Colloidal gold detection card for tetrodotoxin

Abstract

The invention relates to the technical field of biological detection, and discloses a tetrodotoxin colloidal gold detection card, which comprises a detection box used for packaging a detection assembly, the display groove is formed in the detection box and is used for observing a detection result; the liquid dropping groove is positioned at one end of the detection box and is used for accommodating a to-be-detected sample; and the detection paper is arranged in the detection box, comprises a nitrocellulose membrane, an adsorption pad and a diffusion pad, is provided with a detection area and a control area, and is used for displaying a detection result of tetrodotoxin. By optimizing the size of the gold nanoparticles and an antibody labeling process, the sensitivity and the specificity of the detection card are remarkably improved, and the low-concentration tetrodotoxin can be rapidly and accurately detected. Due to the reasonable particle size and the application of the stabilizer, non-specific adsorption is reduced, the false positive rate is reduced, the reliability and consistency of detection results are ensured, clear detection results can be obtained in a shorter time, and the accuracy of rapid judgment in field application is ensured.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a colloidal gold detection card for tetrodotoxin. Background Art

[0002] Tetrodotoxin is an extremely strong neurotoxin that is widely found in pufferfish and related marine life, posing a serious threat to human health. Due to its high toxicity and colorless and odorless characteristics, tetrodotoxin poisoning incidents occur from time to time. Especially in the field of food safety, rapid and accurate detection of tetrodotoxin has become particularly important. Although traditional detection methods such as high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) have high detection sensitivity and accuracy, they require complex sample pretreatment and expensive instruments and equipment, and are time-consuming and technically demanding, making them unsuitable for rapid on-site screening and large-scale application.

[0003] Colloidal gold immunochromatography has become an important means of detecting tetrodotoxin in recent years due to its simple operation, rapid detection and no need for complex equipment. However, existing colloidal gold test cards still have significant deficiencies in sensitivity, specificity and stability.

[0004] To solve the above problems, the present invention proposes an improved tetrodotoxin colloidal gold detection card, which optimizes the preparation of gold nanoparticles and antibody labeling process, and significantly improves the sensitivity, specificity and stability of the detection card by controlling the size of gold nanoparticles, ultrasonic treatment and the rational use of stabilizers. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a colloidal gold detection card for tetrodotoxin, which solves the problem that the gold nanoparticle size and antibody labeling process of the existing detection card are not optimized, resulting in low detection sensitivity and high false positive rate.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: a colloidal gold detection card for tetrodotoxin, comprising:

[0007] A test box, used to encapsulate the test components;

[0008] A display slot, provided on the test box, for observing the test results;

[0009] A drip tank, located at one end of the test box, for containing the sample to be tested;

[0010] The detection paper is arranged inside the detection box, and the detection paper includes a nitrocellulose membrane, an adsorption pad and a diffusion pad, and is provided with a detection area and a control area, and is used to display the detection result of tetrodotoxin.

[0011] Preferably, the method for preparing the detection paper comprises the following steps:

[0012] a) Preparation of gold salt solution: Dissolve 0.01%-0.1% sodium chloroaurate in deionized water and adjust the pH to 5.0-6.0 with sodium carbonate;

[0013] b) Reduction and formation of colloidal gold: slowly dropwise add 10mM-50mM sodium citrate or sodium borohydride solution at 95°C-100°C, and control the dropwise addition time within 15-30 minutes to form 20-40 nanometer gold nanoparticles;

[0014] c) Antibody labeling: Add the specific monoclonal antibody to the colloidal gold solution, keep it at a pH of 5.0-6.0 for labeling, then add 1%-5% polyethylene glycol or bovine serum albumin as a stabilizer, and perform ultrasonic dispersion treatment for 5-10 minutes;

[0015] d) Coating of test paper: evenly coat the labeled colloidal gold antibody solution on the nitrocellulose membrane to form a test line and a control line;

[0016] e) Drying and cutting: Dry the coated nitrocellulose membrane in a constant temperature and humidity environment, and then cut it into test paper strips of appropriate size;

[0017] f) Assembly: Assemble the prepared test paper strip into the test box, making sure one end of the test paper is connected to the drip groove.

[0018] Preferably, the pH value of the sodium chloroaurate solution is precisely controlled at 5.5 by a buffer solution to optimize the formation of gold nanoparticles.

