Method for detecting lactoferrin in milk based on competition method test strip

Abstract

The invention discloses a method for detecting lactoferrin in milk based on a competition method test strip, which comprises the following steps: coupling heparin molecules with bovine serum albumin to obtain a heparin molecule-bovine serum albumin artificial antigen; modifying gold nanoparticles by using an artificial antigen to form a gold label I, and constructing a competition method test strip by matching with a gold label II modified by mouse IgG (Immunoglobulin G); a milk sample is subjected to centrifugal fat removal, pH adjustment and casein precipitation pretreatment and then dropwise added to the test strip, and the LF concentration is calculated through a T line gray value and a working curve. Detection can be completed within about 10-15 min, specificity is high (interference of impure protein is avoided), on-site rapid quantitative / semi-quantitative detection of LF in milk can be achieved, the antiviral activity of LF can be indirectly reflected through the binding strength of heparin and LF, the method is suitable for production quality inspection, dealer sampling inspection and consumer self-inspection, and the detection sensitivity reaches 1 microgram / mL.

Description

Technical Field

[0001] This invention pertains to food component detection methods, specifically a method for detecting lactoferrin in milk based on a competitive test strip. Background Technology

[0002] Lactoferrin (LF) is a core functional component in dairy products. Its antibacterial, antiviral, and immunomodulatory activities directly determine the functional value of dairy products. In particular, LF's ability to block viral invasion of host cells through iron-binding sites has become a core competitive advantage for high-end dairy products (such as infant formula and functional dairy beverages). The market has an urgent need for "accurate content detection + rapid activity assessment" of LF.

[0003] However, current LF detection technologies face numerous insurmountable bottlenecks, severely hindering industry development and meeting consumer demand: While mass spectrometry can determine LF content, it is cumbersome (requiring multiple pretreatment steps such as protein precipitation and column chromatography), has a long detection cycle (ranging from hours to days), and can only detect content without correlating activity. Even if high levels of LF are detected, it's impossible to determine whether it's due to processing inactivation. Furthermore, these methods rely on expensive specialized equipment, making them unsuitable for widespread application in production workshops and sales terminals. While enzyme-linked immunosorbent assay (ELISA) is slightly cheaper, it still requires a microplate reader to read the signal, and impurities such as casein and α-lactalbumin in milk can cause significant detection bias.

[0004] Competitive assay test strips, as a convenient on-site testing tool, are based on a competitive reaction mechanism of specific binding between antigens and antibodies. However, existing competitive assay test strips for LF (sulfate leukemia) face two major drawbacks: first, the lack of highly specific recognition ligands means traditional antibodies cannot avoid matrix interference; second, when sugar molecules (such as heparin) are directly loaded with gold nanoparticles, a stable gold-labeled probe cannot be formed, resulting in weak signals and low sensitivity, failing to meet testing requirements. Furthermore, the methods are complex to operate, highly dependent on equipment, and have poor specificity. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for detecting lactoferrin in milk based on a competitive test strip that is on-site responsive, can correlate activity, is rapid, and has high accuracy.

[0006] Technical solution: The present invention provides a method for detecting lactoferrin in milk based on a competitive test strip, comprising the following steps:

[0007] Step 1: Preparation of heparin-based molecular-bovine serum albumin (BSA) artificial antigen: Heparin-based molecular solution, cross-linking agent, and sodium chloride are stirred at room temperature to activate the antigen; bovine serum albumin solution is added to the activated solution and stirred to react. After the reaction is complete, the antigen is purified by dialysis to remove unreacted substances. After dialysis, excess water is removed to obtain heparin-based molecular-bovine serum albumin artificial antigen.

[0008] Step 2, Preparation of Gold Label 1: Adjust the pH of the gold nanoparticle solution to 5.5-6.5, add heparin-based molecules-bovine serum albumin artificial antigen solution, and shake to react; then add bovine serum albumin molecule solution to block unbound sites on the colloidal gold surface, and continue shaking; after the reaction, centrifuge, discard the supernatant, resuspend the precipitate with buffer, repeat centrifugation to obtain Gold Label 1, i.e., artificial antigen-colloidal gold probe;

[0009] Step 3, Preparation of Gold Label II: Adjust the pH of the gold nanoparticle solution to 5.5-6.5, add mouse IgG solution, and shake to react; add bovine serum albumin solution to block non-specific sites, and continue shaking; after centrifugation, discard the supernatant, resuspend the precipitate with buffer, repeat centrifugation, and obtain Gold Label II, i.e., mouse IgG-colloidal gold probe;

[0010] Step 4, prepare the test strip: Draw the detection line (T line) and the control line (C line) separately and dry them. Spray a mixed gold labeling solution containing gold labeling I and gold labeling II onto the gold labeling pad to obtain the test strip.

[0011] Step 5, Pre-treatment of milk samples: Take the milk sample to be tested, remove the upper fat layer after the first centrifugation, and keep the middle liquid; adjust the pH of the middle liquid to 4.6~4.8, centrifuge a second time, and take the supernatant as the sample solution to be tested;

[0012] Step 6, prepare the working curve: Add different concentrations of lactoferrin standards to the sample solution to be tested prepared in Step 5, prepare whey simulated samples, and drop them onto the test strips in Step 4. After standing at room temperature, read the gray value of the T line of the test strips. Plot the working curve with the logarithm of the lactoferrin concentration as the abscissa and the gray value of the T line as the ordinate.

[0013] Step 7, test the actual sample: Take the sample solution obtained in Step 5, drop it onto the test strip in Step 4, let it stand at room temperature, and read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the actual concentration of lactoferrin in the sample to be tested.

