Preparation method of 40nm colloidal gold for immunodetection
By controlling the preparation conditions through low-temperature reaction and buffer substances, the problems of instability and non-uniform particle size of gold nanoparticles in traditional methods have been solved. This has enabled high reproducibility and uniformity of colloidal gold, improved the stability of binding with antibodies or proteins, reduced the risk of false positives, and enhanced the accuracy of detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- URIT MEDICAL ELECTRONICS CO LTD
- Filing Date
- 2024-04-11
- Publication Date
- 2026-06-02
Smart Images
Figure CN118226023B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of colloidal gold preparation technology, and in particular to a method for preparing 40nm colloidal gold for immunoassay. Background Technology
[0002] Gold nanoparticles, also known as colloidal gold, refer to gold sols with dispersed phase particle diameters between 1 and 100 nm. They belong to a multiphase heterogeneous system and range in color from orange-red to purplish-red. As an important raw material for immunogold technology, their unique stability, small size effect, surface effect, and biocompatibility make them widely applicable in fields such as optics, electronics, catalysis, biomedicine, and immunoassay.
[0003] The preparation methods of colloidal gold can be divided into two categories: physical methods and chemical methods. Physical methods mainly include thermal decomposition, photochemical methods, sonicochemical methods, and radiation decomposition. Although the technology for preparing colloidal gold using physical methods is relatively mature, it is difficult to control the particle size and quality, making the prepared colloidal gold inconvenient to use. Chemical methods for preparing colloidal gold particles use chloroauric acid as the main reducing agent, and commonly use white phosphorus, trisodium citrate, ethanol, and tannic acid-sodium citrate as reducing agents. The technology for preparing colloidal gold particles using chemical methods is relatively stable, and the particle size is related to the dosage of the reducing agent. Currently, chemical methods are the main method used for preparing colloidal gold particles.
[0004] In chemical methods, trisodium citrate is the most commonly used traditional reducing agent for preparing colloidal gold particles. The process of preparing colloidal gold particles with trisodium citrate is simple, and the prepared colloidal gold solution has high transparency and good visual effect. However, the traditional method for preparing gold nanoparticles has problems such as unstable preparation results and non-uniform particle size. Summary of the Invention
[0005] The purpose of this invention is to provide a method for preparing 40nm colloidal gold for immunoassay, which solves the problems of unstable preparation results and uneven particle size in traditional methods for preparing gold nanoparticles.
[0006] To achieve the above objectives, the present invention provides a method for preparing 40nm colloidal gold for immunoassay, comprising the following steps:
[0007] Place the conical flask containing ultrapure water in a constant temperature water bath. After the water temperature reaches the set temperature, adjust the rotor speed to the set value and begin adding the medicine.
[0008] Add a measured amount of buffer solution, stir evenly for about 30 seconds, then add a measured amount of chloroauric acid, stir evenly for a set time, and then add a measured amount of reducing agent. The chloroauric acid, reducing agent and buffer solution should be added dropwise to the liquid surface between the rotor and the bottle wall, and continue to maintain the set water temperature for a period of time.
[0009] Transfer the conical flask to an electric magnetic stirrer and heat it to boiling. Stop heating and bring the volume to a final volume.
[0010] The process includes placing a conical flask containing ultrapure water in a constant-temperature water bath. Once the water temperature reaches the set temperature, the rotor speed is adjusted to the set value, and the addition of the medicine begins. The steps also include:
[0011] The water temperature rises to the set temperature of 45℃.
[0012] The process includes adding a measured amount of buffer solution, stirring evenly for about 30 seconds, then adding a measured amount of chloroauric acid, stirring evenly for a set time, and then adding a measured amount of reducing agent. The chloroauric acid, reducing agent, and buffer solution should be added dropwise onto the liquid surface between the rotor and the bottle wall. The set water temperature is maintained for a period of time. The steps also include:
[0013] The buffer solution is a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate.
