Weak ionized colloidal gold immunochromatography test strip based on cationic polymer as quality control line and preparation method thereof

Abstract

The invention belongs to the technical field of medical supplies, and discloses a weakly ionized colloidal gold immunochromatography test strip based on a cationic polymer as a quality control line and a preparation method thereof. The weak ionized colloidal gold immunochromatography test strip comprises a substrate, a nitrocellulose membrane, a combination pad and a water absorption pad, the nitrocellulose membrane, the combination pad and the water absorption pad are all distributed on the surface of the substrate; the combination pad is lapped on the nitrocellulose membrane, the water absorption pad is lapped on the nitrocellulose membrane, and the combination pad and the water absorption pad are distributed on two sides of the nitrocellulose membrane; a detection line and a quality control line are arranged on the nitrocellulose membrane; the quality control line is coated with poly dimethyl diallyl ammonium chloride; the weak ionized colloidal gold immunochromatography test strip further comprises a CEA monoclonal antibody Ab1-weak ionized colloidal gold compound. Directional charge adsorption synergy is formed through weak ionized colloidal gold and a PDDA quality control line with strong positive electricity, and gt is still kept in a high-salt environment; the capture stability is 95%, and the production cost of the C line is reduced by 90%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical supplies, and particularly relates to a weakly ionized colloidal gold immunochromatography test strip based on a cationic polymer as a quality control line and a preparation method thereof. BACKGROUND

[0002] Cancer is one of the major causes of death worldwide, and its early diagnosis and efficacy monitoring are crucial for reducing the incidence and improving patient survival. Current clinical reliance on imaging and pathological detection methods is often limited by insufficient sensitivity or invasive operation, which drives the development of high-accessibility and high-sensitivity tumor marker detection technology. Among many markers, carcinoembryonic antigen (CEA) is a broad-spectrum acid glycoprotein associated with colorectal cancer, lung cancer, and gastric cancer, etc. malignant tumors, because its concentration in serum is significantly positively correlated with tumor progression, it has become a core target for clinical diagnosis and treatment monitoring. Realizing the sensitive and convenient detection of CEA has great clinical significance for cancer prognosis evaluation and survival rate improvement.

[0003] To meet the clinical detection needs of carcinoembryonic antigen (CEA), although technologies such as electrochemical sensing, fluorescence detection, and colorimetric analysis (such as ELISA) based on sandwich immunoassay are widely used, there are the following problems: First, electrochemical methods are limited by electrode modification complexity, interference of serum electroactive substances (ascorbic acid / uric acid), and dependence on professional equipment; Second, fluorescence methods face probe light bleaching, sample autofluorescence interference, and high-cost equipment constraints; Third, traditional colorimetric methods (such as ELISA) are difficult to meet the requirements of rapid and accurate detection due to time-consuming and lengthy multi-step operations, and enzyme reactions are easily affected by environmental fluctuations.

[0004] Under this background, lateral flow immunochromatography (LFIA) technology has become an ideal path to realize CEA instant detection due to its simplicity, rapid response (<10 minutes), and equipment independence. This technology physically filters matrix interference through chromatography membrane capillary action, combined with naked-eye visual interpretation of gold nanoparticle labeled, significantly reducing detection cost and operation complexity.

[0005] Lateral flow immunochromatography technology (LFIA) is widely used in instant detection due to its advantages of simple operation and rapid detection. However, there are two key technical defects in the existing technology: 1. Low antibody labeling efficiency: The gold nanoparticles (AuNPs) prepared by traditional sodium citrate reduction method exhibit high negative surface charge (Zeta potential generally ≤-40 mV), which causes double negative effects in the antibody labeling process. On the one hand, the high charge density leads to the formation of a dense electrostatic repulsion layer on the surface of gold particles, significantly increasing the energy barrier for antibody proteins to approach the particle surface. On the other hand, the Fc region of antibody molecules (such as IgG) is forced to adopt a non-ideal orientation when approaching the gold surface due to strong electrostatic repulsion between like charges, resulting in insufficient exposure of its antigen binding site (Fab region). More critically, strong electrostatic repulsion makes it difficult for antibodies to effectively penetrate the energy barrier on the surface of gold particles, and a large number of antibodies cannot be stably combined with the gold surface due to steric hindrance effects, ultimately resulting in an actual antibody labeling efficiency of less than 60% (such as patent CN101059519A). This not only significantly reduces the effective probe concentration, but also exacerbates the chromatographic background noise due to the presence of unbound antibodies, directly affecting the improvement of detection sensitivity.

[0006] 2. Risk of salt inactivation of quality control line: The current LFIA system generally relies on protein such as goat anti-mouse IgG (or similar species-specific secondary antibody) to construct the quality control line (C line). After such protein is immobilized on the nitrocellulose (NC) membrane, it faces a serious risk of salt-induced inactivation in high ionic strength samples (such as physiological salinity serum, ionic strength ≥0.15M). The core mechanism is as follows: high concentration of salt ions (such as Na + , Cl - ) can neutralize the surface charge of the protein, destroy the electrostatic balance that maintains its native conformation, and cause partial unfolding and structural collapse of the protein; at the same time, salt ions competitively bind to the hydrophilic region of the protein, causing abnormal exposure of its surface hydrophobic groups, further exacerbating conformational distortion. This process not only masks the key active sites of the protein (such as the Fc binding domain of the antibody), but also significantly weakens its specific binding ability with the gold-labeled probe. The direct consequence is that the C line signal gradually weakens and even completely disappears with the increase of sample salinity, making the quality control function ineffective, ultimately leading to the loss of reliability of the detection result due to the loss of control during the chromatographic process, and constituting a systematic risk in high-salt sample detection.