[0019] Preferably, the reducing agent is added to the gold salt solution at a rate of 0.5 mL to 1.5 mL per minute to ensure the uniformity and stability of the gold nanoparticles.

[0020] Preferably, the polyethylene glycol added after antibody labeling has a molecular weight of 2000-4000 to further enhance the stability of the colloidal gold particles.

[0021] Preferably, the concentration of bovine serum albumin used in the antibody labeling process is 2%-3% to ensure uniform distribution of the antibody on the surface of the colloidal gold particles.

[0022] Preferably, the drying temperature of the test line and the control line on the nitrocellulose membrane after coating is controlled at 37° C.-42° C., and the humidity is controlled at 40%-60%.

[0023] Preferably, the coated nitrocellulose membrane is sterilized with ultraviolet light before cutting to prevent contamination and ensure the sterility of the test paper.

[0024] Preferably, the cutting width of the detection paper strip is controlled within a range of 2.5 mm to 3.5 mm to ensure its stability in the detection box.

[0025] Preferably, the frequency of the ultrasonic dispersion treatment is set at 40kHz-60kHz to ensure uniform dispersion of the gold nanoparticles.

[0026] The present invention provides a colloidal gold detection card for tetrodotoxin, which has the following beneficial effects:

[0027] 1. The present invention significantly improves the sensitivity and specificity of the test card by optimizing the gold nanoparticle size and antibody labeling process, and can quickly and accurately detect low-concentration tetrodotoxin. The application of reasonable particle size and stabilizer reduces nonspecific adsorption, reduces the false positive rate, ensures the reliability and consistency of the test results, and can obtain clear test results in a shorter time, ensuring the accuracy of rapid judgment in field applications.

[0028] 2. The preparation process of the present invention improves the stability of the test card, so that it can still maintain high performance under different environments. The improved coating and drying process, as well as the optimized use of stabilizers, prevent particle aggregation and failure, significantly extend the shelf life of the test card, and ensure consistent color reaction and detection effect after long-term storage.

[0029] 3. The test card of the present invention is simple and fast in design, with clear color development, which significantly improves the detection efficiency. The results can be obtained within a few minutes without complicated operations, and it is suitable for use by non-professionals. The optimized structural design ensures smooth sample flow, fast and consistent color development, and is suitable for on-site rapid detection and large-scale screening, greatly saving detection time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a stereogram of the present invention.

[0031] Among them, 1. detection box; 2. display slot; 3. drip slot; 4. detection paper. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Please see attached Figure 1 The embodiment of the present invention provides a colloidal gold detection card for tetrodotoxin, comprising:

[0034] Detection box 1: used to encapsulate various detection components to ensure the closedness and stability of the detection process and effectively avoid interference of the external environment on the detection results.

[0035] Display slot 2: It is located at the upper part of the test box 1. Through the transparent window design, the user can clearly observe the test results. The color changes of the test line and the control line in the display slot 2 are used to determine the presence or absence of tetrodotoxin;

[0036] Dropping tank 3: located at one end of the test box 1, used to hold the sample to be tested. The dripping tank 3 is designed to be tightly connected to the test paper 4 to ensure that the sample liquid can flow quickly and evenly to the test area;

[0037] Test paper 4: Test paper 4 is the core component of the test card, including nitrocellulose membrane, adsorption pad and diffusion pad. The test paper is provided with a test area and a control area, which can visually display the test results of tetrodotoxin.

[0038] The preparation process of the test paper 4 includes the following steps:

[0039] Step a, preparation of gold salt solution

[0040] Sodium chloroaurate with a concentration of 0.01%-0.1% is dissolved in deionized water, and the pH value is precisely adjusted to 5.0-6.0 by a buffer solution, preferably controlled at 5.5. This treatment can optimize the reduction reaction conditions of gold ions and lay the foundation for the formation of stable gold nanoparticles.

[0041] Step b, reduction and formation of colloidal gold

[0042] Under the condition of maintaining the reaction temperature at 95℃-100℃, a 10mM-50mM sodium citrate or sodium borohydride solution is slowly added at a rate of 0.5mL-1.5mL per minute using a precision titration device. By controlling the drop rate and time (15-30 minutes), gold nanoparticles with a particle size of 20-40 nanometers are formed. The precisely controlled reduction process ensures the uniformity of the particle size of the colloidal gold, further improving the detection sensitivity.

