakly acidic state. This causes most background proteins (such as amylase, IgA, and other components with pI>5) to become positively charged under these conditions, thus being retained by the subsequent cation exchange membrane. Therefore, the anion and cation exchange membranes respectively "deplete" the main negatively charged and positively charged non-target proteins, allowing the target molecules to remain in a free state as much as possible throughout the process. Consequently, by the time the saliva sample flows to the detection section 2, the interfering substances it contains have been greatly reduced, and it can, to a certain extent, enrich the analyte, thus effectively reducing background interference and improving the capture efficiency and detection sensitivity of the target protein.

[0047] The second functional layer 13, located after the anion exchange membrane, consists of a fiber pad. The fiber pad is pre-treated with a weakly acidic second sample treatment solution (such as an acetate-sodium acetate buffer system, concentration 20-50 mM) with a pH of 2. This treatment involves soaking the fiber pad in the second sample treatment solution or coating its surface before drying. Its function is to neutralize the pH of the sample solution after anion exchange to the 2nd pH value. The second functional layer 13 overlaps and connects with the anion exchange membrane, ensuring a smooth transition of liquid flow. As the sample passes through this pad, the buffer salt dissolves and mixes rapidly with the sample solution, raising the pH of the solution and creating suitable conditions for the subsequent cation exchange. The size and shape of the membrane can be comparable to the width of the test strip and conform well to the upstream and downstream materials.

[0048] In other possible implementations, the first ion exchange membrane 12 is a cation exchange membrane, and the second ion exchange membrane 14 is an anion exchange membrane. The second value is less than the first value, and the difference between the two is not less than 2 and not greater than 3. Correspondingly, the first sample processing solution can be 25mM acetate-sodium acetate buffer (pH 5.0); while the second sample processing solution can use the following components: 7.5mM sodium citrate-citric acid buffer (pH 3.0), 100mM NaCl, 1% Triton X-100, 0.5% sodium caseinate, and 0.5% N-acetyl-L-cysteine. Therefore, when the sample passes through the first functional layer 11, since the pH of the first functional layer 11 is the first value, and the first value is greater than the second value, the sample will be weakly acidic when flowing through the first functional layer 11. This will cause most background proteins (such as amylase, IgA, and other components with pI>5) to become positively charged under these conditions, thus being retained by the subsequent cation exchange membrane. Subsequently, the sample flows through the second functional layer 13, which significantly acidifies the pH of the entire sample system. This causes highly acidic, negatively charged impurities such as lactobacillus to become negatively charged, allowing them to be captured and retained by the subsequent anion exchange membrane. Therefore, it effectively retains both positively and negatively charged non-target proteins in the sample, thereby improving the capture efficiency and detection sensitivity of the target protein in the detection unit 2.

[0049] In some embodiments, the conjugate pad 21 of the present invention is immediately following the second ion exchange membrane 14 and can be a conventional conjugate pad 21 found in lateral flow immunochromatographic test strips (such as glass fiber or polyester material). The conjugate pad 21 of the present invention is pre-sprayed and dried with a colloidal gold-labeled probe of the antibody to be detected (e.g., if the analyte is HCG, the probe is an anti-HCG antibody probe). When the sample, after the aforementioned purification process, flows into the conjugate pad 21, the antibody molecules contained therein can rapidly bind with the gold-labeled antibody to form an antigen-antibody-colloidal gold complex. Since the major interfering proteins in the sample have been selectively removed by the preceding anion and cation exchange membranes, the target can fully bind to the labeled probe in an environment with lower background interference. The colloidal gold label in the conjugate pad 21 is usually stored in a neutral or weakly alkaline buffer system, so the pH of the liquid phase environment of the incoming sample is slightly adjusted during reconstitution, which is beneficial to the antigen-antibody affinity. The action time and duration of the conjugate pad 21 can be consistent with conventional test strips because the upstream treatment does not significantly slow down the flow rate. After the gold-labeled antibody captures the antibody to be tested, it continues to migrate with the liquid flow into the downstream nitrocellulose membrane.