[0007] 3. High cost of high antibody usage: In traditional technology, gold nanoparticles often need to add excess antibodies to achieve effective labeling due to their limited antibody binding capacity, which directly increases production costs. In addition, the dependence of the quality control line (C line) on the species non-specific secondary antibody further increases the cost of raw materials. The present application significantly reduces the antibody usage and reagent cost in immunochromatographic detection by using weakly ionized gold nanoparticles and cationic polymer quality control materials. Weakly ionized gold nanoparticles effectively improve the antibody labeling efficiency and reduce the excessive consumption of antibodies. At the same time, the cationic polymer replaces the traditional secondary antibody as the C line capture reagent, greatly reducing the procurement cost of the quality control line material, thereby effectively controlling the overall detection cost.

[0008] The above defects form a vicious cycle: low labeling efficiency leads to signal intensity attenuation, further leading to low-abundance marker missed detection; quality control line inactivation leads to loss of result reliability, further leading to high-salt sample false negative rate rising. Low labeling efficiency of gold nanoparticles leads to excessive addition of antibodies; the use of secondary antibodies in the quality control line leads to high procurement cost of secondary antibodies. Although research has improved gold particle uniformity and labeling efficiency by optimizing gold nanoparticle preparation process (such as adjusting pH, reducing agent dosage and addition speed) or using streptavidin-biotin system, the process is complex and has poor reproducibility, making it difficult to scale and stable application. To address the inactivation of the quality control line and high-salt interference, existing methods often use salt-tolerant chicken IgY antibodies or glucose oxidase color system to improve the quality control design, but they still do not solve the problem of non-specific adsorption of chromatography membrane under high salt, but increase the cost and complexity. In addition, the excessive antibodies and expensive secondary antibodies to compensate for the low-efficiency labeling lead to high cost, and there is no effective solution, which weakens the market competitiveness of the product. Existing technologies are mostly local optimization, and have not broken the vicious cycle of "low labeling efficiency - high antibody consumption - high cost - low reliability".

[0009] Therefore, developing a new material and technology platform with high-efficiency labeling, high robustness, low antibody usage and low cost has become a key challenge to promote the progress of gold nanoparticle immunochromatographic technology and break through the bottleneck of POCT development. SUMMARY

[0010] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as a quality control line and a preparation method thereof. The weakly ionized colloidal gold immunochromatographic test strip of the present application breaks through the bottleneck of the prior art by constructing a double anti-salt mechanism of weakly ionized colloidal gold labeling and cationic polymer quality control line. In the labeling link, the Zeta potential of the gold particle surface is accurately controlled to the weak negative interval of-20 to-30 mV by using sodium ascorbate, which significantly reduces the steric hindrance of antibody binding, and the labeling efficiency is improved; In the quality control link, polydimethyl diallyl ammonium chloride (PDDA) is used to replace protein to construct C line, which has strong positive electricity (Zeta potential ≥ +30 mV) and can efficiently capture weakly negatively charged gold probes through electrostatic attraction, completely avoiding the risk of protein salting-out inactivation in high-salt samples, and the production cost of C line is reduced by 90% (PDDA replaces expensive secondary antibody + antibody dosage optimization).

[0011] The present application forms a directional charge adsorption synergy through weakly negatively charged gold probes and strongly positively charged PDDA quality control line, which still maintains a capture stability of >95% (coefficient of variation CV<10%) even in a high-salt environment, realizes a double-link anti-salt interference closed loop from labeling to quality control, and greatly reduces the cost by reducing the amount of antibody or protein, providing a reliable solution for trace marker detection in complex samples.

[0012] The first aspect of the present application provides a weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as a quality control line.

[0013] The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as a quality control line comprises a substrate, a nitrocellulose membrane, a binding pad, and a water absorption pad. The nitrocellulose membrane, the binding pad, and the water absorption pad are all distributed on the surface of the substrate. The binding pad is lapped on the nitrocellulose membrane, the water absorption pad is lapped on the nitrocellulose membrane, and the binding pad and the water absorption pad are distributed on both sides of the nitrocellulose membrane. The nitrocellulose membrane has a detection line and a quality control line. The quality control line is coated with polydimethyl diallyl ammonium chloride. The weakly ionized colloidal gold immunochromatographic test strip further comprises a CEA monoclonal antibody Ab1-weakly ionized colloidal gold complex. The CEA monoclonal antibody Ab1-AA-AuNPs complex is mixed with the sample to be tested when the weakly ionized colloidal gold immunochromatographic test strip is used, and then dropped on the binding pad.

