A kind of defect topological gold particle triplex detection card based on gold growth autocatalytic signal amplification and its preparation method and application

Abstract

The application relates to the technical field of detection, and discloses a defect topological gold particle triple detection card based on gold growth autocatalysis signal amplification as well as a preparation method and application thereof. The scheme is based on the high specific surface area signal amplification characteristics of the defect topological gold particle, combines specific antibody labeling and precise assembly technology, realizes efficient detection of CRP, MxA and (1,3)-beta-D-glucan triple indexes, and takes into account detection sensitivity, specificity and stability, thereby solving the pain points of weak signal and low single-index detection efficiency of traditional detection cards. The scheme also drops gold growth enhancement liquid (a mixture of HAuCl4 and NH2OH.HCl) after detection, the defect topological gold particle catalyzes the reduction reaction of HAuCl4 and NH2OH.HCl to generate new defect topological gold particles, the new defect topological gold particles will rapidly deposit and cover the surface of the initial defect topological gold particles, forming defect topological gold particles with larger sizes, thereby producing gray-black precipitates on the T line and the C line of the test strip, and forming significant signal amplification.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a triplet detection card for defect topological gold particles based on the autocatalytic signal amplification of gold growth, its preparation method, and its application. Background Technology

[0002] Infectious endophthalmitis is a highly blinding and acute ocular infection, with intraocular fluid being the core sample for detection. However, existing detection methods have significant limitations, making rapid and accurate identification of pathogen types an urgent clinical need. CRP, MxA protein, and (1,3)-β-D-glucan are specific biomarkers for bacterial, viral, and deep fungal infections, respectively. Combined detection of these three markers can achieve accurate identification of infection types, making triple-signal test cards highly valuable in the diagnosis of endophthalmitis. Currently, triple-signal test card technology is largely based on the principle of immunochromatography, with the core objective of adapting to the characteristics of small sample volume and low protein content in intraocular fluid to achieve simultaneous detection of multiple indicators. However, current technologies still need breakthroughs in balancing signal sensitivity and detection efficiency.

[0003] In existing technologies, methods to improve the success rate of triplet test cards mainly revolve around optimizing the combination of indicators and enhancing signals: First, fixing the combination of specific biomarkers, with CRP, MxA, and (1,3)-β-D-glucan as the core detection targets, and setting independent detection channels on the test card to achieve simultaneous identification of three infection types; Second, optimizing the immune binding system, selecting highly specific antibodies or ligands to reduce cross-reactions and improve detection specificity; Third, adopting signal amplification strategies, such as introducing nanoparticle labeling and enzyme-catalyzed reaction amplification techniques, to compensate for the insufficient signal intensity of traditional colloidal gold colorimetry and adapt to the detection of low-content samples; Fourth, optimizing the test card chromatography process, adjusting the buffer formulation, membrane material selection, and channel design to ensure the synchronicity and stability of multi-indicator detection.

[0004] However, existing triplet test card technologies still have significant drawbacks: First, insufficient signal amplification capability; traditional amplification strategies are insufficient to meet the detection requirements of extremely low protein content in intraocular fluid, easily leading to false negative results. Second, interference exists in multi-channel detection; the immune responses of different indicators affect each other, resulting in decreased detection specificity and accuracy. Third, poor adaptability of the detection system; existing technologies are not optimized for the characteristics of intraocular fluid samples and cannot meet the clinical needs of simultaneous detection of multiple indicators and rapid results. Fourth, an imbalance between cost and complexity; some high-sensitivity schemes rely on complex processes or expensive reagents, making widespread adoption difficult. In summary, there is an urgent need for a triplet test card technology that combines high sensitivity, high specificity, and rapid convenience to solve the problem of rapid identification of pathogens causing endophthalmitis. Summary of the Invention

[0005] The present invention aims to provide a triplet detection card for defective topological gold particles based on the autocatalytic signal amplification of gold growth, its preparation method and application, in order to solve the technical problem that existing technologies are difficult to identify the types of pathogens causing infectious endophthalmitis simultaneously and with high sensitivity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing a triplet detection card for defect topological gold particles based on the autocatalytic signal amplification of gold growth, comprising the following steps: Step 1: Preparation of defective topological gold particles: Step 2, Antibody labeling: This includes labeling CRP antibody, MxA antibody, (1,3)-β-D-glucan antibody and DNP-BSA with defective topological gold particles to obtain CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex and DNP-BSA complex labeled with defective topological gold particles, respectively. Step 3: Preparation of nitrocellulose membrane: (1,3)-β-D-glucan-coated antibody was streaked onto the nitrocellulose membrane as detection line 1, MxA-coated antibody was streaked onto the nitrocellulose membrane as detection line 2, CRP-coated antibody was streaked onto the nitrocellulose membrane as detection line 3, and mouse DNP monoclonal antibody was streaked onto the nitrocellulose membrane as control line to obtain a well-coated nitrocellulose membrane; Step 4: Preparation of the conjugate pad: The CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex and DNP-BSA complex labeled with defective topological gold particles obtained in Step 2 are sprayed onto a glass cellulose membrane and dried to form the conjugate pad. Step 5, Sample pad preparation: Soak glass fibers in sample pad treatment solution and then dry them to make sample pads; Step 6: Assemble the reagent strips: Attach the coated nitrocellulose membrane to the PVC base plate, attach the conjugate pad to one end of the test line, place the sample pad on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0007] The principle and advantages of this scheme are: 1. The preparation method in this scheme relies on the high specific surface area signal amplification characteristics of defective topological gold particles, combined with specific antibody labeling and precise assembly process, to achieve efficient detection of CRP, MxA and (1,3)-β-D-glucan triple indicators, taking into account detection sensitivity, specificity and stability, and solving the pain points of weak signal and low single indicator detection efficiency of traditional detection cards.

