A lateral flow immunochromatographic test strip based on enzyme / magnetic driven magnetic gold nanoprobe
By employing magnetic gold nanomaterials combined with nanomotor properties in lateral flow immunochromatography, and utilizing enzyme-driven and magnetic-driven methods, the problems of low sensitivity and long detection time in existing technologies have been solved, achieving rapid and accurate detection results.
Patent Information
- Application Number
- CN202510207430.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2026-08-25
AI Technical Summary
Existing lateral flow immunochromatography techniques suffer from low sensitivity, inability to perform quantitative analysis, and long detection times, especially with insufficient research on nanomotor technology to shorten detection time.
Using magnetic gold nanomaterials as probes and combining them with the characteristics of nanomotors, the diffusion enhancement and magnetic drive of the nanoprobes are achieved through enzyme-driven and magnetic-driven methods. This is achieved by utilizing the glucose oxidase activity of gold nanostars and the superparamagnetism of Fe3O4, thereby shortening the detection time.
It enables faster and more accurate detection, significantly shortens detection time, and improves detection sensitivity and accuracy.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of immunoassay technology, specifically relating to a lateral flow immunochromatographic test strip based on an enzyme / magnetically driven magnetic gold nanoprobe. Background Technology
[0002] Lateral flow immunochromatography (LFIA) has been widely used for the point-of-care testing of disease biomarkers due to its simplicity, low cost, and unique ability to provide visually observable results. LFIA combines nanomaterial labeling technology with the specific reaction between antigens and antibodies to detect target analytes in complex matrices. Therefore, nanomaterials play a crucial role in LFIA, enabling visual detection of analytes based on colorimetric reactions caused by nanomaterial aggregation. Traditional lateral flow immunochromatography uses gold nanoparticles as nanoprobes, but its low sensitivity and inability to quantify limit its widespread application. To address these issues, various nanomaterials have been prepared for LFIA. Nanomotors can achieve autonomous movement by converting energy from various sources, such as internal chemical fuels (e.g., hydrogen peroxide, glucose, urea, and water) or external fields (e.g., light, ultrasound, electricity, and magnetism), into mechanical force. Existing research mainly focuses on the role of other nanomaterials in improving sensitivity and solving quantification problems, with limited research on the role of combining nanomaterials with nanomotor technology in shortening detection time.
[0003] Therefore, it is proposed to use magnetic gold nanomaterials as probes and combine them with the properties of nanomotors to shorten the detection time in LFIA. Summary of the Invention
[0004] The main objective of this invention is to provide a faster and more accurate lateral flow immunochromatographic test strip based on enzyme / magnetically driven magnetic gold nanoprobes, taking into account the autonomous motion characteristics of LFIA technology and nanomotors, in order to shorten the detection time.
[0005] The technical solution of the present invention is as follows:
[0006] This invention provides a lateral flow immunochromatographic test strip based on an enzyme / magnetically driven magnetic gold nanoprobe, the immunochromatographic test strip comprising:
[0007] Magnetic gold nanocomposite material is used as a nanoprobe, wherein the nanoprobe includes a core (Fe3O4)-shell (PDA) structure and is surface-modified with gold nanostars (AuNS) in the form of magnetic gold nanocomposite material (Fe3O4@PDA@AuNS, MPAuNS).
[0008] A self-assembled test strip, comprising a sample pad, a conjugation pad, a nitrocellulose membrane, an absorbent pad, and a base plate. IgG (1 mg / mL) and 560 cc (1 mg / mL) of anti-cTnI monoclonal antibody were sprayed onto the nitrocellulose membrane at a spray rate of 1 μL / cm to construct the test line (T line) and control line (C line), respectively.
[0009] A cTnI target analyte solution containing glucose, wherein the target analyte solution comprises cTnI, glucose, and PBS buffer.
[0010] Preferably, the glucose-containing cTnI target analyte solution has a glucose concentration of 0, 10, 25, 50, or 100 mM, with 50 mM being the most preferred.
[0011] This invention also provides a lateral flow immunochromatographic test strip based on enzyme / magnetically driven magnetic gold nanoprobes, comprising the following steps:
[0012] S1: Soak the sample pad and conjugate pad in buffer solution, then dry them at 37°C for 3 hours for the next step. Attach the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad to the substrate, overlapping them by 2 mm to construct the LFIA test strip. Cut the assembled test strip into 3 mm wide strips for use.
[0013] S2: Add the magnetic gold nanoprobe conjugated with 560cc of antibody to the analytical solution, mix well, incubate, and then drop it onto the sample pad. It flows forward under capillary suction. Finally, record the results on the camera and measure the gray intensity of the T line using an immunoassay analyzer.
[0014] S3: Add glucose to the cTnI analysis solution. Relying on the glucose oxidase activity of gold nanostars, the glucose in the cTnI solution is decomposed by gold nanostars to achieve enzyme-driven operation.