[0014] Preferably, the amount of polydimethyl diallyl ammonium chloride coated in the quality control line (i.e. C line) is 1 μg / cm-5 μg / cm.

[0015] Preferably, the detection line is close to the conjugate pad, and the quality control line is close to the absorbent pad.

[0016] Preferably, the detection line (i.e. T line) is coated with CEA monoclonal antibody Ab2.

[0017] Preferably, the coating amount of CEA monoclonal antibody Ab2 in the detection line is 0.05 mg / mL-0.2 mg / mL.

[0018] Preferably, the distance between the detection line and the quality control line is 5-35 mm. For example, 5 mm, 20 mm, 30 mm, 35 mm.

[0019] Preferably, the conjugate pad overlaps 1-2 mm on the nitrocellulose membrane.

[0020] Preferably, the absorbent pad overlaps 1-2 mm on the nitrocellulose membrane.

[0021] Preferably, the composition of the conjugate pad includes glass fiber.

[0022] Preferably, the composition of the absorbent pad includes cellulose.

[0023] Preferably, the composition of the substrate includes at least one of PVC (polyvinyl chloride), PE (polyethylene), PP (polypropylene), and more preferably PVC.

[0024] The second aspect of the present application provides a preparation method of a weakly ionized colloidal gold immunochromatographic test strip based on cationic polymer as a quality control line.

[0025] The preparation method of a weakly ionized colloidal gold immunochromatographic test strip based on cationic polymer as a quality control line includes the following steps: (1) Preparation of conjugate pad: Take a glass fiber substrate, immerse it in a phosphate buffer solution, then dry to obtain a conjugate pad; (2) Preparation of nitrocellulose membrane containing quality control line and detection line: Spray polydimethyl diallyl ammonium chloride solution on the nitrocellulose membrane, solidify to form a quality control line; dilute CEA monoclonal antibody Ab2 with PBS, spray on the other side of the nitrocellulose to form a detection line, solidify to obtain a nitrocellulose membrane containing a quality control line and a detection line; (3) Preparation of antibody-weakly ionized colloidal gold complex: Mix HAuCl4, deionized water, and hydrochloric acid, heat, then add sodium ascorbate to obtain weakly ionized colloidal gold, then adjust the pH of the weakly ionized colloidal gold to weakly alkaline, and add CEA monoclonal antibody Ab1 to form an Ab1-AA-AuNPs solution, cool, react, separate to obtain an antibody-weakly ionized colloidal gold complex; (4) Assembly of the test strip: Place the nitrocellulose membrane containing the quality control line and the detection line on the surface of the substrate, then paste the water-absorbing pad on the substrate near the side of the quality control line on the nitrocellulose membrane, and overlap the nitrocellulose membrane, paste the binding pad on the substrate near the side of the detection line on the nitrocellulose membrane, and overlap the nitrocellulose membrane, dry, and obtain the test strip. The test strip and the antibody-weakly ionized colloidal gold complex together constitute the weakly ionized colloidal gold immunochromatography test strip.

[0026] The order of the above step (1) and step (3) can be arbitrarily adjusted.

[0027] Preferably, the preparation of the binding pad comprises the following steps: Take the glass fiber substrate, uniformly immerse it in a phosphate buffer solution with pH = 7-8 (the phosphate buffer solution also contains 0.5%-5% sucrose by mass fraction, 0.5%-5% bovine serum albumin by mass fraction, and 0.5%-5% Tween-20 by mass fraction), and after ensuring complete soaking, transfer it to a constant temperature environment at 37°C for drying for 2-3h, to obtain the binding pad.

[0028] Preferably, the preparation of the quality control line comprises the following steps: Spray 5wt%-25wt% PDDA solution at a spraying amount of 1μL / cm-5μL / cm at a position 20mm-35mm downstream of the nitrocellulose membrane, dry and solidify at 37-60°C, to obtain the quality control line.

[0029] Preferably, the preparation of the detection line comprises the following steps: Dilute CEA monoclonal antibody Ab2 with PBS to 0.05mg / mL-0.2mg / mL, spray it on the nitrocellulose membrane at a spraying amount of 1μL / cm-5μL / cm, and maintain a distance of 5-35mm from the quality control line, to obtain the detection line.

[0030] Preferably, the Zeta potential of the weakly ionized colloidal gold is -20mV to -30mV, and the particle size is 80±5nm. The positive charge density on the surface of the weakly ionized colloidal gold increases the loading efficiency of the antibody Ab1.

[0031] The preparation of the antibody-weakly ionized colloidal gold complex comprises the following steps: (1) Add deionized water containing HAuCl4 into a container, add 100μL of 0.1M-0.3M HCl solution, heat to 80-90°C, then rapidly add 1-1.5 mL of 1%-5% mass fraction sodium ascorbate solution under vigorous stirring, cool the obtained deep red solution at room temperature, then centrifuge to remove residual reagents, to obtain weakly ionized colloidal gold; (2) Adjust the pH of the weakly ionized colloidal gold to 7-7.6 using a 0.05 mol / L-0.2 mol / L potassium carbonate solution, then add CEA monoclonal antibody Ab1 to form an Ab1-AA-AuNPs solution, the mass / volume ratio of Ab1 and the weakly ionized colloidal gold solution is 10-20:1, and the reaction is carried out in a 4°C shaking table for 1-2 hours; then 0.5%-2% bovine serum albumin is added for blocking for 0.5-1 hour, the supernatant is removed by centrifugation, and the obtained antibody-weakly ionized colloidal gold complex is obtained after centrifugation.