[0050] In some implementations, the NC membrane 22 (nitrocellulose membrane) serves as the main carrier for the detection reaction, with pre-defined detection lines (T-lines) and control lines (C-lines). The detection line region is immobilized with capture antibodies (typically monoclonal antibodies) targeting the analyte, while the control line is immobilized with antibodies capable of capturing free gold-labeled probes (e.g., anti-mouse IgG). When a sample containing the analyte-gold-labeled complex flows through the NC membrane 22, the HCG in the complex is captured on the lines, enriching and developing color along with the colloidal gold, forming a visible red T-line. The remaining unbound gold-labeled probes continue forward, being captured by another antibody at the C-line and developing color, indicating a normal flow. The rest of the NC membrane 22 can be identical to traditional test strips, but this approach allows the antibodies on the NC membrane 22 to be more "focused" on capturing the target, rather than competing with impurities, thereby improving the specificity and sensitivity of the detection.

[0051] In some embodiments, the absorbent pad 23 is located at the very end of the test strip and is used to continuously attract the sample liquid flow to complete the chromatography process. For example, the absorbent pad 23 can be made of thick cellulose paper, as long as it can absorb liquid far exceeding the sample volume. In this invention, the material and size of the absorbent pad 23 are selected according to conventional design, which can ensure sufficient capillary suction even with the multi-layered structure at the front end, allowing the entire test strip to flow smoothly until the test is completed.

[0052] In some embodiments, the ends of the first functional layer 11, the first ion exchange membrane 12, the second functional layer 13, and the second ion exchange membrane 14 are flush. That is, the ends of each layer are on the same plane along the length direction, which makes the structure of the sample injection section 1 stable and does not require the length of the sample injection section 1 to be too long, so as not to affect the shape of existing test strips, etc.

[0053] In other possible implementations, the ends of the first functional layer 11, the first ion exchange membrane 12, the second functional layer 13, and the second ion exchange membrane 14 are arranged in a stepped manner, and the distance from the detection unit 2 decreases sequentially. That is, a certain stepped shape is also formed at the ends of each layer, but unlike the starting end, it grows sequentially towards the detection unit 2, thereby facilitating the flow of the sample liquid forward layer by layer.

[0054] In some embodiments, the system further includes an outer casing with a liquid inlet 24 disposed in the region where the first functional layer 11 is located. In some embodiments, the liquid inlet 24 is located at the end of the first functional layer 11 furthest from the absorbent pad 23, i.e., at the starting point of the test strip.

[0055] In some embodiments, a detection window 25 is also included, which is set in the area where the NC film 22 is located.

[0056] In some embodiments, the test strips of the present invention are prepared as follows: On the backplate 3, the dried components are sequentially and overlappingly pasted: NC membrane 22 → gold-labeled pad → second ion exchange membrane 14 → second functional layer 13 → anion exchange membrane → first functional layer 11 → absorbent pad 23. The second ion exchange membrane 14, conjugate pad 21, NC membrane 22, and absorbent pad 23 overlap by approximately 1-2 mm. The first functional layer 11, anion exchange membrane, second functional layer 13, and second ion exchange membrane 14 become progressively shorter from top to bottom. At the distal left end, the upper membrane completely covers the lower membrane, and at the proximal end, they are completely aligned or each membrane is exposed, ensuring smooth and uninterrupted chromatography of the solution. The assembled plate is then cut into test strips of uniform width 4 mm using a strip cutter. The card case, saliva cotton, and test strips are stacked in sequence and pressed together using a pressing machine, and the card case cap is then closed.

[0057] In other possible implementations, the detection method of the present invention can also be applied to automated detection equipment, and corresponding functional layers can be set in the detection equipment to remove impurities from the samples.

[0058] In other possible embodiments, the present invention also provides a method for detecting a target protein in a biological sample, which uses the aforementioned method to remove interfering substances from the biological sample before detecting the target protein.

[0059] In some implementations, taking HCG as an example, a test strip with both ion exchange and lateral flow immunochromatography functions can be prepared using the above method. Therefore, biological samples can be detected with high sensitivity without adding any additional steps, simply by following the operation method of the existing lateral flow immunochromatography test strip.