[0014] The process includes adding a measured amount of buffer solution, stirring evenly for about 30 seconds, then adding a measured amount of chloroauric acid, stirring evenly for a set time, and then adding a measured amount of reducing agent. The chloroauric acid, reducing agent, and buffer solution should be added dropwise onto the liquid surface between the rotor and the bottle wall. The set water temperature is maintained for a period of time. The steps also include:
[0015] The buffer solution and chloroauric acid are stirred evenly for 2-4 minutes.
[0016] The process includes adding a measured amount of buffer solution, stirring evenly for about 30 seconds, then adding a measured amount of chloroauric acid, stirring evenly for a set time, and then adding a measured amount of reducing agent. The chloroauric acid, reducing agent, and buffer solution should be added dropwise onto the liquid surface between the rotor and the bottle wall. The set water temperature is maintained for a period of time. The steps also include:
[0017] The reducing agent is ascorbic acid, a weak reducing agent.
[0018] The process includes adding a measured amount of buffer solution, stirring evenly for about 30 seconds, then adding a measured amount of chloroauric acid, stirring evenly for a set time, and then adding a measured amount of reducing agent. The chloroauric acid, reducing agent, and buffer solution should be added dropwise onto the liquid surface between the rotor and the bottle wall. The set water temperature is maintained for a period of time. The steps also include:
[0019] The reducing agent is added in 2-4 seconds.
[0020] The process includes adding a measured amount of buffer solution, stirring evenly for about 30 seconds, then adding a measured amount of chloroauric acid, stirring evenly for a set time, and then adding a measured amount of reducing agent. The chloroauric acid, reducing agent, and buffer solution should be added dropwise onto the liquid surface between the rotor and the bottle wall. The set water temperature is maintained for a period of time. The steps also include:
[0021] Maintain the water temperature at 45°C and heat for at least 15 minutes.
[0022] The steps include transferring the conical flask to an electric magnetic stirrer and reheating it to boiling, then stopping heating and bringing the volume to a final depth.
[0023] The specified volume refers to the amount of ultrapure water added.
[0024] This invention discloses a method for preparing 40nm colloidal gold for immunoassay. By controlling the preparation conditions, chloroauric acid and a reducing agent are added to a stable environment to allow the reaction, thus preparing nano-colloidal gold. This method offers higher reproducibility and uniformity compared to traditional methods. Furthermore, the stability of the colloidal gold after being linked to the labeled antibody or protein is improved, avoiding false positives and increasing accuracy. This invention employs a low-temperature reaction method and stabilizes the preparation environment by adding a buffer substance, thereby avoiding the problems of poor reproducibility and uneven particle size distribution caused by boiling and unstable preparation environments in traditional methods. Attached Figure Description
[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.
[0026] Figure 1 This is a graph showing the UV test results of 40nm colloidal gold prepared using the conventional method of this invention.
[0027] Figure 2 This is a graph showing the UV test results of 40nm colloidal gold prepared by the novel method of this invention.
[0028] Figure 3 This is a step diagram illustrating the preparation method of 40nm colloidal gold for immunoassay according to the present invention. Detailed Implementation
[0029] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.
[0030] Please see Figures 1 to 3 ,in, Figure 1 This is a graph showing the UV test results of 40nm colloidal gold prepared using the conventional method of this invention. Figure 2 This is a graph showing the UV test results of 40nm colloidal gold prepared by the novel method of this invention. Figure 3 This is a step-by-step diagram of the preparation method of 40nm colloidal gold for immunoassay according to the present invention. The present invention provides a method for preparing 40nm colloidal gold for immunoassay, comprising the following steps:
[0031] S101: Place the conical flask containing ultrapure water in a constant temperature water bath. After the water temperature reaches the set temperature, adjust the rotor speed to the set value and begin adding the medicine.
[0032] S102: Add a measured amount of buffer solution, stir evenly for about 30 seconds, then add a measured amount of chloroauric acid, stir evenly for the set time, and then add a measured amount of reducing agent. The chloroauric acid, reducing agent and buffer solution should be added dropwise to the liquid surface between the rotor and the bottle wall, and continue to maintain the set water temperature for a period of time.