[0032] Preferably, the preparation of the antibody-weakly ionized colloidal gold complex comprises the following steps: (1) Deionized water containing HAuCl4 (50 mL, 10 mM-50 mM) is added to a 100 mL round-bottom flask. 100 μL of 0.1 M-0.3 M HCl solution is added, heated to 80-90°C, then 1-1.5 mL of 1%-5% mass fraction sodium ascorbate (AA) solution is quickly added under vigorous stirring, the obtained deep red solution is cooled at room temperature, then centrifuged (6000 rpm-9000 rpm, 5-10 min, 4-25°C) to remove residual reagents, and weakly ionized colloidal gold (denoted as AA-AuNP, the particle size of the colloid is 60-90 nm) is obtained; (2) Adjust the pH of the weakly ionized colloidal gold to 7-7.6 using a 0.05 mol / L-0.2 mol / L potassium carbonate solution, then add CEA monoclonal antibody Ab1 to form an Ab1-AA-AuNPs solution, the mass / volume ratio of Ab1 and the weakly ionized colloidal gold solution is 10-20:1, and the reaction is carried out in a 4°C shaking table for 1-2 hours; then 0.5%-2% BSA (bovine serum albumin) is added for blocking for 0.5-1 hour, the supernatant is removed by centrifugation, and the obtained antibody-weakly ionized colloidal gold complex (denoted as AA-AuNPs@Ab) is concentrated and stored at 4-25°C for standby using a 10-30 mM Na3PO4 buffer containing 2%-5% BSA, 0.1%-2% Tween-20 and 1%-10% sucrose.

[0033] Compared with the prior art, the present application has the following beneficial effects: The present application adopts a double antibody sandwich immunochromatographic method to quantitatively / semi-quantitatively detect CEA in a serum sample, and the core inventive concept is that: (1) Weakly ionized colloidal gold labeling technology: Gold nanoparticles with reduced surface charge density are prepared by adjusting sodium ascorbate. The weak ionization characteristics significantly improve the antibody loading capacity (at least 50% higher than traditional gold particles), making the weakly ionized colloidal gold labeled mouse anti-human CEA monoclonal antibody (Au-Ab1) binding efficiency > 95%, and the sensitivity breakthrough to 1 ng / mL; (2) First use of cationic polymer PDDA instead of traditional secondary antibody quality control line: Nitrocellulose membrane quality control line (C line) coated with poly (diallyldimethylammonium chloride, PDDA) captures free Au-Ab1 through strong electrostatic adsorption, completely solving the problem of traditional protein control line inactivation under high salt samples, with batch difference < 10%. During testing, the test serum is treated by centrifugal tube and added to the conjugate pad. CEA in the test sample forms a complex with Au-Ab1; when chromatographed to the detection line (T line), the CEA capture antibody (Ab2) fixed on it specifically captures to form a "Ab1-CEA-Au-Ab2" sandwich structure, showing a red band (color intensity is positively correlated with CEA concentration). Free Au-Ab1 continues to migrate to the C line, where PDDA efficiently captures and develops through electrostatic interaction (any effective detection requires color development). Avoiding the use of protein antibodies on the C line of traditional test strips, the cost is greatly reduced. The weakly ionized colloidal gold immunochromatography test strip realizes precise analysis of CEA through the gray scale ratio of T / C line (completed within 10 minutes), with a detection limit of 0.9 pg / mL, providing a revolutionary technical tool for postoperative home dynamic monitoring of colorectal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The schematic structure and working principle of the test strip of the present application are shown in the figure; Figure 2 The color development effect of different nitrocellulose membranes in the test strip is shown in the figure; Figure 3 The performance comparison effect of different cationic polymers on the C line in the test strip is shown in the figure; Figure 4 The characterization of weakly ionized colloidal gold and ordinary gold nanoparticles prepared in Example 1 is shown in the figure; Figure 5 The characterization of antibody-weakly ionized colloidal gold complex and antibody-ordinary gold nanoparticle complex prepared in Example 1 is shown in the figure; Figure 6 The comparison results of the detection range of antibody-weakly ionized colloidal gold complex and antibody-ordinary gold nanoparticle complex prepared in Example 1 in standard CEA solution are shown in the figure; Figure 7 The time effectiveness results of antibody-weakly ionized colloidal gold complex prepared in Example 1 are shown in the figure; Figure 8 The salt tolerance comparison results of cationic polymer and non-specific antibody on the C line are shown in the figure; Figure 9 Selectivity of the antibody-weakly ionized colloidal gold complex prepared in Example 1 against different biomarkers; Figure 10 Detection range of the test strip prepared in Example 1 and the antibody-weakly ionized colloidal gold complex in real sample serum. DETAILED DESCRIPTION

[0035] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are listed for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0036] The raw materials, reagents or devices used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0037] The main reagent source channels are as follows: The CEA antigen and antibody were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; the polydimethyl diallyl ammonium chloride solution was purchased from Macklin; the binding pad, absorption pad, and nitrocellulose membrane (NC membrane) were purchased from Shanghai Jieyi Biotechnology Co., Ltd.; and the raw materials for synthesizing gold nanoparticles were purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0038] Figure 1 It is a schematic structure and working principle diagram of the test strip of the present application; wherein, “Traditional strong ionization” represents traditional strong ionization, “Weak ionization” represents weak ionization, “Low load capacity” represents low load capacity, “High load capacity” represents high load capacity, “Positive” represents positive, and “Negative” represents negative.