[0043] Step c, antibody labeling

[0044] The screened specific monoclonal antibody is added to the colloidal gold solution, and the environmental pH is maintained at 5.0-6.0 to ensure that the activity of the antibody is not impaired. Subsequently, polyethylene glycol (PEG, molecular weight 2000-4000) or bovine serum albumin (BSA, concentration 2%-3%) is added at a mass concentration of 1%-5% as a stabilizer. Ultrasonic treatment at a frequency of 40kHz-60kHz is used for 5-10 minutes to ensure the uniform distribution of the antibody on the colloidal gold surface and further stabilize the gold nanoparticles to prevent their aggregation and precipitation.

[0045] Step d, testing the coating of paper

[0046] The labeled colloidal gold antibody solution is evenly coated on the nitrocellulose membrane by an automatic coating device to form a test line and a control line. The test line is used to capture the colloidal gold labeled antibody bound to tetrodotoxin, and the control line is used to show the effectiveness of the test.

[0047] Step e, drying and cutting

[0048] The coated nitrocellulose membrane is dried under constant temperature (37°C-42°C) and constant humidity (40%-60%) to ensure the uniformity and stability of the coating layer. After drying, the membrane is cut into 2.5mm-3.5mm wide test paper strips by a precision cutting device, and ultraviolet disinfection is performed before cutting to prevent contamination and ensure the sterility of the test paper.

[0049] Step f, assembly

[0050] The prepared test paper strip is assembled with an adsorption pad, a diffusion pad, etc. into a test box (1), ensuring that one end of the test paper is well connected to the drip groove (3), thereby achieving smooth flow and detection of the sample liquid.

[0051] Embodiment 1:

[0052] The embodiment of the present invention provides a method for preparing a detection paper of a colloidal gold detection card for tetrodotoxin, comprising the following steps:

[0053] 1. Preparation of Gold Salt Solution

[0054] Sodium chloroaurate at a concentration of 0.05% was dissolved in deionized water and the pH was adjusted to 5.5 using sodium carbonate.

[0055] 2. Reduction and formation of colloidal gold

[0056] At 98° C., 30 mM sodium citrate solution was slowly added dropwise at a rate of 0.8 mL per minute, and the addition time was controlled to be 20 minutes, so as to form gold nanoparticles with an average particle size of 30 nanometers.

[0057] 3. Antibody Labeling

[0058] The monoclonal antibody was added to the colloidal gold solution, the pH was maintained at 5.5, 2% polyethylene glycol (PEG, molecular weight 3000) was added, and ultrasonic treatment was performed for 8 minutes, and the ultrasonic frequency was set to 50 kHz.

[0059] 4. Detection of paper coating

[0060] The colloidal gold labeled antibody solution was coated on the nitrocellulose membrane using an automatic coating device to form a detection line and a control line.

[0061] 5. Drying and cutting

[0062] The coated film was dried in an environment of 40°C and 50% humidity, cut into 3 mm wide test strips, and sterilized with ultraviolet light.

[0063] 6. Assembly

[0064] Assemble the cut test paper strips into the test box, making sure one end of the test paper is connected to the drip groove.

[0065] Embodiment 2:

[0066] The embodiment of the present invention provides a method for preparing a detection paper of a colloidal gold detection card for tetrodotoxin, comprising the following steps:

[0067] 1. Preparation of Gold Salt Solution

[0068] Sodium chloroaurate at a concentration of 0.08% was dissolved in deionized water and the pH was adjusted to 5.3.

[0069] 2. Reduction and formation of colloidal gold

[0070] At 95° C., 20 mM sodium borohydride solution was added dropwise at a rate of 1 mL per minute, and the addition time was controlled to be 25 minutes to form 35 nm gold nanoparticles.

[0071] 3. Antibody Labeling

[0072] The monoclonal antibody was added with colloidal gold solution in a pH 5.3 environment, and after labeling, 3% bovine serum albumin (BSA) was added as a stabilizer, and ultrasonic treatment was performed for 10 minutes at 45 kHz.

[0073] 4. Detection of paper coating

[0074] The labeled antibody solution was coated on a nitrocellulose membrane and cut into 2.8 mm wide strips after drying.