[0014] Further, in step one, the heparin-like molecule is any one of heparin sulfate, heparin sodium, fondaparinux sodium, dalteparin sodium, nadroparin calcium, and enoxaparin sodium, and the cross-linking agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS). Fondaparinux sodium is preferred as it has a stable structure and molecular weight, which can better ensure the consistency of the test strip's effect in practical applications.

[0015] Further, in step one, the final concentration of bovine serum albumin solution is 0.5~2 mg / mL, the final concentration of N-hydroxythiosuccinimide is 0.01~0.05 M, and the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.05-0.2 M. Dialysis purification is carried out in a dialysis bag with a molecular weight cutoff of 12000~14000 Da.

[0016] Furthermore, in steps two and three, the gold nanoparticles are any one of colloidal gold, gold nanostars, gold nanoflowers, and gold nanorods.

[0017] Furthermore, in step two, the amount of heparin-based molecules-bovine serum albumin artificial antigen solution added is 10~100 μg / mL of gold nanoparticles.

[0018] Furthermore, in step four, the volume ratio of gold standard one to gold standard two in the mixed gold standard solution is 1~3:1, and the spraying amount is 5~10 μL / cm. 2 .

[0019] Furthermore, in step four, the T line of the test strip is a lactoferrin solution with a concentration of 0.5~2 mg / mL, and the C line is a goat anti-mouse IgG solution with a concentration of 0.5~2 mg / mL.

[0020] Specifically, the test strip typically consists of a sample pad, a gold-labeled pad, a nitrocellulose membrane (containing the test line (T line) and the control line (C line), and an absorbent pad. When the sample is added to the sample pad, it migrates towards the absorbent pad during chromatography, first encountering the gold-labeled ligand on the gold-labeled pad. The LF in the sample specifically binds to the gold-labeled ligand, forming an "LF-gold-labeled ligand" complex. As the mixture continues to migrate to the T line, the free gold-labeled ligands that have not bound to the LF in the sample bind to the fixed LF antigen on the T line, causing the T line to show a corresponding signal (the color of the gold nanoparticles). The gold-labeled ligands that have formed the complex, because their binding sites are occupied, cannot bind to the T line antigen and will continue to migrate towards the C line, binding to it and producing a color change.

[0021] The final result is determined by the intensity of the T-line signal: the higher the LF content in the sample, the higher the proportion of LF binding to the gold-labeled ligand, resulting in fewer free gold-labeled ligands that can bind to the T-line, and a weaker T-line signal (or even no signal); conversely, the lower the LF content, the stronger the T-line signal. The color development of the C-line is used to verify the validity of the test strip. The C-line should develop color regardless of the presence of LF in the sample; otherwise, the test is invalid.

[0022] Furthermore, in step five, the first centrifugation is performed at a speed of 6000~10000 rpm for 10~20 minutes; the second centrifugation is performed at a speed of 4000~6000 rpm for 10~20 minutes.

[0023] Furthermore, in step six, the concentration of the lactoferrin standard is 0–500 μg / mL, and the incubation time at room temperature is 10–20 minutes. The concentration of the lactoferrin standard is set to 0 μg / mL because, in actual competitive testing, a concentration of 0 μg / mL corresponds to the strongest colorimetric value. Without 0 μg / mL, it would be impossible to accurately distinguish whether the detection limit is true or undetectable.

[0024] Furthermore, in step seven, the standing time at room temperature is 10-15 minutes. The test results of different samples are then compared.

[0025] Detection Principle: Utilizing an indirect competitive colloidal gold method, an artificial antigen is prepared by coupling heparin-like molecules to bovine serum albumin (BSA) via EDC and sulfur-NHS. This artificial antigen is then physically adsorbed and immobilized onto the colloidal gold surface to construct gold label 1 (heparin analog-BSA-colloidal gold probe). This is combined with gold label 2 (mouse IgG-colloidal gold probe), a nitrocellulose membrane (NC membrane), and other test strip components to complete the detection system construction and sealing. Tween-20 and sodium chloride are added to the whey solution obtained after centrifugation to remove fat and pre-treating casein in milk samples (target molecule: lactoferrin LF) to adjust pH. The solution is then dropped onto the colloidal gold test strip sample pad containing gold label 1 and gold label 2, allowing for real-time dynamic investigation of the specific electrostatic interaction between heparin-like molecules and lactoferrin.

[0026] From the perspective of altering the reaction principle, the constructed heparin-BSA-colloidal gold probe structure can better simulate the specific binding mechanism of heparin molecules and lactoferrin in in vitro detection systems. Furthermore, this method uses BSA to block unbound sites on the colloidal gold surface and hydrophobic sites on the test strip sample pad; Tween-20 further blocks the remaining hydrophobic sites between the gold probe and the NC membrane; sodium chloride adjusts the ionic strength of the system to weaken non-specific electrostatic interference; and the milk pretreatment step removes matrix interference components such as fat and casein. Therefore, the method comprehensively shields against non-specific interactions between non-specific proteins in the milk matrix and the gold probe or test strip components. The prerequisite for identifying the specific electrostatic interaction between heparin molecules and lactoferrin is the suppression of non-specific interactions, thus enabling the accurate detection of lactoferrin in complex matrices such as milk.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0028] 1. It can solve the problems of poor ligand specificity and inability to associate LF antiviral and other biological activities in traditional LF detection. By selecting heparin molecules as highly specific recognition ligands for LF, and utilizing the precise complementary electrostatic interaction between its linear negatively charged group chain and the positive potential point of the iron binding domain on the LF surface, it can simultaneously achieve specific recognition of LF (avoiding cross-reaction with contaminating proteins) and activity correlation judgment (binding strength reflects the level of activity) in vitro, thus solving the core pain points of traditional anti-LF antibodies being easily interfered with and only able to detect the content.