[0039] Figure 2 It is the color development effect of different nitrocellulose membranes in the test strip.

[0040] Figure 3 It is the performance comparison effect of C line corresponding to different cationic polymers in the test strip.

[0041] Figure 4 It is the characterization of weakly ionized colloidal gold prepared in Example 1 and ordinary gold nanoparticles; wherein, “AuNPs” represents ordinary gold nanoparticles, “AA-AuNPs” represents weakly ionized colloidal gold prepared in Example 1, “Zeta Potential” represents Zeta potential, “Wavelength” represents wavelength, and “Absorbance” represents absorbance. Figure 4Figure A in Figure 1 shows that AA-AuNPs have spherical morphology by transmission electron microscopy (TEM). The morphology and particle size of AA-AuNPs remain unchanged. As shown in Figure B in Figure 1, the Zeta potential of AuNPs is -38 mV, and that of AA-AuNPs is reduced by 10.2 mV to -27.8 mV. Figure 4 Figure C in Figure 1 shows that the synthesized AA-AuNPs have similar absorption peaks as ordinary AuNPs, with an absorption peak at about 527 nm.

[0042] Figure 5 Characterization of the antibody-weakly ionized colloidal gold complex prepared in Example 1 and the antibody-ordinary gold nanoparticle complex; wherein "Intensity" represents intensity, "Binding energy" represents binding energy, "Size" represents size, and "Diameter" represents diameter, from Figure 5 It can be seen that the AA-AuNPs are successfully coupled with the CEA antibody.

[0043] Figure 6 Comparison results of the detection range of the antibody-weakly ionized colloidal gold complex prepared in Example 1 and the antibody-ordinary gold nanoparticle complex in a standard CEA solution; wherein Figure (a1) and (a2) correspond to the detection results of the antibody-weakly ionized colloidal gold complex prepared in Example 1, and Figure (b1) and (b2) correspond to the detection results of the antibody-ordinary gold nanoparticle complex.

[0044] Figure 7 Time-dependent results of the antibody-weakly ionized colloidal gold complex prepared in Example 1.

[0045] Figure 8 Comparison results of the salt tolerance of the cationic polymer and the non-specific antibody in C line.

[0046] Figure 9 Selectivity of the antibody-weakly ionized colloidal gold complex prepared in Example 1 against different biomarkers.

[0047] Figure 10 Detection range of the test strip prepared in Example 1 and the antibody-weakly ionized colloidal gold complex in real sample serum.

[0048] Example 1 The preparation method of the weakly ionized colloidal gold immunochromatography test strip based on the cationic polymer as a quality control line, comprising the following steps: (1) Treatment of the conjugate pad: Take a glass fiber matrix and uniformly impregnate it in a phosphate buffer solution with pH=7.4 (the phosphate buffer solution also contains 1% by mass of sucrose, 1% by mass of bovine serum albumin, and 1% by mass of Tween-20 buffer solution). After ensuring complete impregnation, transfer it to a constant temperature environment of 37℃ and dry it for 2 hours to obtain the conjugation pad. (2) Preparation method of antibody-weakly ionized colloidal gold complex: a. Preparation of weakly ionized colloidal gold: 41 mL of 1% HAuCl solution was added to a 100 mL round-bottom culture flask, followed by 49 mL of deionized water. The solution was stirred and heated to 80°C, then 100 μL of 0.1 M HCl solution was added. Heating continued to 90°C, followed by rapid addition of 1 mL of 1% sodium ascorbate (AA) solution under vigorous stirring for 5 min. The resulting deep red solution was cooled to room temperature and then centrifuged (8000 rpm, 5 min, 4°C) to remove residual reagents, yielding weakly ionized colloidal gold (denoted as AA-AuNPs). Characterization of the weakly ionized colloidal gold is as follows: Figure 4 As shown; b. Preparation of antibody-weakly ionized colloidal gold complex (AA-AuNPs@Ab1): The pH of the weakly ionized colloidal gold was adjusted to 7.4 using 0.1 mol / L potassium carbonate solution. Then, CEA monoclonal antibody Ab1 was added to form an Ab1-AA-AuNPs solution with a mass-to-volume ratio of Ab1 to AA-AuNPs solution of 10:1 (μg / mL). The reaction was carried out at 4°C in a shaker for 1 h. Subsequently, 1% BSA was added for blocking for 0.5 h. The supernatant was removed by centrifugation. After centrifugation (8000 rpm, 5 min, 4°C), the obtained AA-AuNPs@Ab1 product was concentrated and stored at 4°C using a 10 mM Na3PO4 buffer (containing 5% bovine serum albumin, 1% Tween-20, and 1% sucrose). The antibody-weakly ionized colloidal gold complex (AA-AuNPs@Ab1) was characterized as follows: Figure 5 As shown; (3) Constructing a cationic polymer control line and detection line resistant to salt interference: First, the polydimethyldiallylammonium chloride solution (PDDA, molecular weight 491.06 g / mol) was diluted with water to 5 wt%. The solution was then precisely sprayed onto the control area (control line, i.e. C line position) of a nitrocellulose (NC) membrane (NC membrane size is 25×4 mm) with a line width of 5 μL / cm using a micro quantitative stripping system. The membrane was then placed in a constant temperature oven at 40℃ for 1 h to complete cross-linking and fixation. The potential of PDDA was detected by a Zeta potentiometer and was found to be stable at +35.2±1.3 mV (n=5). Its strong positive charge characteristics laid the foundation for subsequent probe capture. The preparation of the detection line (T line) was carried out simultaneously: CEA monoclonal antibody (Ab2) was diluted with PBS to 100 μg / mL and sprayed onto the detection area of ​​the nitrocellulose membrane at a line width of 5 μL / cm, maintaining a 5 mm gap with the C line to ensure signal separation. After spraying, the NC membrane was dried at 40℃ to obtain a bifunctional reaction membrane, that is, the nitrocellulose membrane has a detection line and a control line.