[0075] 5. Drying and cutting

[0076] The drying temperature was set at 38°C and the humidity was 45% to ensure a uniform coating layer.

[0077] 6. Assembly

[0078] Assemble the sterilized test strips into the test box.

[0079] Embodiment three:

[0080] The embodiment of the present invention provides a method for preparing a detection paper of a colloidal gold detection card for tetrodotoxin, comprising the following steps:

[0081] 1. Preparation of Gold Salt Solution

[0082] Prepare a 0.03% sodium chloroaurate solution and adjust the pH to 5.0.

[0083] 2. Reduction and formation of colloidal gold

[0084] At 100° C., 40 mM sodium citrate solution was added dropwise at a rate of 1.2 mL / min for 15 minutes to generate gold nanoparticles of 40 nm in size.

[0085] 3. Antibody Labeling

[0086] The antibody was labeled with colloidal gold at pH 5.0, 1.5% PEG (molecular weight 2500) was added, and ultrasonic dispersion treatment was performed for 6 minutes at a frequency of 55 kHz.

[0087] 4. Detection of paper coating

[0088] Using a precision coating device, the antibody-colloidal gold solution was evenly coated onto the nitrocellulose membrane to form the detection and control lines.

[0089] 5. Drying and cutting

[0090] The paper was dried at 37°C and 40% humidity and then cut into 3.2 mm wide paper strips.

[0091] 6. Assembly

[0092] The test strips are sterilized with ultraviolet light and then assembled into the test box.

[0093] Embodiment 4:

[0094] The embodiment of the present invention provides a method for preparing a detection paper of a colloidal gold detection card for tetrodotoxin, comprising the following steps:

[0095] 1. Preparation of Gold Salt Solution

[0096] The pH was adjusted to 5.7 using 0.06% sodium chloroaurate solution.

[0097] 2. Reduction and formation of colloidal gold

[0098] At 97°C, 25 mM sodium borohydride solution was added at a rate of 0.7 mL / min for 18 minutes to form 25 nm gold nanoparticles.

[0099] 3. Antibody Labeling

[0100] The pH during antibody labeling was 5.7, 2.5% BSA was added as a stabilizer, and ultrasonic treatment was performed at a frequency of 48 kHz for 7 minutes.

[0101] 4. Detection of paper coating

[0102] The solution was coated onto the membrane, dried and cut into 3.0 mm test strips.

[0103] 5. Drying and cutting

[0104] The drying temperature is 41°C and the humidity is 50%.

[0105] 6. Assembly

[0106] The test strips are sterilized by ultraviolet light and then assembled into the test box.

[0107] Embodiment five:

[0108] The embodiment of the present invention provides a method for preparing a detection paper of a colloidal gold detection card for tetrodotoxin, comprising the following steps:

[0109] 1. Preparation of Gold Salt Solution

[0110] Prepare 0.07% sodium chloroaurate solution and adjust the pH to 5.6.

[0111] 2. Reduction and formation of colloidal gold

[0112] At 99°C, the dropping rate of 30 mM sodium citrate was 0.6 mL / min, the dropping time was controlled at 22 minutes, and the particle size was about 28 nanometers.

[0113] 3. Antibody Labeling

[0114] The pH condition was controlled at 5.6, 2% PEG (molecular weight 3500) was added, and ultrasonic treatment was performed for 9 minutes at 60 kHz.

[0115] 4. Detection of paper coating

[0116] Evenly spread the labeled colloidal gold antibody to form detection and control lines.

[0117] 5. Drying and cutting

[0118] Drying conditions: 39°C, humidity 55%, cutting width 3.3 mm.

[0119] 6. Assembly

[0120] The sterilized paper strip is assembled into the test box to ensure sterility and sealing.

[0121] Experiment 1: Effect of gold nanoparticle size on detection performance

[0122] a. Experimental purpose:

[0123] To study the effects of gold nanoparticles of different sizes on the performance of tetrodotoxin colloidal gold detection cards, especially on the detection sensitivity, specificity and false positive rate.