[0029] 2. Through the competitive test strip detection platform, combined with the signal amplification effect of "heparin-BSA artificial antigen modified gold nanoparticles", the content and activity of LF can be converted into changes in the gray value of the T line. The higher the LF content, the lighter the T line color. When the activity is normal, the gray value of the T line conforms to the regular range, thus realizing the visual reading of the detection results. The signal can be judged intuitively without the need for professional equipment such as high performance liquid chromatographs and enzyme readers. It provides a new tool for the on-site and rapid detection of LF in milk and dairy products, and the detection can be completed in 10 to 15 minutes.

[0030] 3. The "heparin-BSA artificial antigen" stable loading technology solves the problem of direct sugar loading and shedding of sugar molecules. Combined with the pretreatment of milk samples such as centrifugation to remove fat and pH adjustment to precipitate casein, and the shielding of non-specific effects by Tween-20, the interference of impurities such as fat, casein, and α-lactalbumin in the system is shielded, making it possible to accurately quantify LF in complex milk matrices and rapidly assess antiviral activity, with a detection sensitivity of 1 μg / mL. Attached Figure Description

[0031] Figure 1 This is a flowchart of the preparation process of the present invention;

[0032] Figure 2This is a schematic diagram of the structure of the present invention;

[0033] Figure 3 This is a schematic diagram illustrating the principle of the present invention;

[0034] Figure 4 These are actual images of the present invention when detecting LF in whey matrix;

[0035] Figure 5 The working curve of this invention for detecting LF in whey matrix;

[0036] Figure 6 This invention presents the actual sample test results of milk at different processing stages. Detailed Implementation

[0037] Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0038] Example 1

[0039] like Figure 1 A method for detecting lactoferrin in milk based on a competitive test strip includes the following steps:

[0040] Step 1: Preparation of fondaparinux sodium-BSA artificial antigen: Prepare fondaparinux sodium solution, add 0.1 M MEDC and 0.01 M sulfo-NHS (both final concentrations), add sodium chloride, and stir at room temperature for 15 minutes. Add BSA solution to the activation solution to a final concentration of 1 mg / mL, with a BSA to fondaparinux sodium mass ratio of 1:100, and stir at room temperature for 2 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 13000 Da for dialysis purification. The dialysate is 0.125 M sodium chloride solution, and the dialysis time is 72 hours to remove unreacted substances and excess water.

[0041] Step 2, Preparation of Gold Label 1 (Fondaparinux Sodium-BSA-Colloidal Gold Probe): Take the colloidal gold solution and adjust the pH to 6.0 with 0.2 MK2CO3. Prepare a 50 μg / mL colloidal gold-fondaparinux sodium-BSA artificial antigen solution and vortex for 15 minutes. Then add BSA solution to make the final BSA concentration in the system 1 wt%, and continue vortexing for 15 minutes. After the reaction, centrifuge at 8000 rpm for 15 minutes, discard the supernatant, resuspend the precipitate with 0.01 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold label 1, and add 0.01 M PB buffer (pH 6.0) containing 2 wt% BSA, 0.2 wt% Tween-20, 3 wt% trehalose, and 0.6 M sodium chloride. Store at 4℃ for later use.

[0042] Step 3, Preparation of Gold-Labeled Probe II (Mouse IgG-Gold Nanostar Probe): Take colloidal gold solution and adjust the pH to 6.0 with 0.2 M K2CO3. Add mouse IgG solution and incubate at room temperature with shaking for 15 minutes. Add BSA solution to bring the final BSA concentration in the system to 1 wt%, and continue shaking for 15 minutes. Centrifuge at 8000 rpm for 15 minutes, discard the supernatant, resuspend the precipitate with 0.01 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold-labeled probe II, and add 0.01 M PB buffer (pH 6.0) containing 2 wt% BSA, 0.2 wt% Tween-20, 3 wt% trehalose, and 0.6 M sodium chloride. Store at 4℃ for later use.

[0043] Step 4, prepare the test strip: (e.g.) Figure 2 The basic structure of the test strip was constructed by sequentially overlapping the sample pad, gold-labeled pad, NC membrane, and absorbent pad onto a PVC base plate, with an overlap width of 1.5 mm. The NC membrane was then treated by streaking the T line (1 mg / mL LF solution) and C line (1 mg / mL goat anti-mouse IgG solution) at a concentration of 1 μg / mL, with a line spacing of 6 mm, and drying at 37°C for 4 hours. The gold-labeled pad was then treated by mixing gold label one and gold label two at a volume ratio of 2:1, and spraying 7 μL of the mixed gold label solution per square centimeter of glass fiber membrane. Finally, all components were assembled, cut into 4 mm wide test strips, sealed, and stored at 4°C.

[0044] Step 5, Pretreatment of milk samples: Take the milk sample to be tested, centrifuge at 8000 rpm for 15 minutes to remove the upper fat layer, and retain the middle layer liquid. Adjust the pH of the middle layer liquid to 4.7 with 0.1 M HCl. Centrifuge at 5000 rpm for 15 minutes, and take the supernatant (whey) as the sample solution to be tested.

[0045] Step Six, Prepare the Working Curve: Add 0, 1, 5, 20, 100, and 500 μg / mL of LF standard to the blank whey prepared in Step Five to prepare whey simulation samples. Add 100 μL of each concentration of simulation sample to the test strip prepared in Step Four, let it stand at room temperature for 15 minutes, and then read the gray value of the T line. Plot the working curve with the commonly used logarithm of the LF concentration in whey as the x-axis and the gray value of the T line as the y-axis, as shown below. Figure 4 , Figure 5 As shown, the fitting equation R 2 =0.984 (≥0.98).