[0015] S4: Place an external magnet with the same magnetic induction intensity on the absorbent pad of the test strip, and rely on the superparamagnetism of Fe3O4 to achieve magnetic drive.
[0016] Preferably, step S3 contains a cTnI target analyte solution containing glucose, with a glucose concentration of 0, 10, 25, 50, or 100 mM, preferably 50 mM.
[0017] The principle of this invention is as follows: AuNS can be coupled to monoclonal antibodies (mAbs) through electrostatic adsorption. Furthermore, the quinone groups of the PDA coating actively participate in the nucleophilic addition reactions of the -SH and -NH2 residues of the anti-cTnI monoclonal antibody 560cc, enhancing the immobilization of the anti-cTnI monoclonal antibody 560cc. Bovine serum albumin and methoxy polyethylene glycol thiol (MPEG-SH) are then used to block residual binding sites on the MPAuNS surface, increasing the dispersibility of the conjugate in solution and reducing non-specific adsorption between the conjugate and cTnI, thereby improving the accuracy of LFIA. During detection, the analytical solution to be tested is added to the nanoprobe solution, mixed, incubated, and then dropped onto the sample pad. The solution flows through the test strip via capillary action, forming a colored region on the strip with a labeled immune complex.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0019] 1) In this invention, glucose is added to the cTnI analysis solution. Relying on the glucose oxidase activity of gold nanostars, the diffusion of the nanoprobe is enhanced by the decomposition of glucose in the cTnI solution by gold nanostars, thereby achieving enzyme-driven diffusion.
[0020] 2) In this invention, an external magnet with the same magnetic induction intensity is placed on the absorption pad of the test strip, and the magnetic drive of the nanoprobe is achieved by relying on the superparamagnetism of Fe3O4. Attached Figure Description
[0021] Figure 1 This is a schematic diagram of the preparation of the MPAuNS nanoprobe in Example 1 of the present invention and a schematic diagram of the application of the MPAuNS nanoprobe in LFIA.
[0022] Figure 2 The diagram shows the diffusion coefficient distribution of the MPAuNS nanoprobe measured by dynamic light scattering (DLS) in glucose solutions of different concentrations in Example 2 of this invention (left) and the diffusion coefficient values of the MPAuNS nanoprobe at different glucose concentrations (right).
[0023] Figure 3 This is an example of how the immunoassay analyzer recorded the change in the gray intensity of the T line on the test strip over time when it was exposed to 0 mM and 50 mM glucose.
[0024] Figure 4 The left image shows the hysteresis loop diagram of Fe3O4, Fe3O4@PDA and MPAuNS nanoparticles in Example 4 of the present invention, and the right image shows the MPAuNS nanoparticles collected using an external magnetic field.
[0025] Figure 5 In Embodiment 5 of the present invention, the immunoassay analyzer records the change in gray intensity of the T line of the test strip over time under the action of different magnets; Detailed Implementation
[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
[0027] This invention utilizes AuNS to couple with monoclonal antibodies (mAbs) via electrostatic adsorption. Furthermore, the quinone groups on the PDA coating actively participate in the nucleophilic addition reactions of the -SH and -NH2 residues of the anti-cTnI monoclonal antibody 560cc, enhancing the immobilization of the anti-cTnI monoclonal antibody 560cc. Bovine serum albumin and methoxy polyethylene glycol thiol (MPEG-SH) are then used to block residual binding sites on the MPAuNS surface, increasing the dispersibility of the conjugate in solution and reducing non-specific adsorption between the conjugate and cTnI, thereby improving the accuracy of LFIA. During detection, the analytical solution is added to the nanoprobe solution, mixed, incubated, and then dropped onto the sample pad. The solution flows through the test strip via capillary action, forming a colored region on the strip with a labeled immune complex.
[0028] Figure 1 This is a schematic diagram of the detection principle of the present invention. The prepared MPAuNS nanoprobe is mixed with cTnI solution and incubated to form an immune complex (MPAuNS-mAb-cTnI). This complex is then dropped onto the sample pad and further binds to the antibody on the T line to form an immune sandwich complex. Finally, the results are recorded on a camera, and the gray intensity of the T line is measured using an immunoassay analyzer.
[0029] Example 1
[0030] This embodiment defines a specific flowchart of the lateral flow immunochromatographic test strip based on enzyme / magnetically driven magnetic gold nanoprobes (e.g.) Figure 1 The specific experimental method is as follows:
[0031] 1) Preparation of magnetic gold nanoprobes:
[0032] a. After fully dissolving FeCl3·6H2O, trisodium citrate, sodium acetate and H2O, add ethylene glycol and mix well. Transfer to a polytetrafluoroethylene liner and react at 200℃ for 12h. After thoroughly washing the bottom solid material with water, Fe3O4 nanoparticles are obtained.