[0124] b. Experimental materials and equipment:

[0125] Sodium chloroaurate, sodium citrate, sodium borohydride, specific monoclonal antibody, polyethylene glycol (PEG), bovine serum albumin (BSA)

[0126] Constant temperature water bath, ultrasonic processor, precision titration equipment, spectrometer, ultraviolet disinfection equipment

[0127] Spectrophotometer, colloidal gold reader, micropipette

[0128] Test paper prepared in Examples 1-5

[0129] Tetrodotoxin standard solution (0ng / mL, 2ng / mL, 4ng / mL)

[0130] c. Experimental steps:

[0131] 1. Preparation of Gold Nanoparticles

[0132] According to the process parameters of Examples 1-5, gold nanoparticles of different sizes were prepared respectively:

[0133] Example 1: 30 nm (95°C, 0.8 mL / min sodium citrate)

[0134] Example 2: 35 nm (95°C, 1 mL / min sodium borohydride)

[0135] Example 3: 40 nm (100°C, 1.2 mL / min sodium citrate)

[0136] Example 4: 25 nm (97°C, 0.7 mL / min sodium borohydride)

[0137] Example 5: 28 nm (99°C, 0.6 mL / min sodium citrate)

[0138] 2. Antibody Labeling

[0139] Specific monoclonal antibodies were added to respective colloidal gold solutions and labeled according to the conditions of each example:

[0140] Example 1: 2% PEG, 50kHz ultrasonic treatment for 8 minutes

[0141] Example 2: 3% BSA, 45kHz ultrasonic treatment for 10 minutes

[0142] Example 3: 1.5% PEG, 55kHz ultrasonic treatment for 6 minutes

[0143] Example 4: 2.5% BSA, 48kHz ultrasonic treatment for 7 minutes

[0144] Example 5: 2% PEG, 60kHz ultrasonic treatment for 9 minutes

[0145] 3. Coating and assembly of test paper

[0146] a. Use automatic coating equipment to evenly coat the labeled colloidal gold antibody solution on the nitrocellulose membrane to form a test line and a control line.

[0147] b. Dry the coated nitrocellulose membrane under corresponding temperature and humidity conditions, and cut it into test paper strips with a width of 2.5 mm to 3.5 mm.

[0148] c. After UV disinfection of the test paper strip, assemble it into the test box, ensuring that one end is connected to the drip tank.

[0149] 4. Performance Testing

[0150] a. Apply the prepared test card to different concentrations of tetrodotoxin standard solution (0 ng / mL, 2 ng / mL, 4 ng / mL) for testing.

[0151] b. Each group of experiments was repeated 3 times, the color intensity of the detection line was recorded, and the detection sensitivity and false positive rate were calculated.

[0152]

[0153]

[0154] 5. Experimental results analysis:

[0155] a. Relationship between color intensity and particle size:

[0156] Larger gold nanoparticles (such as 40 nm in Example 3) have weaker color intensity, especially in low concentration samples (2 ng / mL), indicating that larger particles may affect detection sensitivity.

[0157] Smaller particles (25 nm in Example 4) showed stronger color development and the highest detection sensitivity (98.5%), but stability may be affected at too small a particle size.

[0158] b. Sensitivity and false positive rate:

[0159] Example 1 (30 nm) and Example 4 (25 nm) showed higher detection sensitivity and lower false positive rate, which indicates that smaller or medium-sized gold nanoparticles are helpful to improve detection performance.

[0160] Although Example 3 (40 nm) showed more obvious color development, the false positive rate was relatively high (6.8%), indicating that larger particles may increase the probability of nonspecific reactions.

[0161] c. Optimal particle size:

[0162] In terms of sensitivity and false positive rate, particles of 30 nm (Example 1) and 28 nm (Example 5) showed a better balance. It is recommended to optimize the preparation conditions and focus on the application of these sizes.

[0163] Experimental conclusion:

[0164] The size of gold nanoparticles significantly affects the performance of the tetrodotoxin test card. Medium-sized particles (25-30nm) provide higher detection sensitivity and lower false positive rate, which is suitable for practical detection. This provides valuable parameter basis for subsequent test card optimization.

[0165] Experiment 2: Effect of gold nanoparticle size on detection performance

[0166] 1. Experimental purpose:

[0167] The effect of gold nanoparticles of different sizes on the performance of tetrodotoxin colloidal gold detection cards was studied, with a focus on evaluating the effect of size changes of gold nanoparticles on detection sensitivity, color intensity and false positive rate.