[0033] S103: Transfer the conical flask to an electric magnetic stirrer and heat it to boiling. Stop heating and bring to a final volume.
[0034] Specifically, the conditions are controlled as follows: Place the conical flask containing ultrapure water in a constant-temperature water bath. Once the water temperature reaches the set temperature, adjust the rotor speed to the set value and begin adding the reagents. The reaction temperature is 45℃. Reagent addition: Add a measured amount of buffer solution, stir evenly for approximately 30 seconds, then add a measured amount of chloroauric acid, stirring evenly for the set time (precisely controlled). Next, add a measured amount of reducing agent. The chloroauric acid, reducing agent, and buffer solution should be added dropwise onto the liquid surface between the rotor and the flask wall. Continue to maintain the set water temperature for a period of time. The buffer solution is a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate. The mixing time between the buffer solution and chloroauric acid is 2-4 minutes, specifically 3 minutes. The reducing agent is the weak reducing agent ascorbic acid. The dropwise addition time of the reducing agent is controlled at 2-4 seconds. After the chloroauric acid solution and reducing agent are added, maintain heating at 45℃ for at least 15 minutes. Elimination of reducing substances: Transfer the conical flask to an electric magnetic stirrer and heat to boiling. Stop heating and bring to a final volume. The fixed volume is the amount of ultrapure water added.
[0035] Figure 1 The results of three UV tests on 40nm colloidal gold prepared using the traditional method are compared with those of 40nm colloidal gold prepared using the new method. Figure 2 The new method produces colloidal gold with advantages in reproducibility and uniformity.
[0036] The test results are shown in the table below:
[0037] Traditional method test results New method test results 1 Abs: 0.952; λmax: 524nm Abs: 0.972; λmax: 525nm 2 Abs: 0.823; λmax: 525nm Abs: 0.967; λmax: 525nm 3 Abs: 0.625; λmax: 528nm Abs: 0.934; λmax: 524nm
[0038] Note: 1. Abs is the value shown on the vertical axis of the above graph, which is defined as the absorbance of the test solution and can represent the relative concentration of the test solution.
[0039] 2. λmax is the value shown on the horizontal axis of the above figure. It is defined as a specific absorption peak of the test solution and can represent the size of the maximum particle size of the solution.
[0040] The method of this invention for preparing colloidal gold nanoparticles exhibits superior reproducibility and uniformity compared to traditional methods: High uniformity: This improves the stability of the colloidal gold after it is linked to the antibody or protein to be labeled, avoiding false positives and resulting in higher accuracy. High reproducibility: This minimizes batch-to-batch variation, reduces production costs, and ensures more stable color development of the product.
[0041] The preparation of 1% chloroauric acid solution:
[0042] Remove chloroauric acid (1.0g / vial) from the refrigerator and allow it to warm to room temperature. Gently tap the ampoule opening with your finger to allow the solid to collect at the bottom. Use a grinding wheel to open the ampoule opening, then pipette 1 mL of ultrapure water into the ampoule. After dissolving, transfer the solution to a brown volumetric flask (100 mL). Rinse the ampoule 10 times, transferring the rinse liquid to the volumetric flask. Finally, dilute to 100 mL with ultrapure water.
[0043] The solution can be stored in a refrigerator at (2-8)℃, away from light and sealed, and has a shelf life of 2 months.
[0044] Preparation of phosphate buffer:
[0045] Dissolve a certain amount of disodium hydrogen phosphate in ultrapure water to obtain reagent solution 1; take a clean beaker, fill it with ultrapure water, and dissolve a certain amount of disodium hydrogen phosphate to obtain reagent solution 2; take another clean beaker, mix the two in a certain proportion to obtain a 0.2M-0.5M buffer solution. It can be stored in a sealed container at (2-8)℃ for 1 day.