[0033] b. Fe3O4 nanoparticles were completely dispersed in phosphate buffer, dopamine hydrochloride was added, and the reaction was carried out at room temperature under an electric stirrer for 4 hours. The solid material after the reaction was thoroughly washed with water to obtain Fe3O4@PDA nanoparticles.
[0034] c. Completely disperse Fe3O4@PDA in HEPES solution, then add HAuCl4 solution and stir to ensure uniform mixing. React the above solution for 1 hour. After the solid material after reaction is thoroughly washed with water, MPAuNS nanoprobe is obtained.
[0035] 2) MPAuNS is applied to LFIA:
[0036] a. Disperse MPAuNS in borate buffer (pH 9.0), then add 560cc of antibody to the above solution and incubate for 4 hours. Add bovine serum albumin solution and continue incubation for 30 minutes, then add MPEG-SH solution and incubate for another 30 minutes. Wash with ultrapure water and resuspend in borate buffer containing 0.2% bovine serum albumin, 1.5% sucrose, and 1% S9 solution to obtain the MPAuNS-mAb complex.
[0037] b. Add the MPAuNS-mAb complex to a solution containing cTnI, mix well, incubate, and then drop it onto the sample pad. It flows through the test strip via capillary action, forming a sandwich structure at point T.
[0038] Example 2
[0039] This embodiment determines the diffusion-enhanced behavior of MPAuNS nanoprobes under glucose fuel and the optimal glucose concentration (e.g., ...). Figure 2 The specific experimental method is as follows:
[0040] 1) Dynamic light scattering study on the diffusion enhancement behavior of glucose concentration and nanoprobe: The probe was mixed with glucose solutions of different concentrations (0, 10, 25, 50, 100 mM) at a volume ratio of 1:1, and the diffusion coefficient of the probe at different concentrations was recorded by dynamic light scattering.
[0041] Results analysis: such as Figure 2 As shown in a and 2b, with increasing glucose concentration, the diffusion coefficient distribution of the MPAuNS nanoprobe gradually shifts to a higher value range to the right. The relevant average diffusion coefficient increases from 1.17 μm at 0 mM glucose. 2 / s (PDI = 0.184) increased to 1.71 μm at 50 mM glucose. 2 / s. However, when the concentration is further increased to 100mM, the viscosity resistance generated by the high-concentration glucose solution is greater than the corresponding driving force, resulting in a decrease in slope and diffusion coefficient. Therefore, the optimal glucose concentration for propelling the MPAuNS nanoprobe is determined to be 50mM.
[0042] Example 3
[0043] This embodiment confirms the feasibility of enzyme-driven detection of cTnI using MPAuNS nanoprobes and glucose-containing cTnI solutions (e.g., Figure 3 The specific experimental method is as follows:
[0044] 1) Enzyme-driven implementation: MPAuNS-mAb nanoprobes were added to 30 μL of a positive sample, which was a cTnI serum solution containing 0 or 50 mM glucose. The solution was added to a sample pad, and the changes in the gray value of the T line were recorded using an immunoassay analyzer to compare the effect of enzyme-driven implementation.
[0045] Results analysis: such as Figure 3 As shown, the grayscale values of the control group with a glucose concentration of 0 mM at minutes 1, 3, and 5 were 180, 432, and 813, respectively. After the addition of glucose, the grayscale values significantly increased to 580, 783, and 900, respectively. This is because when the serum solution containing glucose comes into contact with the nanoprobes, due to the catalytic properties of AuNS, glucose can be continuously converted into gluconic acid and hydrogen peroxide. With the help of enhanced diffusion, MPAuNS-mAb-cTnI flows towards the T line more rapidly. At minute 15, the grayscale values for the presence and absence of glucose were 1881 and 1870, respectively, remaining essentially unchanged. This indicates that the addition of glucose has no effect on the formation of the MPAuNS-mAb-cTnI immune complex, nor does it affect the binding between the immune complex and the antibody on the T line. Therefore, glucose-fueled nanoprobes can be used for LFIA detection of cTnI and have the ability to shorten the detection time in a short period of time.
[0046] Example 4
[0047] This embodiment determines the magnetic response performance of the MPAuNS nanoprobe (e.g., Figure 4 The specific experimental method is as follows:
[0048] 1) Prepare a large number of powder samples of Fe3O4, Fe3O4@PDA and MPAuNS nanoparticles, measure their hysteresis loop parameters, and plot the hysteresis loop diagram.
[0049] 2) Record the motion of MPAuNS nanoparticles under the action of an external magnet to further study the magnetic response performance of MPAuNS nanoprobes.