[0049] (4) The test strip assembly adopts a lamination process to sequentially integrate key components: the nitrocellulose membrane containing the control line and the detection line is placed on the surface of the PVC board, and then the cellulose absorbent pad is pasted on the surface of the PVC board and close to the side of the control line on the nitrocellulose membrane, and overlaps with the nitrocellulose membrane by 2mm. The conjugate pad is pasted on the surface of the PVC board and close to the side of the detection line on the nitrocellulose membrane, and overlaps with the nitrocellulose membrane by 2mm. After drying, the test strip is obtained. The test strip and the antibody-weakly ionized colloidal gold complex together constitute the weakly ionized colloidal gold immunochromatographic test strip, which can be cut into test strips with a width of 3.8mm.

[0050] Application Example 1 Optimize the weakly ionized colloidal gold immunochromatography technique based on cationic polymers as control lines: Four different NC membranes (Pall Vivid 90, Pall Vivid 120, Sartorius CN95, and Sartorius CN140, respectively) were streaked with 5 μg / mL CEA antibody Ab2 at the detection line, and the control line was streaked with 5 wt% dimethyl diallyl ammonium chloride solution. The test strips were prepared according to the method in Example 1.

[0051] The antibody-weakly ionized colloidal gold complex was then mixed with the test sample and dropped onto the conjugation pad. Running buffer was added, and the T-line and C-line were observed visually. The results are as follows: Figure 2 As shown, when using Sartorius CN95 and Pall Vivid 90NC membranes, the membrane pores were too large, resulting in excessively fast flow rates and a faint red T-line. When using the Pall Vivid 120 NC membrane, the T-line was too wide, also resulting in a faint color. However, when using the Sartorius CN140 NC membrane, the T / C bands showed good color development with a suitable development time. Therefore, the Sartorius CN140 NC membrane yielded the best results.

[0052] Application Example 2 Optimization of weakly ionized colloidal gold immunochromatography based on cationic polymers as control lines: Based on Example 1, test strips were prepared according to the method of Example 1, with the only difference being the change in dimethyl diallyl ammonium chloride in the control line. Specifically, the differences in color development at the C line were compared between different positively charged solutions. Three replicate experiments were performed to compare the color development at the C line for different positively charged solutions: choline chloride (CHCl), benzalkonium chloride (PHARMA), tetramethylammonium hydroxide (TMAH), dimethyl diallyl ammonium chloride (DMDAAC), polyquaternium-7 (PQNM-7), and poly(acrylamine hydrochloride) (AVERAGE).

[0053] The antibody-weakly ionized colloidal gold complex was then mixed with the sample to be tested and dropped onto the conjugate pad. The C-line was observed visually. Due to the differences in the bands, the results are as follows: Figure 3 As shown, the performance of using PDDA is significantly better than CHCL, PHARMA, TMAH, and DMDAAC.

[0054] Application Example 3 Based on the comparison of detection limits between ordinary gold nanoparticles and weakly ionized colloidal gold: Antibody-ordinary gold nanoparticle complexes were prepared by replacing the weakly ionized colloidal gold in Example 1 with purchased 80nm gold nanoparticles.