[0046] Step 7: Test the actual sample and analyze the results: Take 100 μL of the whey sample solution obtained in Step 5, add it to the test strip in Step 4, let it stand at room temperature for 12 minutes, and then read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the LF concentration in the sample. Perform three parallel tests, and the results are as follows. Figure 6 As shown.

[0047] Figure 6 This paper presents the detection results of lactoferrin content in 13 actual milk samples (including 9 commercial milk samples and 4 raw milk samples) using this test strip. The results show a significant difference in lactoferrin concentration between raw and commercial milk: the concentrations in commercial milk samples (numbered 1-9) were generally lower, ranging from 4.50 μg / mL to 106.86 μg / mL; while the concentrations in raw milk samples (numbered 10-13) were significantly higher, ranging from 110.54 μg / mL to 409.42 μg / mL, generally much higher than the commercial milk group. This difference is mainly attributed to the heat sensitivity of lactoferrin. The high-temperature sterilization treatment (such as pasteurization or UHT) during the processing of commercial milk leads to protein denaturation and loss, while raw milk retains a higher natural content. This result not only conforms to theoretical expectations but also verifies that this method has good applicability and a wide detection range in complex matrices.

[0048] Example 2

[0049] A method for detecting lactoferrin in milk based on a competitive test strip includes the following steps:

[0050] Step 1: Preparation of Heparin Sulfate-BSA Artificial Antigen: Prepare a heparin sulfate solution, add 0.05 M EDC and 0.01 M sulfo-NHS (both final concentrations), add sodium chloride, and stir at room temperature for 10 minutes. Add BSA solution to the activation solution to a final concentration of 0.5 mg / mL, with a BSA to heparin sulfate mass ratio of 1:100, and stir at room temperature for 2 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 12000 Da for dialysis purification. The dialysate is 0.125 M sodium chloride solution, and the dialysis time is 48 hours to remove unreacted substances and excess water.

[0051] Step 2, Preparation of Gold Label 1 (Heparin Sulfate-BSA-Gold Nanostar Probe): Take the gold nanostar solution and adjust the pH to 6.0 with 0.2 MK2CO3. Prepare a 10 μg / mL gold nanostar heparin sulfate-BSA artificial antigen solution and vortex for 15 minutes. Then add BSA solution to make the final BSA concentration in the system 0.5 wt%, and continue vortexing for 15 minutes. After the reaction, centrifuge at 6000 rpm for 15 minutes, discard the supernatant, resuspend the precipitate with 0.01 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold label 1, and add 0.01 M PB buffer (pH 6.0) containing 1 wt% BSA, 0.1 wt% Tween-20, 1 wt% trehalose, and 0.6 M sodium chloride. Store at 4℃ for later use.

[0052] Step 3, Preparation of Gold-Labeled Probe II (Mouse IgG-Colloidal Gold Probe): Take the gold nanostar solution and adjust the pH to 6.0 with 0.2 M K2CO3. Add mouse IgG solution and vortex at room temperature for 15 minutes. Add BSA solution to bring the final BSA concentration in the system to 0.5 wt%, and continue vortexing for 15 minutes. Centrifuge at 6000 rpm for 15 minutes, discard the supernatant, resuspend the precipitate with 0.01 MPB buffer, repeat centrifugation 3 times, adjust the concentration of gold-labeled probe II, and add 0.01 M PB buffer (pH 6.0) containing 1 wt% BSA, 0.1 wt% Tween-20, 1 wt% trehalose, and 0.8 M sodium chloride. Store at 4℃ for later use.

[0053] Step 4, Test Strip Preparation: Construct the basic structure of the test strip by sequentially overlapping the sample pad, gold-labeled pad, NC membrane, and absorbent pad onto a PVC base plate, with an overlap width of 1 mm. Treat the NC membrane by streaking the T line (0.5 mg / mL LF solution) and C line (0.5 mg / mL goat anti-mouse IgG solution) at a concentration of 1 μg / mL and a line spacing of 5 mm. Dry at 37°C for 2 hours. Treat the gold-labeled pad by mixing gold label one and gold label two at a 1:1 volume ratio, and spraying 5 μL of the mixed gold label solution onto each square centimeter of glass fiber membrane. Finally, assemble all components, cut into 3 mm wide test strips, seal, and store at 4°C.

[0054] Step 5, Pretreatment of milk samples: Take the milk sample to be tested, centrifuge at 6000 rpm for 10 minutes to remove the upper fat layer, and retain the middle liquid. Adjust the pH of the middle liquid to 4.6 with 0.1 M HCl. Centrifuge at 4000 rpm for 10 minutes, and take the supernatant (whey) as the sample solution to be tested.

[0055] Step Six: Prepare the working curve: Add 0, 1, 5, 20, 100, and 500 μg / mL of LF standard to the blank whey prepared in Step Five to prepare whey simulation samples. Add 100 μL of each concentration of simulation sample to the test strip prepared in Step Four, let it stand at room temperature for 10 minutes, and then read the gray value of the T line. Plot the working curve with the commonly used logarithm of the LF concentration in whey as the x-axis and the gray value of the T line as the y-axis.

[0056] Step 7: Test the actual sample and analyze the results: Take 100 μL of the whey sample solution obtained in Step 5, add it to the test strip in Step 4, let it stand at room temperature for 10 minutes, and then read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the LF concentration in the sample. Perform three parallel tests.