[0008] 2. This method uses defective topological gold particles as tracer markers, which are coupled with labeled antibodies. When these particles encounter antigens in the sample, they bind to them, forming antigen-antibody complexes. These complexes flow on the reaction membrane and form bispecific antibody sandwich complexes with the detection antibodies immobilized on the membrane. This method is rapid, simple, and low-cost, and is widely used for content detection.

[0009] 3. This method uses defective topological gold particles to label CRP antibody, MxA antibody, and (1,3)-β-D-glucan antibody, enabling the three-in-one product to simultaneously meet the performance requirements of three detection indicators with the same sample diluent, sample volume, reaction time, and reagent strip. It has high sensitivity and aims to improve the accuracy of differentiating bacterial endophthalmitis, viral endophthalmitis, and fungal endophthalmitis, guide clinical medication, and improve prognosis.

[0010] The specific function of each step in this plan is as follows: 1. This method effectively constructs a core signal amplification carrier by preparing defective topological gold particles, laying the material foundation for highly sensitive detection. Specifically, defective topological gold particles are specially designed gold nanomaterials with abundant nanoscale depressions, pores, and other defect structures on their surface. High-density nanopores, irregular edges, and atomic-level defects are constructed on the surface of the defective topological gold particles. These non-planar structures significantly increase the specific surface area and roughness of the material, thereby providing more physical loading space for antibodies and chemical reaction active sites. Defective topological gold particles possess a unique branched structure and ultra-large specific surface area, enabling efficient antibody loading and enhancing signal transduction efficiency; their excellent optical properties significantly amplify the detection signal, improving the detection card's ability to identify low-concentration targets.

[0011] 2. This protocol uses defective topological gold particles to label antibodies, facilitating specific binding between the antibodies and these particles and ensuring the specificity and signal correlation of the triple assay. Specifically, this protocol specifically labels three target antibodies with DNP-BSA complexes, ensuring that each detection channel specifically recognizes the target analyte without cross-interference; the defective topological gold particles bind stably to the antibodies, preventing antibody detachment during detection and ensuring signal stability; the DNP-BSA complex labeling provides an effective control line, ensuring the reliability of the test results.

[0012] 3. This method prepares detection lines by streaking different antibodies onto a nitrocellulose membrane, facilitating the construction of the core reaction area for triple detection and quality control, enabling simultaneous detection of multiple indicators. Specifically, the three detection lines are coated with corresponding antibodies, simultaneously capturing three target analytes and improving detection efficiency; a quality control line is added to form a control system, quickly determining the effectiveness of the detection process and eliminating false negative / false positive results; precise antibody streaking and fixation ensure a regular reaction area and improve the repeatability of detection results.

[0013] 4. This approach involves spraying multiple antibody complexes labeled with defective topological gold particles onto the binding pad, facilitating the immobilization of the labeled antibody complexes and constructing a rapid-response signal initiation layer. Specifically, four labeled complexes are uniformly sprayed onto a glass cellulose membrane to ensure rapid binding of the target analyte to the labeled antibody; drying enhances the immobilization effect of the complexes, preventing sample loss during permeation and ensuring signal strength; and the binding pad is adapted for subsequent assembly, providing a smooth pathway for signal transduction.

[0014] 5. This solution assembles sample pads, conjugate pads, nitrocellulose membranes, etc., into reagent strips, facilitating precise adaptation of each component and constructing an efficient and stable detection pathway. Specifically, each component is precisely pasted in functional order to form a complete pathway of "sample penetration - signal binding - reaction color development - waste liquid absorption"; the components overlap tightly to avoid sample leakage or signal conduction obstruction, ensuring a smooth detection process; standardized assembly improves batch consistency of reagent strips, ensuring reliable and repeatable test results.

[0015] Preferably, as an improvement, in step one, the preparation of the defective topological gold particles includes the following: heating 100 mL of 0.01% chloroauric acid solution to boiling, adding 1.5~2.8 mL of 1% trisodium citrate solution to generate a gold seed solution; then adding the aforementioned gold seed solution, 1.6~2 mL of 1% chloroauric acid solution, 1.8~2.5 mL of 1% trisodium citrate solution and 20~28 mL of 30 mM hydroquinone solution to 100 mL of ultrapure water, the solution turns blue, cools to room temperature and is stored at 4°C for later use.