[0168] 2. Experimental materials and equipment:

[0169] a. Chemical reagents: sodium chloroaurate, sodium citrate, sodium borohydride, specific monoclonal antibodies, PEG (polyethylene glycol), BSA (bovine serum albumin)

[0170] b. Experimental equipment: constant temperature water bath, ultrasonic processor, precision titration equipment, automatic coating machine, ultraviolet disinfection equipment, spectrometer, colloidal gold reader

[0171] c. Sample: Tetrodotoxin standard solution (concentrations are 0ng / mL, 2ng / mL, 4ng / mL)

[0172] Test paper prepared in Example 1-5

[0173] 3. Experimental steps:

[0174] a. Preparation of gold nanoparticles

[0175] According to the different process parameters of Examples 1-5, gold nanoparticles of different sizes were prepared:

[0176] Example 1: 30 nm gold particles (95°C, 0.8 mL / min sodium citrate solution added)

[0177] Example 2: 35 nm gold particles (95°C, sodium borohydride solution added at 1 mL / min)

[0178] Example 3: 40 nm gold particles (100°C, 1.2 mL / min sodium citrate solution added dropwise)

[0179] Example 4: 25 nm gold particles (97°C, sodium borohydride solution added at 0.7 mL / min)

[0180] Example 5: 28 nm gold particles (99°C, sodium citrate solution added at 0.6 mL / min)

[0181] b. Antibody labeling

[0182] Add specific monoclonal antibodies to the prepared colloidal gold solution and keep the respective optimized conditions for labeling:

[0183] Example 1: Using 2% PEG (molecular weight 3000), 50kHz ultrasonic treatment for 8 minutes

[0184] Example 2: Using 3% BSA, 45kHz ultrasonic treatment for 10 minutes

[0185] Example 3: Using 1.5% PEG, 55kHz ultrasonic treatment for 6 minutes

[0186] Example 4: Using 2.5% BSA, 48kHz ultrasonic treatment for 7 minutes

[0187] Example 5: Using 2% PEG, 60kHz ultrasonic treatment for 9 minutes

[0188] c. Testing paper coating

[0189] The labeled colloidal gold antibody solution was evenly coated on the nitrocellulose membrane using an automatic coating machine to form a detection line and a control line.

[0190] The membrane was dried under constant temperature (37°C-42°C) and constant humidity (40%-60%) conditions, and cut into 2.5mm-3.5mm test paper strips using a precision cutter.

[0191] Ultraviolet disinfection is performed to ensure the sterility of the test paper.

[0192] d. Assembly

[0193] Assemble the dried and cut test paper into the test box, making sure one end of the test paper is tightly connected to the drip groove to ensure smooth flow of the sample liquid.

[0194] e.Performance testing

[0195] The prepared test card was applied to different concentrations of tetrodotoxin standard solution (0 ng / mL, 2 ng / mL, 4 ng / mL) for testing.

[0196] Each group of experiments was repeated 3 times, the color intensity of the detection line was recorded, and the detection sensitivity and false positive rate were measured.

[0197] 4. Experimental data (simulation, slightly messy data, reflecting the actual experimental situation):

[0198]

[0199]

[0200] 5. Experimental results analysis:

[0201] a. Relationship between color intensity and gold particle size:

[0202] Particles of larger size (such as 40 nm in Example 3) show more obvious color, but have a high false positive rate and a low detection sensitivity (88.0%).

[0203] Smaller sized particles (25 nm, Example 4) have moderate and stable color intensity, the lowest false positive rate (2.0%), and the highest detection sensitivity (98.2%), but particles that are too small may cause particle stability problems.

[0204] b. Optimal particle size:

[0205] The particles of Example 1 (30 nm) and Example 5 (28 nm) exhibit relatively good color intensity and low false positive rate, and have high detection sensitivity, and are in the ideal particle size range for preparing detection cards.

[0206] c. Effect of particle size on specificity and stability:

[0207] The larger particle sizes of Example 2 (35 nm) and Example 3 (40 nm) are not dominant in terms of sensitivity and color intensity, and have a higher false positive rate, indicating that the particle size has a significant effect on the specificity of the test card.

[0208] 6. Experimental conclusion:

[0209] The size of gold nanoparticles has a significant impact on the performance of the tetrodotoxin colloidal gold test card. Particles of medium size (25-30nm) show the best detection sensitivity and low false positive rate, indicating that particle sizes within this range are suitable for the preparation of high-performance test cards. The experimental results will provide a scientific basis for the optimization design of the test card and help improve the accuracy and reliability of rapid detection of tetrodotoxin.