[0057] Example 3

[0058] A method for detecting lactoferrin in milk based on a competitive test strip includes the following steps:

[0059] Step 1: Preparation of Heparin Sodium-BSA Artificial Antigen: Prepare a heparin sodium solution, add 0.2 M EDC and 0.05 M sulfo-NHS as cross-linking agents, add sodium chloride, and stir at room temperature for 30 minutes. Add BSA solution to the activation solution to a final concentration of 2 mg / mL, with a BSA to heparin sodium mass ratio of 1:100, and stir at room temperature for 4 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 14000 Da for dialysis purification. The dialysate is water, and the dialysis time is 72 hours to remove unreacted substances and excess water.

[0060] Step 2, Preparation of Gold Label 1 (Heparin Sodium-BSA-Gold Nanorod Probe): Take the gold nanorod solution and adjust the pH to 6.0 with 0.2 MK2CO3. Add 100 μg / mL of heparin sodium-BSA artificial antigen solution containing gold nanorods and shake for 20 minutes. Then add BSA solution to bring the final BSA concentration in the system to 1 wt%, and continue shaking for 20 minutes. After the reaction, centrifuge at 10000 rpm for 20 minutes, discard the supernatant, resuspend the precipitate in 0.05 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold label 1, and add 0.01 M PB buffer (pH 6.0) containing 2 wt% BSA, 0.2 wt% Tween-20, 5 wt% trehalose, and 0.4 M sodium chloride. Store at 4℃ for later use.

[0061] Step 3, Preparation of Gold-Labeled Probe II (Mouse IgG-Gold Nanorod Probe): Take the gold nanorod solution and adjust the pH to 6.0 with 0.2 M K2CO3. Add mouse IgG solution and incubate at room temperature with shaking for 20 minutes. Add BSA solution to bring the final BSA concentration in the system to 1 wt%, and continue shaking for 20 minutes. Centrifuge at 10,000 rpm for 20 minutes, discard the supernatant, resuspend the precipitate with 0.05 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold-labeled probe II, and add 0.01 MPB, 0.6 M sodium chloride buffer (pH 6.0) containing 2 wt% BSA, 0.2 wt% Tween-20, and 5 wt% trehalose. Store at 4℃ for later use.

[0062] Step 4: Preparation of Colloidal Gold Test Strips: Construct the basic structure of the test strip by sequentially overlapping the sample pad, gold-labeled pad, NC membrane, and absorbent pad onto a PVC base plate, with an overlap width of 2 mm. Treat the NC membrane by streaking the T line (2 mg / mL LF solution) and C line (2 mg / mL goat anti-mouse IgG solution) at a concentration of 1 μg / mL and a line spacing of 8 mm. Dry at 37°C for 4 hours. Treat the gold-labeled pad by mixing gold label one and gold label two at a volume ratio of 3:1, and spraying 10 μL of the mixed gold label solution onto each square centimeter of glass fiber membrane. Finally, assemble all components, cut into 5 mm wide test strips, seal, and store at 4°C.

[0063] Step 5, Pre-treatment of milk samples: Take the milk sample to be tested, centrifuge at 10,000 rpm for 20 minutes to remove the upper fat layer, and retain the middle layer liquid. Adjust the pH of the middle layer liquid to 4.8 (upper limit of pH) with 0.1 M HCl. Centrifuge at 6,000 rpm for 20 minutes, and take the supernatant (whey) as the sample solution to be tested.

[0064] Step Six: Prepare the working curve: Add 0, 1, 5, 20, 100, and 500 μg / mL of LF standard to the blank whey prepared in Step Five to prepare whey simulation samples. Add 100 μL of each concentration of simulation sample to the test strip prepared in Step Four, let it stand at room temperature for 20 minutes, and then read the gray value of the T line. Plot the working curve with the commonly used logarithm of the LF concentration in whey as the x-axis and the gray value of the T line as the y-axis.

[0065] Step 7: Test the actual sample and analyze the results: Take 100 μL of the whey sample solution obtained in Step 5, add it to the test strip in Step 4, let it stand at room temperature for 15 minutes, and then read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the LF concentration in the sample. Perform three parallel tests.

[0066] Example 4

[0067] A method for detecting lactoferrin in milk based on a competitive test strip includes the following steps:

[0068] Step 1: Preparation of Dalteparin Sodium-BSA Artificial Antigen: Prepare a dalteparin sodium solution, add 0.08 M EDC and 0.02 M sulfo-NHS (both final concentrations), add sodium chloride, and stir at room temperature for 15 minutes. Add BSA solution to the activation solution to a final concentration of 0.8 mg / mL, with a BSA to dalteparin sodium mass ratio of 1:200, and stir at room temperature for 2.5 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 12000 Da for dialysis purification. The dialysate is water, and the dialysis time is 50 hours to remove unreacted substances and excess water.

[0069] Step 2, Preparation of Gold Label 1 (Dalteparin Sodium-BSA-Gold Nanoflower Probe): Take the gold nanoflower solution and adjust the pH to 6.0 with 0.2 MK2CO3. Add 30 μg / mL of the gold nanoflower dalteparin sodium-BSA artificial antigen solution and shake for 16 minutes. Then add BSA solution to make the final BSA concentration in the system 0.6 wt%, and continue shaking for 16 minutes. After the reaction, centrifuge at 7000 rpm for 16 minutes, discard the supernatant, resuspend the precipitate with 0.01 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold label 1, and add 0.01 M PB buffer (pH 6.0) containing 1.2 wt% BSA, 0.12 wt% Tween-20, and 2 wt% trehalose. Store at 4℃ for later use.