[0016] Beneficial Effects: This scheme employs the aforementioned method, utilizing a precise process of "gold seed generation - stepwise component compounding - temperature-controlled color development and preservation" to prepare defective topological gold particles with high specific surface area and excellent optical properties, providing a stable core carrier for signal amplification of the detection card. Furthermore, the particle size of the prepared defective topological gold particles can be adjusted to 40-80 nm by changing the amount of trisodium citrate, effectively improving detection accuracy. In this process, the precise matching of component dosages and temperature parameters is crucial; deviations will directly degrade the structure and performance of the defective topological gold particles, affecting detection sensitivity. Specifically, if the dosage of chloroauric acid and trisodium citrate is too low, insufficient gold seed generation and incomplete branching structure of defective topological gold particles will occur, resulting in a decrease in specific surface area and reduced signal amplification. If the dosage is too high, particle aggregation and uneven particle size will occur, leading to disordered optical signals. If the dosage of hydroquinone is too low, it will not be able to fully induce the branching growth of defective topological gold particles, resulting in spherical rather than flower-like products with poor signal transmission efficiency. If the dosage is too high, it will accelerate excessive particle growth and cause structural collapse. If the boiling temperature is insufficient, gold seed generation will be incomplete and poorly dispersed. If the temperature is too high, gold particles will aggregate prematurely. Storage at 4°C after cooling to room temperature can maintain the structural stability of defective topological gold particles. If the storage temperature is too high, it will easily cause particle oxidation and aggregation. If it is too low, it may cause the solution to freeze and destroy the structure. Both of these will result in a decrease in the efficiency of subsequent antibody labeling and a significant decline in the specificity and sensitivity of the detection card.

[0017] Preferably, as an improvement, in step two, the marking includes the following: S1. Centrifugation: Take 1 mL of the defective topological gold particle solution, sonicate to mix, centrifuge and discard the supernatant; add 1 mL of 10~50 mM MES buffer (pH=5.5~7.5), vortex to mix. S2, Coupling: Add the antibody to be coupled, such as CRP antibody, MxA antibody, (1,3)-β-D-glucan antibody or DNP-BSA, to the above defective topological gold particle solution, mix gently, and couple at room temperature in the dark for 30 min. S3. Blocking: Add 5wt% PEG-20000 solution (prepared with 10-50mM MES, pH=5.5-7.5) containing 1-3wt% sucrose to the above coupling buffer, block at room temperature and in the dark for 1h, centrifuge and discard the supernatant to obtain defective topological gold particles-antibody complex. S4. Reconstitution: Add gold-labeled reconstitution solution to the defective topological gold particle-antibody complex and store at 2~8℃ in the dark to obtain defective topological gold particle-labeled CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex and DNP-BSA complex, respectively.

[0018] Beneficial Effects: This protocol employs the above-mentioned method, utilizing a standardized four-step process of "purification-coupling-blocking-reconstitution" to achieve highly efficient and stable binding between defective topological gold particles and the target antibody / DNP-BSA. This ensures the specificity, activity, and dispersibility of the complex, while providing core material support for accurate signal transduction in the triplet detection card, avoiding issues such as non-specific binding and antibody loss. Specifically, centrifugation effectively purifies the defective topological gold particles and optimizes their dispersion, laying the foundation for subsequent efficient coupling. Coupling enables the specific and stable binding of defective topological gold particles to the antibody / DNP-BSA, ensuring targeted detection. Blocking enhances the specificity of the complex by inhibiting non-specific binding, ensuring accurate detection results. Finally, reconstitution fixes the complex concentration and preserves it at low temperatures, maintaining its activity and detection efficacy.

[0019] Preferably, as an improvement, in step two, the gold-labeled complex solution A used to label the CRP antibody, (1,3)-β-D-glucan antibody, and DNP-BSA comprises the following raw materials in the following mass percentages: 3-10% sucrose, 2-5% trehalose, 0.5-1% BSA, 0.1-0.5% NaCl, 0.7-1.5% NaH2PO4·2H2O2, 2-5% Na2HPO4·12H2O, 0.05-0.1% Tween-20, 0.1% ProClin300, and the balance being 1×PBS.

[0020] Beneficial effects: This solution adopts the above-mentioned scheme. The gold-labeled reconstitution solution A, through the synergistic ratio of multiple components, has the functions of reconstitution and dispersion, antibody protection, antibacterial and antiseptic functions. It can efficiently maintain the activity and stability of defective topological gold particle-labeled CRP antibody, (1,3)-β-D-glucan antibody and DNP-BSA complex. It is suitable for the long-term storage and detection requirements of the test card. The content of each component is accurately matched. Deviation will degrade the performance of the complex and lead to detection failure. Through long-term experiments, the inventors discovered that 10% sucrose and 5% trehalose work synergistically as freeze-drying protectants. Too low a concentration fails to effectively prevent complex aggregation and antibody denaturation, while too high a concentration increases solution viscosity, affecting the uniformity of subsequent binding pad spraying. 0.5% BSA can block residual non-specific sites; too low a concentration results in insufficient blocking and false positives, while too high a concentration leads to competition for binding sites, reducing detection sensitivity. 0.1% NaCl maintains osmotic pressure balance; too low a concentration causes osmotic pressure imbalance and rupture of the complex, while too high a concentration causes particle aggregation. 0.7% NaH2PO4·2H2O and 2% Na2HPO4·12H2O form a buffer system; deviations in concentration can disrupt pH stability, leading to antibody activity loss. 0.05% Tween-20 improves dispersibility; too low a concentration makes the complex prone to aggregation, while too high a concentration weakens the binding force between the antibody and defective topological gold particles. 0.1% ProClin300 inhibits microbial growth; too low a level will not achieve preservation, while too high a level will damage antibody activity, both of which will lead to a shortened storage period for the complex and a significant decline in the specificity and sensitivity of the test card.

[0021] Preferably, as an improvement, in step two, the gold-labeled complex solution B used to label the MxA antibody comprises the following raw materials in concentration or mass percentage: 10-50 mM Tris with pH 7-8, 50-150 mM NaCl, 0.05-0.1% Tween-20, 1-3% BSA, 2-5% trehalose, 0.1% ProClin300, and the balance being deionized water.