[0060] Example: Preparation of the ion-exchange lateral flow immunochromatographic coupling test strip of the present invention Using a first ion exchange membrane 12 as an anion exchange membrane and a second ion exchange membrane 14 as a cation exchange membrane, and with the second value being greater than the first value and the difference between the two not less than 2 and not greater than 3, the test strip of this application is prepared. Specifically, the test strip of the present invention is prepared according to the following steps: 1.1 Processing and preparation of each functional layer of the test strip: The glass fiber membrane (first functional layer 11) and the anion exchange membrane (ASTOM, AXP-D quaternary ammonium modified anion exchange membrane) were immersed in the first sample processing solution (7.5 mM sodium citrate-citric acid buffer (pH 3.0, 100 mM NaCl, 1% Triton X-100, 0.5% sodium caseinate, 0.5% N-acetyl-L-cysteine) and then dried at 37°C for 12 hours.

[0061] The glass fiber membrane (second functional layer 13) and the second ion exchange membrane 14 (ASTOM, CXP-S sulfonic acid modified cation exchange membrane) were immersed in the second sample processing solution (25 mM acetate-sodium acetate buffer (pH 5.0)) and then dried at 37°C for 12 hours.

[0062] 1.2 Preparation of colloidal gold solution: Take 100 ml of 0.01% chloroauric acid aqueous solution and heat it to boiling. While stirring, accurately add 0.7 ml of 1% trisodium citrate aqueous solution. The golden yellow chloroauric acid aqueous solution turns purple-red within 2 minutes. Continue boiling for 15 minutes. After cooling, restore the original volume with distilled water. The gold sol prepared in this way has a highest absorption peak in the visible light region of 535 nm, and the measured value OD535 is about 1.1.

[0063] 1.3 Colloidal gold-antibody labeling and purification: Adjust the pH of 1 mL of colloidal gold solution to 8.0 using 0.1 M K₂CO₃ solution. Then, add 1 mg of the HCG antibody to be labeled dropwise to the colloidal gold solution and mix at low speed at room temperature for 60 minutes. Add 1 mL of blocking solution (100 mM Tris-HCl (pH 8.5), 1% BSA, 5% skim milk, 0.5% PEG 8000, 0.1% Proclin 300), and continue mixing by rotation for 30 minutes. Centrifuge at 4 °C / 14,000 rpm for 5 minutes, discard the supernatant, and resuspend the precipitate in reconstitution solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 7.5% trehalose, 0.1% Tween 20). Repeat twice. Resuspend the precipitate in 1 mL of reconstitution solution and store at 4 °C for later use.

[0064] 1.4 Scrapping process for the T-line (test line) and C-line (quality control line): The glass fiber membrane was immersed in sample processing solution (formulation: 50mM Tris-HCl (pH 8.0), 100mM NaCl, 1% Triton X-100, 0.5% sodium caseinate, 0.5% N-acetyl-L-cysteine) and then dried at 37°C for 12 hours. The labeled and purified gold-labeled antibody solution was resuspended and fixed onto a polyester fiber membrane using a quantitative streak spray method at 2 μL / cm, and then dried at 37°C for 12 hours. The detection line (T line) was sprayed with another monoclonal antibody (1 mg / mL) targeting the analyte; the control line (C line) was sprayed with a secondary antibody against the labeled antibody (e.g., goat anti-mouse IgG, 1 mg / mL), fixed onto a nitrocellulose membrane (NC membrane 22) using a quantitative streak spray method at 1 μL / cm, and then dried at 37°C for 12 hours.

[0065] 1.5 Preparation of test strips: On the PVC backing plate 3, attach the dried components in the following order from bottom to top, overlapping each other: NC membrane 22 → gold label pad → second ion exchange membrane 14 → second functional layer 13 → first ion exchange membrane 12 → first functional layer 11 → absorbent pad 23. The cation exchange membrane, conjugate pad 21, NC membrane 22, and absorbent pad 23 overlap by approximately 1-2 mm. The first functional layer 11, anion exchange membrane, second functional layer 13, and second ion exchange membrane 14 become progressively shorter from top to bottom. At the distal left end, the upper membrane completely covers the lower membrane, while at the proximal end, they are either completely aligned or each membrane is exposed, ensuring smooth and uninterrupted chromatography. Cut the assembled plate into test strips of uniform width (4 mm) using a strip cutter. Stack the cartridge, saliva cotton, and test strips in sequence, press them together using a cartridge press, and then close the cartridge lid.