[0070] Step 3, Preparation of Gold-Labeled Probe II (Mouse IgG-Gold Nanoflower Probe): Take the gold nanoflower solution and adjust the pH to 6.0 with 0.2 M K2CO3. Add mouse IgG solution and incubate at room temperature with shaking for 16 minutes. Add BSA solution to bring the final BSA concentration in the system to 0.6 wt%, and continue shaking for 16 minutes. Centrifuge at 7000 rpm for 16 minutes, discard the supernatant, resuspend the precipitate with 0.01 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold-labeled probe II, and add 0.01 M PB buffer (pH 6.0) containing 1.2 wt% BSA, 0.12 wt% Tween-20, 2 wt% trehalose, and 0.6 M sodium chloride. Store at 4℃ for later use.

[0071] Step 4, Test Strip Preparation: Construct the basic structure of the test strip by sequentially overlapping the sample pad, gold-labeled pad, NC membrane, and absorbent pad onto a PVC base plate, with an overlap width of 1.2 mm. Treat the NC membrane by streaking the T line (0.8 mg / mL LF solution) and C line (0.8 mg / mL goat anti-mouse IgG solution) at a concentration of 1 μg / mL and a line spacing of 5.5 mm. Dry at 37°C for 2.5 hours. Treat the gold-labeled pad by mixing gold label one and gold label two at a 1:1 volume ratio, and spraying 5 μL of the mixed gold label solution onto each square centimeter of glass fiber membrane. Finally, assemble all components, cut into 3 mm wide test strips, seal, and store at 4°C.

[0072] Step 5, Pretreatment of milk samples: Take the milk sample to be tested, centrifuge at 7000 rpm for 12 minutes to remove the upper fat layer, and retain the middle layer liquid. Adjust the pH of the middle layer liquid to 4.65 with 0.1 M HCl. Centrifuge at 4500 rpm for 12 minutes, and take the supernatant (whey) as the sample solution to be tested.

[0073] Step Six, Prepare the Working Curve: Add 0, 1, 5, 20, 100, and 500 μg / mL of LF standard to the blank whey prepared in Step Five to prepare whey simulation samples. Add 100 μL of each concentration of simulation sample to the test strip prepared in Step Four, let it stand at room temperature for 15 minutes, and then read the gray value of the T line. Plot the working curve with the commonly used logarithm of the LF concentration in whey as the x-axis and the gray value of the T line as the y-axis.

[0074] Step 7: Test the actual sample and analyze the results: Take 100 μL of the whey sample solution obtained in Step 5, add it to the test strip in Step 4, let it stand at room temperature for 15 minutes, and then read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the LF concentration in the sample. Perform three parallel tests.

[0075] Example 5

[0076] A method for detecting lactoferrin in milk based on a competitive test strip includes the following steps:

[0077] Step 1: Preparation of nadroparin calcium-BSA artificial antigen: Prepare nadroparin calcium solution, add 0.15 M MEDC and 0.04 M sulfo-NHS (both final concentrations), add sodium chloride, and stir at room temperature for 25 minutes. Add BSA solution to the activation solution to a final concentration of 1.5 mg / mL, with a BSA to nadroparin calcium mass ratio of 1:80, and stir at room temperature for 3.5 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 14000 Da for dialysis purification. The dialysate is 0.125 M sodium chloride solution, and the dialysis time is 72 hours to remove unreacted substances and excess water.

[0078] Step 2, Preparation of Gold Label 1 (Nadroparin Calcium-BSA-Colloidal Gold Probe): Take the colloidal gold solution and adjust the pH to 6.0 with 0.2 MK2CO3. Add 80 μg / mL of colloidal gold nadroparin calcium-BSA artificial antigen solution and vortex for 19 minutes. Then add BSA solution to bring the final BSA concentration in the system to 0.9 wt%, and continue vortexing for 19 minutes. After the reaction, centrifuge at 9000 rpm for 19 minutes, discard the supernatant, resuspend the precipitate in 0.05 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold label 1, and add 0.01 M PB buffer (pH 6.0) containing 1.8 wt% BSA, 0.18 wt% Tween-20, and 4 wt% trehalose. Store at 4℃ for later use.

[0079] Step 3, Preparation of Gold-Labeled Probe II (Mouse IgG-Colloidal Gold Probe): Take the colloidal gold solution and adjust the pH to 6.0 with 0.2 M K2CO3. Add mouse IgG solution and incubate at room temperature with shaking for 19 minutes. Add BSA solution to bring the final BSA concentration in the system to 0.9 wt%, and continue shaking for 19 minutes. Centrifuge at 9000 rpm for 19 minutes, discard the supernatant, resuspend the precipitate in 0.05 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold-labeled probe II, and add 0.01 M PB buffer (pH 6.0) containing 1.8 wt% BSA, 0.18 wt% Tween-20, 4 wt% trehalose, and 0.6 M sodium chloride. Store at 4℃ for later use.

[0080] Step 4, Test Strip Preparation: Construct the basic structure of the test strip by sequentially overlapping the sample pad, gold-labeled pad, NC membrane, and absorbent pad onto a PVC base plate, with an overlap width of 1.8 mm. Treat the NC membrane by streaking the T line (1.8 mg / mL LF solution) and C line (1.8 mg / mL goat anti-mouse IgG solution) at a concentration of 1 μg / mL and a line spacing of 7.5 mm. Dry at 37°C for 4 hours. Treat the gold-labeled pad by mixing gold label one and gold label two at a volume ratio of 2.5:1, and spraying 10 μL of the mixed gold label solution onto each square centimeter of glass fiber membrane. Finally, assemble all components, cut into 5 mm wide test strips, seal, and store at 4°C.

[0081] Step 5, Pretreatment of milk samples: Take the milk sample to be tested, centrifuge at 9000 rpm for 18 minutes to remove the upper fat layer, and retain the middle liquid. Adjust the pH of the middle liquid to 4.75 with 0.1 M HCl. Centrifuge at 5500 rpm for 18 minutes, and take the supernatant (whey) as the sample solution to be tested.