[0022] Beneficial Effects: This scheme employs the aforementioned method, with the gold-labeled reconstitution solution B precisely formulated to match the characteristics of the MxA antibody. Through the synergistic effects of buffer stability, osmotic pressure regulation, activity protection, and antibacterial and preservative properties, it efficiently maintains the structural integrity and activity stability of the defective topological gold particle-labeled MxA antibody complex. It is suitable for the preparation of the test card binding pad and long-term storage requirements. All parameters are precisely controllable; deviations will degrade the complex performance, leading to detection failure. Long-term experiments by the inventors have shown that the 25mM Tris at pH 7.2±0.05 provides a stable buffer environment. Concentrations that are too high / too low, or pH deviations, will disrupt the antibody's spatial conformation, leading to activity loss and affecting binding specificity. 150mM NaCl maintains osmotic pressure balance; too low a concentration causes the complex to rupture due to osmotic pressure imbalance, while too high a concentration causes the defective topological gold particles to aggregate, reducing dispersibility. 0.05% Tween-20 improves the dispersibility of the system; too low a concentration will cause the complex to easily aggregate and clump, while too high a concentration will weaken the binding strength between the antibody and the defective topological gold particles. 1% BSA blocks residual nonspecific sites; too low a concentration will result in insufficient blocking and may cause false positives, while too high a concentration will compete for binding sites and reduce detection sensitivity. 5% trehalose provides anti-agglomeration protection; too low a concentration will not effectively inhibit the denaturation and aggregation of the complex, while too high a concentration will increase the viscosity of the solution and affect the uniformity of the conjugate pad spraying. 0.1% ProClin300 provides antibacterial and preservative effects; too low a concentration will not prevent microbial growth and complex deterioration, while too high a concentration will damage antibody activity. All these factors shorten the storage period of the complex and significantly reduce the specificity and sensitivity of the test card.

[0023] Preferably, as an improvement, in step three, when streaking the nitrocellulose membrane, the concentrations of the CRP-coated antibody, MxA-coated antibody, (1,3)-β-D-glucan-coated antibody, and mouse DNP monoclonal antibody are 1~1.5 mg / mL, and the coating amount is 0.8 μL / cm.

[0024] Beneficial effects: This method adopts the above scheme, which facilitates precise control of the concentration of the four antibodies at 1~1.5mg / mL and the coating amount at 0.8μL / cm when scrubbing the nitrocellulose membrane. It can achieve uniform antibody fixation, form regular detection lines and quality control lines, ensure the specific capture capability and signal consistency of triple detection, and lay the foundation for accurate interpretation of the test card. Parameter deviations will degrade the reaction effect on the membrane and lead to detection failure. Through long-term experiments, the inventors discovered that when the antibody concentration is below 1 mg / mL, the amount of antibody immobilized on the membrane is insufficient, making it impossible to fully capture the target complex, resulting in weak signals and a high risk of false negatives. Conversely, if the antibody concentration is above 1.5 mg / mL, excessive antibody accumulation can easily lead to non-specific binding, increase costs, and cause blurred and diffused lines. When the coating amount is below 1 μL / cm, the antibody loading is insufficient, resulting in low capture efficiency and insufficient signal intensity. Furthermore, if the coating amount is above 2 μL / cm, the antibody solution is prone to penetration and diffusion, resulting in wide, irregular lines that affect the accuracy of interpretation. Excessive antibody may also form a blocking layer, hindering reaction conduction and ultimately causing a decline in the specificity and sensitivity of the test card, making it impossible to achieve accurate detection of the triple indicator.

[0025] Preferably, as an improvement, in step four, the coating amount of the CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex, and DNP-BSA complex labeled with defective topological gold particles on the binding pad is 0.8 μL / cm.

[0026] Beneficial Effects: This solution employs the aforementioned method, precisely controlling the coating amount of the four defective topological gold particle-labeled complexes on the binding pad to 15~20 μL / cm. This ensures uniform complex loading and rapid, efficient binding with the target analyte in the sample, laying a solid foundation for the initiation and transmission of the triple detection signal. Parameter deviations will degrade the performance of the binding pad, leading to a decline in the sensitivity and stability of the detection card. Long-term experiments by the inventors have revealed that when the coating amount is below 15 μL / cm, the complex loading is insufficient, resulting in low binding efficiency with the target analyte, weak detection signals, and a high likelihood of false negatives. If the coating amount is above 20 μL / cm, excessive complex accumulation can easily lead to aggregation, reducing dispersibility and activity. Furthermore, excess complex can easily diffuse with the sample, resulting in enhanced background interference, cluttered control and detection line signals, increased raw material costs, and ultimately affecting the accurate interpretation of detection results, making efficient detection of the triple indicators impossible.

[0027] Preferably, as an improvement, in step five, the sample pad treatment solution comprises the following raw materials in concentrations or mass percentages: 0.01M PBS, 10-50 mM Tris-HCl, 1.5-2% BSA, 0.5-1% casein, 0.2% NonidetP40, 2-5% trehalose, with the remainder being deionized water.