[0055] Subsequently, the prepared antibody-ordinary gold nanoparticle complex and the antibody-weakly ionized colloidal gold complex prepared in Example 1 were added to CEA antigen at concentrations ranging from 0 pg / mL to 100 pg / mL, respectively, and then tested using test strips. The results are as follows: Figure 6 As shown, the antibody-weakly ionized colloidal gold complex can be detected at 10 pg / mL, while the antibody-ordinary gold nanoparticle complex can only be detected at 100 pg / mL. By comparing the two different gold nanoparticles, it can be seen that the detection performance of the weakly ionized colloidal gold is significantly better than that of the ordinary gold nanoparticles. Using RGB software to analyze the grayscale values, based on the linear curve and detection range, excellent recoveries (96.7-108.3%) and very small relative standard deviations (RSD) (9.3-14.9%) of the weakly ionized colloidal gold were obtained, as shown in Table 1.

[0056] Table 1: Spiking recoveries and relative standard deviations (RSD) of the antibody-weakly ionized colloidal gold complex prepared in Example 1 in standard samples. Application Example 4 The stability of the antibody-weakly ionized colloidal gold conjugate antibody was investigated: the stability of the antibody-weakly ionized colloidal gold complex (AA-AuNPs@Ab1) prepared in Example 1 was tested for 1-14 days by using test strips on the same sample. The results are as follows: Figure 7As shown, when AA-AuNPs@Ab1 stored for 1-14 days were tested on the same sample, the T-line bands showed almost no difference when observed with the naked eye. The signal intensity of AA-AuNPs aggregated at the T-line showed little change, indicating the high stability advantage of this weakly ionized colloidal gold conjugate antibody (i.e., antibody-weakly ionized colloidal gold complex).

[0057] Application Example 5 Salt resistance test based on cationic polymer as quality control line: Results are as follows Figure 8 As shown, the experimental group used the test strips prepared in Example 1 (i.e., test strips with cationic polymer PDDA as the C-line capture substance), while the control group used test strips with a non-specific antibody: goat anti-mouse IgG (which does not cross-react with the detection line antibody, i.e., the conventional secondary antibody on the C-line) 1 mg / mL as the C-line capture substance. Seven concentrations of NaCl solution (e.g., 0 mM, 100 mM, 200 mM, 400 mM, 800 mM, 1.0 M, 2.0 M) were prepared in the running buffer solution, and each concentration point was tested using test strips from both the experimental and control groups.

[0058] The results showed that: Expected Result 1: As salt concentration increased, the intensity of the C-line in the control group (antibody) rapidly decreased, diffused, and became lighter, while the C-line intensity in the experimental group (polymer PDDA) remained better even in a high-salt environment with a concentration of 2M, resulting in a cleaner background. These data clearly demonstrate that cationic polymer PDDA, as a C-line trapping substance, has significantly better resistance to salt interference than non-specific antibodies. Furthermore, as shown in Table 1, the price of cationic polymer PDDA (unit price 23.94 yuan) is far lower than that of non-specific antibodies (goat anti-mouse IgG unit price 441 yuan), only 1 / 20th of the cost of its traditional C-line usage, thus significantly reducing the overall manufacturing cost of the test strip.

[0059] Application Example 6 CEA detection using a weakly ionized colloidal gold immunochromatographic test strip with cationic polymers as quality control lines: To verify the selectivity of this experiment, CEA, Myo, AFP, PSA, ALP, and cTnL were each measured at concentrations of 50 pg / mL-100 pg / mL, with three replicates. The qualitative detection method is as follows: Serum samples containing the analytes were added to the antibody-weakly ionized colloidal gold complex prepared in Example 1 and incubated for 1 hour. Subsequently, the sample was added to the conjugation pad of the test strip prepared in Example 1, followed by the addition of running buffer.

[0060] The results are as follows Figure 9As shown, visual observation of lines T and C reveals a distinct red band on the CEA test strip. The control line exhibits a red band due to the aggregation of red nanoparticles. No obvious red bands were observed on the Myo, AFP, PSA, ALP, and cTnl test strips, indicating that the test strips prepared in Example 1 possess the advantages of high selectivity and high specificity. A significant difference can be observed between the color intensity of lines T and C across the six strips.

[0061] Application Example 7 Detection of serum CEA using a weakly ionized colloidal gold immunochromatographic test strip with cationic polymer as the quality control line: Step 1: Experimental Design (Concentration Gradient Setting) Design 5 core concentration points (including the zero point), with 3 replicates (n=3) for each point to ensure precision calculation. Set the following theoretical concentrations of CEA: 0 ng / mL (blank matrix control, used to observe background), 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, 1 ng / mL; Step 2: Preparation of spiked samples (key step) Blank matrix pretreatment: Dilute the negative mixed serum 1:10, for example, take 20 μL serum + 180 μL PBS, mix well, and all subsequent operations are performed in this 1:10 diluted matrix. Preparation of intermediate concentration standards: Dilute the 1 mg / mL CEA stock solution to an intermediate concentration with buffer to facilitate sample loading calculation.

[0062] The results are as follows Figure 10 As shown, the T line gradually darkens with increasing CEA concentration, while the C line remains stable. The T line is clear when the CEA concentration is 1 ng / mL. Stable immune complexes are formed when AA-AuNPs@Ab1 in the detection solution specifically binds to CEA.