[0082] Step Six: Prepare the working curve: Add 0, 1, 5, 20, 100, and 500 μg / mL of LF standard to the blank whey prepared in Step Five to prepare whey simulation samples. Add 100 μL of each concentration of simulation sample to the test strip prepared in Step Four, let it stand at room temperature for 20 minutes, and then read the gray value of the T line. Plot the working curve with the commonly used logarithm of the LF concentration in whey as the x-axis and the gray value of the T line as the y-axis.

[0083] Step 7: Test the actual sample and analyze the results: Take 100 μL of the whey sample solution obtained in Step 5, add it to the test strip in Step 4, let it stand at room temperature for 20 minutes, and then read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the LF concentration in the sample. Perform three parallel tests.

[0084] Example 6

[0085] A method for detecting lactoferrin in milk based on a competitive test strip includes the following steps:

[0086] Step 1: Preparation of enoxaparin sodium-BSA artificial antigen: Prepare enoxaparin sodium solution, add 0.1 M MEDC and 0.03 M sulfo-NHS (both final concentrations), add sodium chloride, and stir at room temperature for 22 minutes. Add BSA solution to the activation solution to a final concentration of 1 mg / mL, with a BSA to enoxaparin sodium mass ratio of 1:60, and stir at room temperature for 3 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 13000 Da for dialysis purification. The dialysate is water, and the dialysis time is 55 hours to remove unreacted substances and excess water.

[0087] Step 2, Preparation of Gold Label 1 (Enoxaparin Sodium-BSA-Gold Nanorod Probe): Take the gold nanorod solution and adjust the pH to 6.0 with 0.2 MK2CO3. Add 60 μg / mL colloidal gold enoxaparin sodium-BSA artificial antigen solution and shake for 17 minutes. Then add BSA solution to make the final BSA concentration in the system 0.7 wt%, and continue shaking for 17 minutes. After the reaction, centrifuge at 8500 rpm for 17 minutes, discard the supernatant, resuspend the precipitate with 0.03 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold label 1, and add 0.01 M PB buffer (pH 6.0) containing 1.5 wt% BSA, 0.15 wt% Tween-20, 3 wt% trehalose, and 0.6 M sodium chloride. Store at 4℃ for later use.

[0088] Step 3, Preparation of Gold-Labeled Probe II (Mouse IgG-Gold Nanorod Probe): Take the gold nanorod solution and adjust the pH to 6.0 with 0.2 M K2CO3. Add mouse IgG solution and incubate at room temperature with shaking for 17 minutes. Add BSA solution to bring the final BSA concentration in the system to 0.7 wt%, and continue shaking for 17 minutes. Centrifuge at 8500 rpm for 17 minutes, discard the supernatant, resuspend the precipitate in 0.03 M PB buffer, repeat centrifugation 3 times, adjust the concentration of gold-labeled probe II, and add 0.01 M PB buffer (pH 6.0) containing 1.5 wt% BSA, 0.15 wt% Tween-20, 3 wt% trehalose, and 0.6 M sodium chloride. Store at 4℃ for later use.

[0089] Step 4: Preparation of Colloidal Gold Test Strips: Construct the basic structure of the test strip by sequentially overlapping the sample pad, gold-labeled pad, NC membrane, and absorbent pad onto a PVC base plate, with an overlap width of 1.5 mm. Treat the NC membrane by streaking the T line (1.5 mg / mL LF solution) and C line (1.5 mg / mL goat anti-mouse IgG solution) at a concentration of 1 μg / mL and a line spacing of 6 mm. Dry at 37°C for 3 hours. Treat the gold-labeled pad by mixing gold label one and gold label two at a volume ratio of 2:1, and spraying 8 μL of the mixed gold label solution onto each square centimeter of glass fiber membrane. Finally, assemble all components, cut into 4 mm wide test strips, seal, and store at 4°C.

[0090] Step 5, Pretreatment of milk samples: Take the milk sample to be tested, centrifuge at 8500 rpm for 16 minutes to remove the upper fat layer, and retain the middle liquid. Adjust the pH of the middle liquid to 4.7 with 0.1 M HCl. Centrifuge at 5200 rpm for 16 minutes, and take the supernatant (whey) as the sample solution to be tested.

[0091] Step Six: Prepare the working curve: Add 0, 1, 5, 20, 100, and 500 μg / mL of LF standard to the blank whey prepared in Step Five to prepare whey simulation samples. Add 100 μL of each concentration of simulation sample to the test strip prepared in Step Four, let it stand at room temperature for 16 minutes, and then read the gray value of the T line. Plot the working curve with the commonly used logarithm of the LF concentration in whey as the x-axis and the gray value of the T line as the y-axis.

[0092] Step 7: Test the actual sample and analyze the results: Take 100 μL of the whey sample solution obtained in Step 5, add it to the test strip in Step 4, let it stand at room temperature for 13 minutes, and then read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the LF concentration in the sample. Perform three parallel tests.

[0093] Of the above embodiments, Embodiment 1 is the best embodiment.

[0094] Comparative Example 1

[0095] A comparative method for detecting lactoferrin in milk based on competitive test strips includes the following steps:

[0096] Steps one through four are completely identical to those in Example 1.

[0097] Step 5: Prepare simulated samples containing other proteins. Take the blank whey from Example 1 and add bovine serum albumin (200 μg / mL), α-lactalbumin (200 μg / mL), and β-lactoglobulin (200 μg / mL) to prepare three groups of simulated samples containing non-target proteins. Separately, use the blank whey as a blank control group.