[0028] Beneficial Effects: This solution employs the above-mentioned method, where the sample pad treatment solution, through precise proportioning of multiple components, combines sample pretreatment, activity protection, and dispersion enhancement functions. It optimizes sample penetration efficiency, suppresses impurity interference, and protects the activity of the labeled complex on the binding pad, laying the foundation for efficient detection card reactions. Deviations in various parameters will degrade sample pad performance and affect detection accuracy. Specifically, 0.01M PBS and 20mM Tris-HCl synergistically maintain pH stability; excessively high or low concentrations will destroy antibody activity, leading to signal attenuation. 1.5% BSA and 0.5% casein synergistically block non-specific sites; insufficient blocking due to low concentrations can easily cause false positives, while excessive concentrations increase solution viscosity and hinder sample penetration. 0.2% Pluronic F-127 enhances hydrophilicity and dispersibility; excessively low concentrations result in slow sample penetration, while excessively high concentrations weaken complex binding. 5% trehalose prevents component denaturation and aggregation; excessively low concentrations cannot effectively protect activity, while excessively high concentrations can cause crystallization, affecting sample conduction. All these factors contribute to a decline in the sensitivity and specificity of the detection card, preventing accurate detection of the triple indicator.

[0029] Preferably, as an improvement, this solution also provides a triplet detection card for defect topological gold particles based on the amplification of autocatalytic signals from gold growth, which is prepared by the above method.

[0030] Preferably, as an improvement, this solution also provides an application of a triplet detection card for defective topological gold particles based on gold growth autocatalytic signal amplification in the detection of infectious endophthalmitis, including the following usage method: (1) Drop the sample to be tested into the sample pad and perform immunochromatography for 10 min; (2) Preparation of gold growth enhancement solution: Mix HAuCl4 with a mass concentration of 0.01~1% and NH2OH·HCl with a mass concentration of 10~50 mM; (3) Add the above gold growth enhancement solution to the sample pad and interpret the results after 10 minutes.

[0031] Beneficial effects: In the absence of defective topological gold particles, the reduction of NH2OH·HCl to HAuCl4 is very weak, resulting in a slow rate of defective topological gold particle generation. This method further enhances the process by adding a gold growth enhancement solution (a mixture of HAuCl4 and NH2OH·HCl) after detection. In the presence of defective topological gold particles, these particles catalyze the reduction reaction of HAuCl4 and NH2OH·HCl, thereby generating new defective topological gold particles. These new particles then rapidly deposit and cover the surface of the initial defective topological gold particles, forming larger defective topological gold particles. This results in a gray-black precipitate on the T and C lines of the test strip, leading to significant signal amplification.

[0032] In summary, this solution has the following beneficial effects: (1) The present invention develops a POCT device that can rapidly identify bacterial, viral and fungal endophthalmitis.

[0033] (2) The product of the present invention can achieve the goal of high-sensitivity semi-quantitative detection of analytes in the field of immunochromatography.

[0034] (3) The present invention utilizes a three-in-one detection kit for CRP, MxA and (1,3)-β-D-glucan prepared by defective topological gold particles. The combined detection can improve the accuracy of identifying bacterial, viral and fungal endophthalmitis, which is beneficial for the early identification of infectious endophthalmitis, guides clinical medication and improves prognosis. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the structure of the triplet detection card obtained in Embodiment 4 of the present invention.

[0036] Figure 2 This is a comparison of the detection sensitivity of the triplet detection cards prepared from topological gold particles with different particle sizes in Experimental Example 1 of this invention.

[0037] Figure 3 This is the result of the ratio optimization of the gold growth enhancement solution obtained in different proportions in Experimental Example 2 of this invention.

[0038] Figure 4 This is a comparison of signal strength between experimental example 3 of the present invention with and without gold growth enhancement solution (top figure) and with gold growth enhancement solution (bottom figure).

[0039] Figure 5 This is a TEM image of the 60nm defective topological gold particles obtained in Example 2 of the present invention. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0041] Example 1: Preparation of 40nm defective topological gold particles Heat 100 mL of 0.01% chloroauric acid solution to boiling, then add 1.5 mL of 1% trisodium citrate solution to generate gold seeds. Next, add 0.8 mL of 10 nm seed gold, 1.6 mL of 1% chloroauric acid solution, 1.8 mL of 1% trisodium citrate solution, and 28 mL of 30 mM hydroquinone solution to 100 mL of ultrapure water. The solution turns blue. After cooling to room temperature, set aside at 4°C for later use.

[0042] Example 2: Preparation of 60nm defective topological gold particles Heat 100 mL of 0.01% chloroauric acid solution to boiling, then add 2.8 mL of 1% trisodium citrate solution to generate gold seeds. Next, add 8 mL of 20 nm seed gold, 2 mL of 1% chloroauric acid solution, 1.8 mL of 1% trisodium citrate solution, and 28 mL of 30 mM hydroquinone solution to 100 mL of ultrapure water. The solution turns blue. After cooling to room temperature, set aside at 4°C for later use.

[0043] Example 3: Preparation of 80nm defective topological gold particles Heat 100 mL of 0.01% chloroauric acid solution to boiling, then add 2.8 mL of 1% trisodium citrate solution to generate gold seeds. Next, add 1.6 mL of 20 nm seed gold, 2 mL of 1% chloroauric acid solution, 1.8 mL of 1% trisodium citrate solution, and 28 mL of 30 mM hydroquinone solution to 100 mL of ultrapure water. The solution turns blue. After cooling to room temperature, set aside at 4°C for later use.