[0046] Preparation of ascorbic acid solution:
[0047] Weigh a certain amount of ascorbic acid into a clean container, add ultrapure water to dissolve it, and dilute to volume with ultrapure water to prepare a 1%-3% ascorbic acid solution. The solution can be stored in a sealed container at (2-8)℃ for 4 hours.
[0048] Reagent filtration:
[0049] The prepared 1% chloroauric acid solution, phosphate buffer, and ascorbic acid solution must be filtered before use. The syringes and filter membranes used for filtration are dedicated to this purpose and should not be mixed.
[0050] Take a disposable syringe (10 mL), attach the filter membrane (0.22 μm, 50 mm microporous membrane), rinse three times with ultrapure water to remove all liquid from the syringe, then draw 10 mL of reagent solution and filter it into a 10 mL centrifuge tube using the disposable syringe. The syringe and filter membrane are for single use only and should be discarded after use.
[0051] Production of colloidal gold solution:
[0052] Glassware processing:
[0053] The conical flasks and rotors used in the production of colloidal gold solutions require processing: Take the cleaned conical flasks and rotors, measure 100g of ultrapure water into the conical flasks, place them on an electric magnetic stirrer, turn on the stirrer (700 rpm), add 500μL of phosphate buffer and stir for about 30s, then add 1mL of 1% chloroauric acid solution, continue stirring for 3min, then add 550μL of ascorbic acid solution (the solution should be added dropwise to the liquid surface between the rotor and the flask wall), and immediately turn on the heating switch, heating until the liquid boils and then stopping the heating. Discard the solution in the conical flasks, rinse 3-4 times with ultrapure water, and place them in an oven to dry for later use.
[0054] Preparation of colloidal gold solution:
[0055] Measure 100g of ultrapure water into a treated conical flask, place it in a heat-collecting magnetic stirrer, adjust the temperature to 47℃, turn on the stirrer (speed 700±10r / min), adjust the iron frame so that the liquid level in the conical flask is lower than the liquid level in the stirrer, and the rotor rotates stably (the rotor is located at the center of the bottom of the flask and there is no turbulence). Insert a thermometer to measure the temperature. After the water temperature rises to 45℃±0.5℃, add 500μL of 0.5M phosphate buffer and stir evenly for about 30s. Then add 1mL of 1% chloroauric acid solution and stir evenly for 3min (time precisely controlled). Then add 550μL of 1% ascorbic acid solution (the addition should not be too fast, and should be completed in about 2-4s). The solution should be added dropwise to the liquid surface between the rotor and the bottle wall. Continue to maintain the water temperature at 45℃ for 15min. Then transfer the conical flask to an electric magnetic stirrer and heat it to boiling at a speed of 700±10r / min. Stop heating and make up to 100g.
[0056] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.
Claims
1. A method for preparing 40nm colloidal gold for immunoassay, characterized in that, Includes the following steps: Place the conical flask containing ultrapure water in a constant temperature water bath. After the water temperature rises to 45°C, adjust the rotor speed to the set value and begin adding the medicine. Add a measured amount of buffer solution, stir evenly for about 30 seconds, then add a measured amount of chloroauric acid, stir evenly for 2-4 minutes, and then add a measured amount of reducing agent. The chloroauric acid, reducing agent, and buffer solution should be added dropwise to the liquid surface between the rotor and the bottle wall. Continue to maintain the water temperature at 45°C and heat for at least 15 minutes. The buffer solution is a 0.2M-0.5M mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, the chloroauric acid is a 1% chloroauric acid solution, the reducing agent is a weak reducing agent ascorbic acid, the reducing agent is added dropwise over 2-4 seconds, and the ascorbic acid is a 1%-3% ascorbic acid solution. Transfer the conical flask to an electric magnetic stirrer and heat it to boiling. Stop heating and bring the volume to a final volume.
2. The method for preparing 40nm colloidal gold for immunoassay as described in claim 1, characterized in that, Transfer the conical flask to an electric magnetic stirrer and reheat to boiling. Stop heating and bring to a final volume. The steps also include: The specified volume refers to the amount of ultrapure water added.