[0098] The results showed that all three groups of samples containing bovine serum albumin, α-lactalbumin, and β-lactoglobulin, as well as the blank control group, exhibited complete and clear T and C lines, proving that the test strip of this invention has no cross-reactivity with other proteins in milk and has good specificity.

[0099] Comparative Example 2

[0100] A comparative method for detecting lactoferrin in milk based on competitive test strips includes the following steps:

[0101] Steps one through four are completely identical to those in Example 1.

[0102] Step 5: Prepare untreated milk samples. Take fresh milk, without centrifugation to remove fat or pH adjustment, and directly use the undiluted milk as the sample to be tested. Simultaneously, take the whey from this milk that has been pretreated according to Step 5 of Example 1, and add LF standard to a concentration of 50 μg / mL as a pretreatment control group.

[0103] Step Six: Detection and Result Analysis. Take 100 μL each of the untreated milk stock solution and the pretreated control group sample, and add them to the test strip from Step Four. After standing at room temperature for 15 minutes, read the gray value of the T line. The results show that the T line in the untreated group is not clearly visible and exhibits a tailing phenomenon. This proves that without sample pretreatment, the fat and casein in milk will seriously interfere with the detection. The pretreatment step of this invention can effectively eliminate matrix interference.

[0104] Comparative Example 3

[0105] A comparative method for detecting lactoferrin in milk based on competitive test strips includes the following steps:

[0106] Steps one through four are completely identical to those in Example 1.

[0107] Step 5: Prepare a blank sample without LF. Take fresh milk, boil it to deactivate the LF, and pretreat it according to step 5 of Example 1 to obtain blank whey without LF.

[0108] Step six is ​​completely consistent with Example 1. The results show that the T line is intact and clear, and the C line is normally colored, proving that in the absence of the target molecule LF, the gold-labeled probe can bind normally to the LF of the T line, and the T line remains intact.

Claims

1. A method for detecting lactoferrin in milk based on a competitive test strip, characterized in that, Includes the following steps: Step 1: Heparin molecule solution, cross-linking agent, and sodium chloride are stirred at room temperature to activate the mixture; bovine serum albumin solution is added to the activated solution and stirred to react. After the reaction is complete, the mixture is purified by dialysis to obtain heparin molecule-bovine serum albumin artificial antigen. Step 2: Adjust the pH of the gold nanoparticle solution to 5.5-6.5, add heparin-based molecule-bovine serum albumin artificial antigen solution, and shake to react; then add bovine serum albumin solution to block unbound sites on the colloidal gold surface, and continue shaking; after the reaction, centrifuge, discard the supernatant, resuspend the precipitate with buffer, repeat centrifugation, and obtain gold label 1; Step 3: Adjust the pH of the gold nanoparticle solution to 5.5-6.5, add mouse IgG solution, and shake to react; add bovine serum albumin solution to block non-specific sites, and continue shaking; after centrifugation, discard the supernatant, resuspend the precipitate with buffer, and repeat centrifugation to obtain gold nanoparticle II; Step 4: Draw T-line and C-line respectively and dry them. Spray a mixed gold labeling solution containing gold labeling one and gold labeling two onto the gold labeling pad to obtain the test strip. Step 5: Take the milk sample to be tested, remove the upper fat layer after the first centrifugation, and keep the middle liquid; adjust the pH of the middle liquid to 4.6~4.8, centrifuge a second time, and take the supernatant as the sample solution to be tested; Step 6: Add lactoferrin standards of different concentrations to the sample solution to be tested prepared in Step 5, prepare whey simulation samples, and drop them onto the test strips in Step 4. After standing at room temperature, read the gray value of the T line of the test strips. Plot the working curve with the logarithm of the lactoferrin concentration as the abscissa and the gray value of the T line as the ordinate. Step 7: Take the sample solution obtained in Step 5 and drop it onto the test strip in Step 4. After standing at room temperature, read the gray value of the T line. Substitute the gray value into the working curve in Step 6 to calculate the actual concentration of lactoferrin in the sample.

2. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In step one, the heparin molecules are any one of heparin sulfate, heparin sodium, fondaparinux sodium, dalteparin sodium, nadroparin calcium, and enoxaparin sodium, and the crosslinking agents are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide.

3. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 2, characterized in that: In step one, the final concentration of bovine serum albumin solution is 0.5-2 mg / mL, the final concentration of N-hydroxythiosuccinimide is 0.01-0.05 M, and the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.05-0.2 M. Dialysis purification is carried out in a dialysis bag with a molecular weight cutoff of 12000-14000 Da.

4. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In steps two and three, the gold nanoparticles are any one of colloidal gold, gold nanostars, gold nanoflowers, and gold nanorods.

5. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In step two, the amount of heparin-based molecules-bovine serum albumin artificial antigen solution added is 10~100 μg / mL of gold nanoparticles.

6. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In step four, the volume ratio of gold standard one to gold standard two in the mixed gold standard solution is 1~3:1, and the spraying amount is 5~10 μL / cm. 2 .

7. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In step four, the T line of the test strip is a lactoferrin solution with a concentration of 0.5~2 mg / mL, and the C line is a goat anti-mouse IgG solution with a concentration of 0.5~2 mg / mL.

8. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In step five, the first centrifugation is performed at a speed of 6000~10000 rpm for 10~20 minutes; the second centrifugation is performed at a speed of 4000~6000 rpm for 10~20 minutes.

9. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In step six, the concentration of the lactoferrin standard is 0-500 μg / mL, and the incubation time at room temperature is 10-20 minutes.

10. The method for detecting lactoferrin in milk based on a competitive test strip according to claim 1, characterized in that: In step seven, the time for standing at room temperature is 10-15 minutes.

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