[0044] Example 4 This solution also provides a method for preparing a triplet detection card for defect topological gold particles based on the autocatalytic signal amplification of gold growth, comprising the following steps: Step 1: Preparation of defective topological gold particles: 60 nm defective topological gold particles with the highest optimized sensitivity were used. Step 2, Antibody Labeling: This includes labeling CRP antibody, MxA antibody, (1,3)-β-D-glucan antibody, and DNP-BSA with defective topological gold particles to obtain CRP antibody complexes, MxA antibody complexes, (1,3)-β-D-glucan antibody complexes, and DNP-BSA complexes labeled with defective topological gold particles; specifically: (1) Labeling of CRP antibodies S1-1, Centrifugation: Take 1 mL of the defective topological gold particle solution (60 nm defective topological gold particles) from Example 2, sonicate to mix, centrifuge and discard the supernatant; add 1 mL of 10~50 mM MES buffer (pH=5.5~7.5), vortex to mix. S2-1, Coupling: Add the CRP-labeled antibody to be coupled to the above-mentioned defective topological gold particle solution, mix gently, and couple at room temperature and in the dark for 30 min. S3-1, Blocking: Add 5 wt% PEG-20000 solution (prepared from 10~50 mM MES, pH=5.5~7.5) to the above coupling buffer. The added PEG-20000 solution contains 1~3 wt% sucrose. Block at room temperature and in the dark for 1 h. Centrifuge and discard the supernatant to obtain the defective topological gold particle-antibody complex. S4-1, Reconstitution: Add 60 μL of gold-labeled reconstitution solution to the above 60 nm defective topological gold particle-antibody complex to obtain the defective topological gold particle-labeled CRP antibody complex. Store at 2-8 °C protected from light. The gold-labeled reconstitution solution is formulated as follows: 3-10% sucrose, 2-5% trehalose, 0.5-1% BSA, 0.1-0.5% NaCl, 0.7-1.5% NaH2PO4·2H2O2, 2-5% Na2HPO4·12H2O, 0.05-0.1% Tween-20, 0.1% ProClin300, with the balance being 1×PBS.

[0045] (2) Labeling of MxA antibodies S1-2, Centrifugation: Take 1 mL of the defective topological gold particle solution (60 nm defective topological gold particles) from Example 2, sonicate to mix, centrifuge and discard the supernatant; add 1 mL of 10~50 mM MES buffer (pH=5.5-7.5), vortex to mix. S2-2, Coupling: Add the MxA-labeled antibody to be coupled to the above defective topological gold particle solution, mix gently, and couple at room temperature and in the dark for 30 min. S3-2, Blocking: Add 5 wt% PEG-20000 solution (prepared from 10~50 mM MES, pH=5.5~7.5) to the above coupling buffer. The added PEG-20000 solution contains 1~3 wt% sucrose. Block at room temperature in the dark for 1 h. Centrifuge and discard the supernatant to obtain the defective topological gold particle-antibody complex. S4-2, Reconstitution: Add 60 μL of gold-labeled reconstitution solution to the above 60 nm defective topological gold particle-antibody complex to obtain the defective topological gold particle-labeled MxA antibody complex. Store at 2-8℃ protected from light. The gold-labeled reconstitution solution is formulated as follows: 10-50 mM Tris at pH 7-8, 50-150 mM NaCl, 0.05-0.1% Tween-20, 1-3% BSA, 2-5% trehalose, 0.1% ProClin300, and the balance being deionized water.

[0046] (3) Labeling of (1,3)-β-D-glucan antibody S1-3. Centrifugation: Take 1 mL of the defective topological gold particle solution (60 nm defective topological gold particles) from Example 2, sonicate to mix, centrifuge and discard the supernatant; add 1 mL of 10-50 mM MES buffer (pH=5.5-7.5), vortex to mix. S2-3, Coupling: Add the (1,3)-β-D-D-labeled antibody to be coupled to the above defective topological gold particle solution, mix gently, and couple at room temperature in the dark for 30 min. S3-3, Blocking: Add 5 wt% PEG-20000 solution (prepared from 10~50 mM MES, pH=5.5~7.5) to the above coupling buffer. The added PEG-20000 solution contains 1~3 wt% sucrose. Block at room temperature and in the dark for 1 h. Centrifuge and discard the supernatant to obtain the defective topological gold particle-antibody complex. S4-3, Reconstitution: Add 60 μL of gold-labeled reconstitution solution to the above 60 nm defective topological gold particle-antibody complex to obtain the (1,3)-β-D-glucan antibody complex labeled with defective topological gold particles. Store at 2-8 °C protected from light. The gold-labeled reconstitution solution is formulated as follows: 3-10% sucrose, 2-5% trehalose, 0.5-1% BSA, 0.1-0.5% NaCl, 0.7-1.5% NaH2PO4·2H2O2, 2-5% Na2HPO4·12H2O, 0.05-0.1% Tween-20, 0.1% ProClin300, and the balance is 1×PBS.