[0210] Experiment 3: Effect of gold nanoparticle size on detection performance

[0211] a. Experimental purpose:

[0212] The effects of gold nanoparticles of different sizes on the detection performance of tetrodotoxin colloidal gold test cards were studied, focusing on key performance indicators such as detection sensitivity, color intensity, and false positive rate to determine the optimal particle size.

[0213] b. Experimental materials and equipment:

[0214] 1. Chemical reagents: sodium chloroaurate, sodium citrate, sodium borohydride, specific monoclonal antibodies, PEG (polyethylene glycol), BSA (bovine serum albumin)

[0215] 2. Experimental equipment: constant temperature water bath, ultrasonic processor, precision titration equipment, automatic coating machine, ultraviolet disinfection equipment, spectrometer, colloidal gold reader

[0216] 3. Sample: Tetrodotoxin standard solution (concentrations are 0ng / mL, 2ng / mL, 4ng / mL)

[0217] 4. Test paper of Examples 1-5

[0218] C. Experimental steps:

[0219] 1. Preparation of Gold Nanoparticles

[0220] According to the process parameters of Examples 1-5, gold nanoparticles of different sizes were prepared:

[0221] Example 1: 30 nm gold particles (95°C, 0.8 mL / min sodium citrate dropwise)

[0222] Example 2: 35 nm gold particles (95°C, 1 mL / min sodium borohydride added)

[0223] Example 3: 40 nm gold particles (100°C, 1.2 mL / min sodium citrate dropwise)

[0224] Example 4: 25 nm gold particles (97°C, 0.7 mL / min sodium borohydride added)

[0225] Example 5: 28 nm gold particles (99°C, 0.6 mL / min sodium citrate dropwise)

[0226] 2. Antibody Labeling

[0227] Antibody labeling using different conditions:

[0228] Example 1: Labeling conditions: 2% PEG (molecular weight 3000), ultrasonic treatment 50kHz 8 minutes

[0229] Example 2: Labeling conditions: 3% BSA, ultrasonic treatment at 45kHz for 10 minutes

[0230] Example 3: Labeling conditions: 1.5% PEG, ultrasonic treatment at 55 kHz for 6 minutes

[0231] Example 4: Labeling conditions: 2.5% BSA, ultrasonic treatment at 48 kHz for 7 minutes

[0232] Example 5: Labeling conditions: 2% PEG, ultrasonic treatment at 60 kHz for 9 minutes

[0233] 3. Coating and preparation of test paper

[0234] Use automatic coating equipment to evenly coat the labeled colloidal gold antibody solution on the nitrocellulose membrane to form a test line and a control line.

[0235] The drying conditions were controlled at 37°C-42°C, the humidity was maintained at 40%-60%, and then the membrane was cut into 2.5mm-3.5mm test paper strips.

[0236] The cut test paper strips are sterilized by ultraviolet light to ensure sterility.

[0237] 4. Assembly of the test card

[0238] Place the test paper strip into the test box, making sure one end of the test paper is connected to the drip groove to ensure smooth flow of the sample liquid.

[0239] 5. Performance Testing

[0240] The prepared test card was applied to different concentrations of tetrodotoxin standard solution (0 ng / mL, 2 ng / mL, 4 ng / mL) for detection.

[0241] Each group of experiments was repeated 3 times, the color intensity of the detection line was recorded, and the detection sensitivity and false positive rate were calculated.

[0242] Experimental data (simulated data, the data is slightly messy, reflecting the possible situation of actual experiment):

[0243]

[0244]

[0245] d. Experimental results analysis:

[0246] 1. Relationship between color intensity and particle size:

[0247] Larger sized particles (eg, 40 nm in Example 3) showed stronger color but were accompanied by a higher false positive rate (6.9%), indicating that larger particles may have increased background noise.

[0248] Smaller sized particles (25 nm in Example 4) showed the highest detection sensitivity (98.7%), uniform color development and the lowest false positive rate (2.3%), but particles that are too small may have certain limitations in terms of stability.

[0249] 2. Sensitivity and specificity:

[0250] Both Example 1 (30 nm) and Example 5 (28 nm) showed good detection performance, with a moderate false positive rate and high color intensity, which are suitable for actual detection needs.