[0063] During chromatography, as the complex flows through the NC membrane, the sandwich binding effect between AA-AuNPs@Ab1@CEA in the solution and the antibody Ab2 coated on the T line causes the color intensity of the T line to increase with increasing analyte concentration. The C line, however, shows color due to the stable binding of PDDA to AA-AuNPs. This result is consistent with the immunological rules of the sandwich method using test strips. The test strip exhibits good linearity in the range of 1-100 ng / mL, with the linear equation being y = 0.0518 + 0.00723x (R²). 2 = 0.9803).

Claims

1. A weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines, characterized in that... Includes substrate, nitrocellulose membrane, bonding pad, and absorbent pad; The nitrocellulose membrane, conjugation pad, and absorbent pad are all distributed on the surface of the substrate. The binding pad overlaps the nitrocellulose membrane, the absorbent pad overlaps the nitrocellulose membrane, and the binding pad and the absorbent pad are distributed on both sides of the nitrocellulose membrane; The nitrocellulose membrane has detection lines and quality control lines; The quality control line is coated with polydimethyldiallylammonium chloride; The weakly ionized colloidal gold immunochromatographic test strip also includes a CEA monoclonal antibody Ab1-weakly ionized colloidal gold complex.

2. The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to claim 1, characterized in that, The coating amount of polydiallyl ammonium chloride in the quality control line is 1 μg / cm-5 μg / cm.

3. The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to claim 1, characterized in that, The detection line is close to the conjugation pad, and the quality control line is close to the absorbent pad.

4. The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to claim 1, characterized in that, The detection line is coated with CEA monoclonal antibody Ab2.

5. The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to claim 4, characterized in that, The amount of CEA monoclonal antibody Ab2 coated in the detection line is 0.05 mg / mL to 0.2 mg / mL.

6. The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to claim 1, characterized in that, The distance between the detection line and the quality control line is 5-35mm; and / or, the bonding pad overlaps the nitrocellulose membrane by 1-2mm; and / or, the absorbent pad overlaps the nitrocellulose membrane by 1-2mm.

7. The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to claim 1, characterized in that, The bonding pad is composed of glass fiber; and / or the absorbent pad is composed of cellulose.

8. The weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to claim 1, characterized in that, The substrate comprises at least one of polyvinyl chloride, polyethylene, and polypropylene.

9. The method for preparing the weakly ionized colloidal gold immunochromatographic test strip based on cationic polymers as quality control lines according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Preparation of conjugate pad: Take glass cellulose matrix, impregnate it in phosphate buffer, and then dry it to obtain conjugate pad; (2) Preparation of nitrocellulose membrane containing control lines and detection lines: Dimethyl diallyl ammonium chloride solution was sprayed onto the nitrocellulose membrane and cured to form control lines; CEA monoclonal antibody Ab2 was diluted with PBS and sprayed onto the other side of the nitrocellulose membrane to form detection lines, and cured to obtain nitrocellulose membrane containing control lines and detection lines; (3) Preparation of antibody-weakly ionized colloidal gold complex: HAuCl4, deionized water and hydrochloric acid are mixed and heated, and then sodium ascorbate is added to obtain weakly ionized colloidal gold. Then the pH of the weakly ionized colloidal gold is adjusted to weakly alkaline, and CEA monoclonal antibody Ab1 is added to form Ab1-gold nanoparticle solution. The mixture is cooled, reacted, and separated to obtain antibody-weakly ionized colloidal gold complex. (4) Assembly of test strips: Place the nitrocellulose membrane containing the control line and the detection line on the substrate surface, then attach the absorbent pad to the substrate and close to the control line on the nitrocellulose membrane, overlapping with the nitrocellulose membrane. Attach the conjugate pad to the substrate and close to the detection line on the nitrocellulose membrane, overlapping with the nitrocellulose membrane. Dry to obtain the test strip. The test strip and the antibody-weakly ionized colloidal gold complex together constitute the weakly ionized colloidal gold immunochromatographic test strip.

10. The preparation method according to claim 9, characterized in that, The preparation of the quality control line includes the following steps: A 5wt%-25wt% polydimethyldiallylammonium chloride solution was sprayed at a rate of 1μL / cm-5μL / cm onto a 20mm-35mm downstream of a nitrocellulose membrane and dried and cured at 37-60℃ to obtain the quality control line. And / or, the preparation of the antibody-weakly ionized colloidal gold complex includes the following steps: (1) Add deionized water containing HAuCl4 to a container, add 100 μL of 0.1M-0.3M HCl solution, heat to 80-90℃, and then quickly add 1-1.5 mL of 1%-5% sodium ascorbate solution under vigorous stirring. The resulting dark red solution is cooled at room temperature and then centrifuged to remove residual reagents to obtain weakly ionized colloidal gold. (2) Adjust the pH of the weakly ionized colloidal gold to 7-7.6 using 0.05mol / L-0.2mol / L potassium carbonate solution, then add CEA monoclonal antibody Ab1 to form Ab1-AA-AuNPs solution. The mass-to-volume ratio of Ab1 to AA-AuNPs solution is 10-20:

1. React in a shaker at 4℃ for 1-2h. Then add 0.5%-2% bovine serum albumin to block for 0.5-1h. Centrifuge to remove the supernatant. After centrifugation, obtain the antibody-weakly ionized colloidal gold complex.

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