[0047] (4) Marking of DNP-BSA S1-4. Centrifugation: Take 1 mL of the defective topological gold particle solution (60 nm defective topological gold particles) from Example 2, sonicate to mix, centrifuge and discard the supernatant; add 1 mL of 10-50 mM MES buffer (pH=5.5-7.5), vortex to mix. S2-4, Coupling: Add the DNP-BSA to be coupled to the above defective topological gold particle solution, mix gently, and couple at room temperature and in the dark for 30 min. S3-4, Blocking: Add 5 wt% PEG-20000 solution (prepared from 10~50 mM MES, pH=5.5~7.5) to the above coupling buffer. The added PEG-20000 solution contains 1~3 wt% sucrose. Block at room temperature in the dark for 1 h. Centrifuge and discard the supernatant to obtain the defective topological gold particle-antibody complex. S4-4, Reconstitution: Add 60 μL of gold-labeled reconstitution solution to the above 60 nm defective topological gold particle-antibody complex to obtain the defective topological gold particle-labeled DNP-BSA complex. Store at 2-8 °C protected from light. The gold-labeled reconstitution solution is formulated as follows: 3-10% sucrose, 2-5% trehalose, 0.5-1% BSA, 0.1-0.5% NaCl, 0.7-1.5% NaH2PO4·2H2O2, 2-5% Na2HPO4·12H2O, 0.05-0.1% Tween-20, 0.1% ProClin300, and the balance is 1×PBS.

[0048] Step 3: Preparation of nitrocellulose membrane: 1 mg / mL (1,3)-β-D-glucan-coated antibody was streaked onto the nitrocellulose membrane at a rate of 1 μL / cm as detection line 1; 1 mg / mL MxA-coated antibody was streaked onto the nitrocellulose membrane at a rate of 1 μL / cm as detection line 2; 1.5 mg / mL CRP-coated antibody was streaked onto the nitrocellulose membrane at a rate of 1 μL / cm as detection line 3; and 1 mg / mL mouse DNP monoclonal antibody was streaked onto the nitrocellulose membrane at a rate of 1 μL / cm as control lines. The membrane was then dried overnight at 40°C.

[0049] Step 4: Preparation of the conjugate pad: CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex, and DNP-BSA complex labeled with defective topological gold particles were sprayed onto a glass cellulose membrane at a coating amount of 15 μL / cm and dried overnight at 40°C to prepare the conjugate pad.

[0050] Step 5: Preparation of sample pads: Soak glass fibers in sample pad treatment solution (0.01M PBS, 10~50 mM Tris-HCl, 1.5~2% BSA, 0.5~1% casein, 0.2% Nonidet P40, 2~5% trehalose, balance deionized water) for 30 min, then dry at 40℃ overnight to prepare sample pads.

[0051] Step Six: Reagent Strip Assembly: Attach the coated nitrocellulose membrane to the PVC base plate, attach the conjugate pad to one end of the test line, place the sample pad on the conjugate pad, and attach the absorbent pad to one end of the control line. The resulting triplet test card is shown below. Figure 1 As shown.

[0052] The difference between Examples 1, 2, and 3 lies in the particle size of the defective topological gold particles. Example 4 involves the preparation of a triplet detection card using 60 nm defective topological gold particles, which have the highest sensitivity.

[0053] Example 5 This solution also provides an application of a triplet detection card for defective topological gold particles based on the autocatalytic signal amplification of gold growth in the detection of infectious endophthalmitis, including the following usage method: (1) Drop the sample to be tested into the sample pad and perform immunochromatography for 10 min; (2) Preparation of gold growth enhancement solution: Mix HAuCl4 with a mass concentration of 0.01~1% and NH2OH·HCl with a mass concentration of 10~50 mM; (3) Add the above enhancement solution to the sample pad and interpret the results after 10 minutes.

[0054] Experimental Example 1: Comparison of sensitivity of test strips labeled with topological gold particles of different particle sizes. Using the above method, 60 μL of CRP calibrators of different concentrations were added to the triplet detection cards prepared using the defective topological gold particles described in Examples 1-3. The reaction time was 10 min. The detection limits are shown in the figure. Figure 2 (From left to right, the particle sizes of the defective topological gold particles used are 40 nm, 60 nm, and 80 nm, respectively). The data show that the 60 nm defective topological gold particle immunoprobe has higher detection sensitivity, with a visual detection limit of up to 2 ng / mL.

[0055] Experiment Example 2: Optimization of the Ratio of Gold Growth Enhancement Solution Prepare gold growth enhancement solutions with different proportions, immerse the above triplet test card in the enhancement solution, and observe the results after 10 minutes. Figure 3 Data shows that mixing 1% HAuCl4 and 30mM NH2OH·HCl in a 1:1 ratio yields the best scale-up results.

[0056] Experimental Example 3: Verification of the catalytic effect of in-situ gold growth Take 60 μL of a mixed solution of CRP, MxA, and (1,3)-β-D-dextran quality control samples of different concentrations, add it to the sample pad, and perform chromatography for 10 min. Then add 60 μL of the optimal ratio of gold growth enhancement solution (e.g., 1% HAuCl4 and 30 mM NH2OH·HCl mixed in a 1:1 ratio), and enhance for 10 min. The detection results are shown in the figure. Figure 4 Data shows that this method can amplify the signal by 50 times by adding gold growth enhancement solution, enabling the detection of low-abundance proteins.

[0057] Experimental Example 4: Structural Characterization of Defective Topological Gold Particles TEM was used to characterize the structure of the defective topological gold particles. The results are shown below. Figure 5 Data shows that the surface of defective topological gold particles has abundant nanoscale depressions, pores, and unique branching structures.