[0066] Comparative Example: Preparation of a standard test strip for direct determination of colloidal gold lateral flow immunochromatography in saliva 2.1 Preparation of colloidal gold solution: Take 100 ml of 0.01% chloroauric acid aqueous solution and heat to boiling. While stirring, accurately add 0.7 ml of 1% trisodium citrate aqueous solution. The golden yellow chloroauric acid aqueous solution turns purple-red within 2 minutes. Continue boiling for 15 minutes. After cooling, restore to the original volume with distilled water. The gold sol prepared in this way has a highest absorption peak in the visible light region of 535 nm, and the measured value OD535 is about 1.1.

[0067] 2.2 Colloidal Gold-Antibody Labeling and Purification: Adjust the pH of 1 mL of colloidal gold solution to 8.0 using 0.1 M K₂CO₃ solution. Then, add 1 mg of the antibody to be labeled dropwise to the colloidal gold solution and mix at low speed at room temperature for 60 minutes. Add 1 mL of blocking agent (100 mM Tris-HCl (pH 8.5), 1% BSA, 5% skim milk, 0.5% PEG 8000, 0.1% Proclin 300), and continue mixing by rotation for 30 minutes. Centrifuge at 4 °C / 14,000 rpm for 5 minutes, discard the supernatant, and resuspend the precipitate in reconstitution solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 7.5% trehalose, 0.1% Tween 20). Repeat twice. Resuspend the precipitate in 1 mL of reconstitution solution and store at 4 °C for later use.

[0068] 2.3 Treatment of each chromatography membrane: The glass fiber membrane was immersed in sample processing solution (formulation: 50mM Tris-HCl (pH 8.0), 100mM NaCl, 1% Triton X-100, 0.5% sodium caseinate, 0.5% N-acetyl-L-cysteine), and then dried at 37°C for 12 hours. The labeled and purified gold-labeled antibody solution was resuspended and fixed onto the polyester fiber membrane using a quantitative spraying method at 2 μL / cm, and then dried at 37°C for 12 hours. Detection line (T line): Spray with another monoclonal antibody against the target analyte (1 mg / mL); Control line (C line): Spray with secondary antibody against the labeled antibody (e.g., goat anti-mouse IgG, 1 mg / mL), and fix onto the nitrocellulose membrane (NC membrane 22) using a quantitative streaking method at 1 μL / cm, and then dried at 37°C for 12 hours.

[0069] Attaching the chromatography membrane and cutting and assembling the test strips: On the PVC backing plate 3, attach the dried components in sequence with overlap: NC membrane 22 → gold label pad → sample pad → absorbent pad 23, with an overlap of approximately 1-2 mm between each component to ensure smooth chromatography without gaps. Cut the assembled plate into test strips of uniform width of 4 mm using a strip cutter. Stack the cartridge, saliva swab, and test strips in sequence, press them together with a cartridge press, and then close the cartridge cover.

[0070] Experimental Example 1: Comparison of detection sensitivity between the test strips of the Example and Comparative Examples To evaluate the effectiveness of the technical approach of this invention on samples, human chorionic gonadotropin (HCG), a highly representative target in saliva with extremely high sensitivity requirements, was selected. Clinically high-concentration positive saliva was used as the sample, diluted with negative biological samples to prepare concentration gradient sensitivity reference standards. Simultaneously, artificial saliva (Yuan Ye, R22154) containing the main interfering proteins in saliva (2 mg / mL mucin, 1 mg / mL α-amylase, 0.5 mg / mL sIgA, 0.1 mg / mL albumin, 20 μg / mL lysozyme) was used as a matrix to prepare concentration gradient sensitivity reference standards. These were used for performance testing of the technical method of this invention, as detailed below: Table 3

[0071] The results are shown in Table 4.

[0072] Table 4

[0073] Based on the test results above, the technical method of coupling ion exchange and lateral flow immunochromatography in this invention can significantly improve the detection sensitivity of HCG in biological samples.

[0074] Experimental Example 2: Comparison of the removal effects of the test strips from the Example and Comparative examples on impurity proteins. The experimental methods and chromatography procedures were as described in Example 1. After chromatography, once the liquid reached equilibrium, 2 μL of sample from the upper layer of the first functional layer (before treatment) and 2 μL of sample from the lower layer of the cation exchange membrane (after treatment) were taken from each experimental group. Protein concentration was determined using the BCA protein quantification kit (Adamas-Life, E8053) according to the kit instructions. The results are as follows: Table 5 \ The test results show that in the conventional method's control group chromatography process, after the sample pad is lysed and filtered, only a small amount of protein is retained, with most entering the subsequent reaction and detection system. After removing impurities through the adsorption of the two ion exchange membranes of this invention, the protein concentration is reduced by approximately 90%, thus greatly controlling interfering factors in the subsequent reaction and detection system.