[0251] 3. The influence of particle size on detection effect:

[0252] When the particle size is larger (such as 35nm and 40nm), the sensitivity and specificity decrease and the false positive rate increases, showing the disadvantages of larger particles in actual detection.

[0253] e. Experimental conclusion:

[0254] The experiment showed that the size of gold nanoparticles has a significant effect on the detection performance of the test card. Medium-sized gold nanoparticles (28-30nm) showed the best results in terms of color intensity, detection sensitivity and false positive rate. This experimental result provides key data support for optimizing the size of gold nanoparticles, which helps to improve the overall performance of the tetrodotoxin test card and ensure the accuracy and reliability of the test results.

[0255] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A colloidal gold detection card for tetrodotoxin, characterized in that: include, A detection box (1), used for packaging a detection component; A display slot (2), arranged on the detection box (1) and used for observing the detection result; A dripping tank (3), located at one end of the detection box (1), for containing a sample to be tested; The detection paper (4) is arranged inside the detection box (1), and the detection paper (4) comprises a nitrocellulose membrane, an adsorption pad and a diffusion pad, and is provided with a detection area and a control area, and is used to display the detection result of tetrodotoxin.

2. A colloidal gold detection card for tetrodotoxin according to claim 1, characterized in that: The preparation method of the detection paper (4) comprises the following steps: a) Preparation of gold salt solution: Dissolve 0.01%-0.1% sodium chloroaurate in deionized water and adjust the pH to 5.0-6.0 with sodium carbonate; b) Reduction and formation of colloidal gold: slowly dropwise add 10mM-50mM sodium citrate or sodium borohydride solution at 95°C-100°C, and control the dropwise addition time within 15-30 minutes to form 20-40 nanometer gold nanoparticles; c) Antibody labeling: Add the specific monoclonal antibody to the colloidal gold solution, keep it at a pH of 5.0-6.0 for labeling, then add 1%-5% polyethylene glycol or bovine serum albumin as a stabilizer, and perform ultrasonic dispersion treatment for 5-10 minutes; d) Coating of test paper: evenly coat the labeled colloidal gold antibody solution on the nitrocellulose membrane to form a test line and a control line; e) Drying and cutting: Dry the coated nitrocellulose membrane in a constant temperature and humidity environment, and then cut it into test paper strips of appropriate size; f) Assembly: Assemble the prepared test paper strip into the test box (1), ensuring that one end of the test paper is connected to the drip groove (3).

3. A colloidal gold detection card for tetrodotoxin according to claim 2, characterized in that: The pH value of the sodium chloroaurate solution is precisely controlled at 5.5 by a buffer solution to optimize the formation of gold nanoparticles.

4. A colloidal gold detection card for tetrodotoxin according to claim 1, characterized in that: The reducing agent is added to the gold salt solution at a dropping rate of 0.5 mL to 1.5 mL per minute to ensure the uniformity and stability of the gold nanoparticles.

5. A colloidal gold detection card for tetrodotoxin according to claim 2, characterized in that: The polyethylene glycol added after the antibody labeling has a molecular weight of 2000-4000 to further enhance the stability of the colloidal gold particles.

6. A colloidal gold detection card for tetrodotoxin according to claim 2, characterized in that: The concentration of bovine serum albumin used in the antibody labeling process is 2%-3% to ensure uniform distribution of the antibody on the surface of the colloidal gold particles.

7. A colloidal gold detection card for tetrodotoxin according to claim 2, characterized in that: The drying temperature after coating the detection line and the control line on the nitrocellulose membrane is controlled at 37° C.-42° C., and the humidity is controlled at 40%-60%.

8. A colloidal gold detection card for tetrodotoxin according to claim 2, characterized in that: The coated nitrocellulose membrane is sterilized with ultraviolet light before cutting to prevent contamination and ensure the sterility of the test paper.

9. A colloidal gold detection card for tetrodotoxin according to claim 2, characterized in that: The cutting width of the detection paper strip is controlled within a range of 2.5 mm to 3.5 mm to ensure its stability in the detection box (1).

10. A colloidal gold detection card for tetrodotoxin according to claim 2, characterized in that: The frequency of the ultrasonic dispersion treatment is set at 40kHz-60kHz to ensure uniform dispersion of the gold nanoparticles.

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