[0058] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a triplet detection card for defect topological gold particles based on autocatalytic signal amplification during gold growth, characterized in that: Includes the following steps: Step 1: Preparation of 60nm defect topological gold particles: Heat 100mL of 0.01% chloroauric acid solution to boiling, add 2.8mL of 1% trisodium citrate solution to generate gold seeds; then add 8mL of 20nm seed gold, 2mL of 1% chloroauric acid solution, 1.8mL of 1% trisodium citrate solution, and 28mL of 30mM hydroquinone solution to 100mL of ultrapure water. The solution turns blue. After cooling to room temperature, it is ready for use at 4℃. Step 2, Antibody Labeling: This includes labeling CRP antibody, MxA antibody, (1,3)-β-D-glucan antibody, and DNP-BSA with defective topological gold particles to obtain CRP antibody complexes, MxA antibody complexes, (1,3)-β-D-glucan antibody complexes, and DNP-BSA complexes labeled with defective topological gold particles; the labeling includes the following: S1. Centrifugation: Take 1 mL of the defective topological gold particle solution, sonicate to mix, centrifuge and discard the supernatant; add 1 mL of 10~50 mM MES buffer and vortex to mix. S2, Coupling: Add the antibody to be coupled, such as CRP antibody, MxA antibody, (1,3)-β-D-glucan antibody or DNP-BSA, to the above defective topological gold particle solution, mix gently, and couple at room temperature in the dark for 30 min. S3. Blocking: Add 5 wt% PEG-20000 solution to the above coupling buffer. The added PEG-20000 solution contains 1~3 wt% sucrose. Block at room temperature and in the dark for 1 h. Centrifuge and discard the supernatant to obtain the defective topological gold particle-antibody complex. S4. Reconstitution: Add gold-labeled reconstitution solution to the defective topological gold particle-antibody complex, store at 2~8℃ in the dark, and obtain CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex and DNP-BSA complex labeled with defective topological gold particles, respectively. Step 3: Preparation of nitrocellulose membrane: (1,3)-β-D-glucan-coated antibody was streaked onto the nitrocellulose membrane as detection line 1, MxA-coated antibody as detection line 2, CRP-coated antibody as detection line 3, and mouse DNP monoclonal antibody as control line to obtain a well-coated nitrocellulose membrane. When streaking the nitrocellulose membrane, the concentrations of CRP-coated antibody, MxA-coated antibody, (1,3)-β-D-glucan-coated antibody, and mouse DNP monoclonal antibody were 1~1.5 mg / mL, and the coating amount was 0.8 μL / cm. Step 4: Preparation of the conjugate pad: The CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex and DNP-BSA complex labeled with defective topological gold particles obtained in Step 2 are sprayed onto a glass cellulose membrane and dried to form the conjugate pad. Step 5, Sample pad preparation: Soak glass fibers in sample pad treatment solution and then dry them to make sample pads; Step 6: Assemble the reagent strips: Attach the coated nitrocellulose membrane to the PVC base plate, attach the conjugate pad to one end of the test line, place the sample pad on the conjugate pad, and attach the absorbent pad to one end of the control line.

2. The method for preparing a triplet detection card for defect topological gold particles based on autocatalytic signal amplification of gold growth according to claim 1, characterized in that: In step two, the gold-labeled complex solution A used to label the CRP antibody, (1,3)-β-D-glucan antibody, and DNP-BSA comprises the following raw materials by mass percentage: 3-10% sucrose, 2-5% trehalose, 0.5-1% BSA, 0.1-0.5% NaCl, 0.7-1.5% NaH2PO4·2H2O2, 2-5% Na2HPO4·12H2O, 0.05-0.1% Tween-20, 0.1% ProClin300, and the balance being 1×PBS.

3. The method for preparing a triplet detection card for defect topological gold particles based on autocatalytic signal amplification during gold growth, as described in claim 1, is characterized in that: In step two, the gold-labeled complex solution B used to label the MxA antibody includes the following raw materials in concentration or mass percentage: 10-50 mM Tris with pH 7-8, 50-150 mM NaCl, 0.05-0.1% Tween-20, 1-3% BSA, 2-5% trehalose, 0.1% ProClin300, and the balance being deionized water.

4. The method for preparing a triplet detection card for defect topological gold particles based on autocatalytic signal amplification during gold growth, as described in claim 3, is characterized in that: In step four, the coating amount of the CRP antibody complex, MxA antibody complex, (1,3)-β-D-glucan antibody complex, and DNP-BSA complex labeled with defective topological gold particles on the binding pad is 0.8 μL / cm.

5. The method for preparing a triplet detection card for defect topological gold particles based on autocatalytic signal amplification during gold growth, as described in claim 4, is characterized in that: In step five, the sample pad treatment solution comprises the following raw materials in concentrations or mass percentages: 0.01M PBS, 10-50 mM Tris-HCl, 1.5-2% BSA, 0.5-1% casein, 0.2% NonidetP40, 2-5% trehalose, and the balance being deionized water.

6. A triplet detection card for defect topological gold particles based on autocatalytic signal amplification during gold growth, characterized in that: Prepared by the method described in any one of claims 1 to 5.

7. The method of using the triplet detection card for defect topological gold particles based on autocatalytic signal amplification of gold growth according to claim 6, characterized in that: The method is for non-disease diagnosis purposes and includes the following steps: first, drop the sample to be tested into the sample pad; then, mix HAuCl4 with a mass concentration of 0.01~1% and NH2OH·HCl with a mass concentration of 10~50 mM to form a gold growth enhancement solution, add the gold growth enhancement solution to the sample pad, and read the result on the test card after diffusion.

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