[0075] Experiment Example 3: Comparison of Background Noise Reduction Effects The concentration gradient reference material prepared from the clinically collected biological samples in Experiment 1 was used as the test sample for background observation, and only the band formation of the control line C of the test strip was compared. Figure 5 To measure the test results of the samples using the test strips prepared using the method in Example 1, four tests were performed as one sample group (corresponding from left to right to 0 IU / mL, 0.5 IU / mL, 2 IU / mL, and 8 IU / mL). Figure 6 To test the sample results using the test strips prepared by the comparative method, four tests were used as one sample group (corresponding to 0 IU / mL, 0.5 IU / mL, 2 IU / mL and 8 IU / mL from left to right).

[0076] The test results show that, compared with the existing technology, the test strips prepared using the method of this invention have a significantly cleaner chromatographic background, a higher signal-to-noise ratio, and clearer and more distinct bands.

[0077] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for removing interfering substances from biological samples, characterized in that, The isoelectric point of the target object is greater than a first value and less than a second value, including the following steps: STEP 01: Adjust the pH of the biological sample to the first value; STEP 02: Pass the biological sample from step STEP 01 through the first ion exchange membrane to retain the first group of impurities; STEP 03: Adjust the pH of the biological sample from step STEP02 to a second value, where the second value is greater than the first value and the difference between the two is not less than 2 and not greater than 3. STEP 04: Pass the biological sample from step STEP 03 through the second ion exchange membrane to retain the second group of impurities. When the isoelectric point of the first impurity group is less than the first value, the isoelectric point of the second impurity group is greater than the second value; When the isoelectric point of the first impurity group is greater than the second value, the isoelectric point of the second impurity group is less than the first value.

2. The method for removing interfering substances from biological samples according to claim 1, characterized in that, The first value is 3.0±0.5, and the second value is 5.0±0.

5.

3. The method for removing interfering substances from biological samples according to claim 1, characterized in that, The first value is 3.0±0.2, and the second value is 5.0±0.

2.

4. The method for removing interfering substances from biological samples according to claim 1, characterized in that, The first value is 3.0, and the second value is 5.

0.

5. The method for removing interfering substances from biological samples according to claim 1, characterized in that, The first impurity group is mucin, and the second impurity group is at least one of amylase and IgA.

6. A method for detecting a target protein in a biological sample, characterized in that, The target protein is detected after removing interfering substances from the biological sample using the method described in any one of claims 1-5.

7. The method for detecting target proteins in biological samples according to claim 6, characterized in that, The biological sample is a saliva sample, and the target substance to be tested is HCG.

8. A detection mechanism for target proteins in biological samples, characterized in that, This method is used to implement the detection method for the target object as described in any one of claims 5-7.

9. The detection mechanism for target proteins in biological samples according to claim 8, characterized in that, The detection mechanism can be used for at least one of a test kit, a test strip card, or a test strip. The detection mechanism includes a sample injection section and a detection section. The sample injection section includes an interference retention area, which includes the following layers through which the sample to be tested passes sequentially: The first functional layer is a membrane layer with a pH value of 1. First ion exchange membrane; The second functional layer is a membrane layer with a pH value of a second value; The second ion exchange membrane is an ion exchange membrane with opposite charges to the first ion exchange membrane. The isoelectric point of the target analyte is between the first and second values. The lengths of the first functional layer, the first ion exchange membrane, the second functional layer, and the second ion exchange membrane decrease sequentially.

10. The method for preparing a test strip for detecting target proteins in biological samples according to claim 9, characterized in that, Includes the following steps: In the region corresponding to the sample injection area of ​​the nitrocellulose membrane with the gold-labeled pad, the second ion exchange membrane, the second functional layer, the first ion exchange membrane, and the first functional layer are sequentially arranged in the direction away from the nitrocellulose membrane, with the lengths of the first functional layer, the first ion exchange membrane, the second functional layer, and the second ion exchange membrane decreasing sequentially.

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