[0050] Results analysis: such as Figure 4 As shown in figure a, no significant remanence or coercivity was observed in the MPAuNS nanoparticles; therefore, MPAuNS retains the superparamagnetism of Fe3O4 and possesses excellent magnetic properties. Figure 4 As shown in b, the MPAuNS nanoprobe was found to move towards the magnet under the action of an external magnet, indicating that the MPAuNS nanoprobe has good magnetic response performance and can be used for subsequent magnetic drive.
[0051] Example 5
[0052] This embodiment confirms the feasibility of achieving magnetic actuation using MPAuNS nanoprobes and external magnets to detect cTnI (e.g., Figure 5 The specific experimental method is as follows:
[0053] 1) Place the magnet on the absorbent pad of the test strip for magnetic drive studies of LFIA.
[0054] 2) By changing the number of magnets, the strength of the magnetic field can be altered, and the effect of magnetic drive can be further studied.
[0055] Results analysis: such as Figure 5 As shown, within a 15-minute detection period, the gray intensity of the T-line on the test strip with the magnet was consistently higher than that on the test strip without the magnet. Furthermore, this difference in gray intensity became more pronounced with an increase in the number of magnets. The presence of the magnets provides a driving force for the rapid inflow of the magnetic MPAuNS-mAb-cTnI immune complex into the T-line, and also reduces the residue of the MPAuNS-mAb-cTnI immune complex on the nitrocellulose membrane. Therefore, this MPAuNS nanoprobe can achieve magnetic actuation under the influence of magnets, which can shorten the detection time within a certain period.
Claims
1. A lateral flow immunochromatographic test strip based on enzyme / magnetically driven magnetic gold nanoprobes, characterized in that, The lateral flow immunochromatographic test strip includes a nanoprobe made of magnetic gold nanocomposite material, a self-assembled test strip, a glucose-containing cTnI target analyte solution, and an external magnet with the same magnetic induction intensity.
2. The lateral flow immunochromatographic test strip as described in claim 1, characterized in that, The magnetic gold nanocomposite material refers to: Fe3O4 nanoclusters with carboxyl functionalization synthesized by a solvothermal method using FeCl3·6H2O as the iron source and trisodium citrate dodecahydrate as the stabilizer; and PDA coating on the surface of the Fe3O4 nanoparticles by solution oxidation, wherein dopamine hydrochloride can self-polymerize on the surface of the carboxyl functionalized Fe3O4 clusters to form -COO-NH3. + -Iron pairs, and through self-polymerization, form a PDA shell with a well-defined core-shell structure; PDA is a natural binder that can attract HAuCl4 to its surface through non-covalent bonding or chelation of hydroxyl-metal complexes. Subsequently, HEPES in solution can reduce gold ions (AuCl4). - AuNSs were synthesized in situ.
3. The lateral flow immunochromatographic test strip as described in claim 1, characterized in that, The nanoprobes rely primarily on the adhesive properties of PDA to bind to antibodies, and use bovine serum albumin and methoxy polyethylene glycol thiol (MPEG-SH) as blocking agents.
4. The lateral flow immunochromatographic test strip as described in claim 1, characterized in that, The self-assembled test strip includes a sample pad, a conjugation pad, a nitrocellulose membrane, an absorbent pad, and a base plate. IgG (1 mg / mL) and 560 cc (1 mg / mL) of anti-cTnI monoclonal antibody were sprayed onto the nitrocellulose membrane at a spray rate of 1 μL / cm to construct the test line (T line) and control line (C line), respectively.
5. The lateral flow immunochromatographic test strip as described in claim 1, characterized in that, The glucose-containing cTnI target analyte solution utilizes the glucose oxidase activity of gold nanostars to decompose glucose in the cTnI solution, thereby achieving enzyme-driven processing.
6. The lateral flow immunochromatographic test strip as described in claim 1, characterized in that, The magnetically driven motion is mainly achieved by utilizing the superparamagnetism of Fe3O4 under the action of an external magnet.
7. The lateral flow immunochromatographic test strip based on enzyme / magnetically driven magnetic gold nanoprobes as described in any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Soak the sample pad and conjugate pad in buffer solution, then dry them at 37°C for 3 hours for the next step. Attach the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad to the substrate, overlapping them by 2 mm to construct the LFIA test strip. Cut the assembled strip into 3 mm wide strips for use. S2: Add 560cc of antibody-conjugated magnetic gold nanoprobe (MPAuNS-mAb) to the analytical solution, mix well, incubate, and then drop it onto the sample pad. The nanoprobe flows forward under capillary action. Finally, record the results on a camera and measure the gray intensity of the T line using an immunoassay analyzer.
8. The preparation method according to claim 7, characterized in that, The sample pad, in which the treatment solution comprises 12.1 g / L tris(hydroxymethyl)aminomethane, 10 g / L polyvinylpyrrolidone, 10 g / L Tetronic 1307 and 5 g / L casein.