Colloidal gold rapid detection card for saxitoxin

Abstract

The present invention relates to the technical field of biochemical detection, and discloses a saxitoxin colloidal gold rapid detection card, which comprises: a detection box, the middle of which is provided with a detection groove for placing other components; the sample pad is arranged on one side in the detection box and is used for absorbing a sample to be detected; the combination pad is mounted on one side of the sample pad, and the combination pad comprises a labeled antibody combined with saxitoxin; the detection line is connected with the combination pad and located on one side of the interior of the detection box, a specific antibody capable of being combined with saxitoxin is fixed to the outer wall of the detection line, and the detection line is used for forming visible color lines after being in contact with a target antigen. Through genetic engineering modification and a double marking strategy, the detection capability of a specific antibody on a detection line is remarkably improved, accurate detection can still be achieved even if low-concentration saxitoxin exists, and the problems that a traditional detection method is insufficient in sensitivity and prone to false positive or false negative are effectively solved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical detection, in particular to a saxitoxin colloidal gold rapid detection card. Background Art

[0002] Paralytic shellfish toxins (PSTs) mainly include a series of toxins, among which saxitoxin (STX) is the most toxic one.

[0003] This type of toxin is mainly produced by dinoflagellates (such as Alexandrium) and gradually accumulates in shellfish through the food chain. Saxitoxin can act on the nervous system, block sodium ion channels, and cause central nervous system paralysis. In severe cases, it can cause respiratory failure or even death. Due to the extremely high toxicity of STX, its rapid detection in shellfish and related products is particularly important.

[0004] Although chromatographic assays (e.g., liquid chromatography-mass spectrometry) and bioassays (e.g., mouse bioassay) perform well in detecting STX, they both have some significant disadvantages:

[0005] The detection process is complicated and the operation is cumbersome: Chromatographic analysis technology requires professional experimental equipment and operators. The detection process is complicated and time-consuming, and is not suitable for rapid on-site detection. The mouse bioassay involves animal experiments, which is not only ethically controversial, but also takes a long time to detect, and cannot meet the needs of rapid screening.

[0006] High equipment cost and poor portability: Precision instruments such as chromatography are expensive, bulky, and poorly portable, limiting their application in field environments. At the same time, these methods usually require complex sample pretreatment steps, further increasing the time and cost of testing.

[0007] Sensitivity and specificity limitations: Although existing chromatographic and biological detection methods can provide high sensitivity detection under certain conditions, in complex matrix samples (such as environmental water samples or untreated shellfish extracts), sensitivity and specificity are often affected by interfering substances, which can easily lead to false positive or false negative results. In addition, these methods may not be sensitive enough when detecting extremely low concentrations of toxins and cannot meet the strict requirements of food safety monitoring.

[0008] To this end, those skilled in the art provide a saxitoxin colloidal gold rapid detection card to solve the problems raised in the above background technology. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention provides a saxitoxin colloidal gold rapid detection card, which solves the problems of the prior art such as complex detection process, high equipment cost, poor portability, and limited sensitivity and specificity.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solutions: a saxitoxin colloidal gold rapid detection card, comprising:

[0011] A test box, wherein a test slot is provided in the middle for placing other components;

[0012] A sample pad is arranged at one side of the detection box and is used to absorb the sample to be tested;

[0013] A conjugate pad installed on one side of the sample pad, wherein the conjugate pad comprises a labeled antibody that binds to saxitoxin;

[0014] The detection line is connected to the conjugate pad and is located on one side of the inner part of the detection box. The outer wall of the detection line is fixed with specific antibodies that can bind to saxitoxin and is used to form a visible color line after contacting the target antigen;

[0015] The control line is in contact with the test line and is disposed on one side of the inner part of the test box. An antibody that binds to the Fc segment of the labeled antibody is fixed on its outer wall and is used to verify the effectiveness of the test;

[0016] The absorbent pad is installed on one side of the inner part of the test box to absorb excess liquid.

[0017] Preferably, the labeled antibody is combined with colloidal gold particles, wherein the colloidal gold particles have a particle size of 20-40 nm.

[0018] Preferably, the specific antibody preparation method is:

[0019] According to the structure of saxitoxin, multiple epitope fragments are designed and conjugated with a carrier protein, wherein the carrier protein is selected from one or more of key pore protein, ovalbumin or bovine serum albumin and is chemically modified;

[0020] The antigen conjugate is used to immunize an animal model, and a strategy of gradually increasing the antigen dose is adopted, and the immunization process lasts for 6-8 weeks;

[0021] High-throughput microfluidics technology was used to screen monoclonal antibodies with high affinity and specificity for saxitoxin in the antibody library, and surface plasmon resonance technology was used to monitor the binding kinetics of antibodies and antigens;

[0022] Finally, the screened monoclonal antibodies are genetically engineered and site-directed mutations are performed on the amino acid sequences of the antibodies.

[0023] Preferably, the specific antibody fixed on the detection line is a mouse monoclonal antibody or a genetically engineered antibody, and is optimized by affinity maturation technology.

[0024] Preferably, the modified antibody is labeled by a double labeling strategy, wherein the colloidal gold label is used for naked eye visual detection, and the nanomaterial label is used to enhance the optical detection signal, and the nanomaterial is quantum dots or carbon nanotubes.

[0025] Preferably, a treatment agent is introduced into the inner cavity of the detection line before use, and the treatment agent includes a nanofiber membrane and an enzyme, and the nanofiber membrane is surface-modified.

[0026] Preferably, the enzyme in the treating agent is a specific protease, which is used to decompose non-target proteins in the sample.

[0027] A saxitoxin colloidal gold rapid test method, used for a saxitoxin colloidal gold rapid test card, comprises the following steps:

[0028] S1. Prepare samples: collect shellfish extract and environmental water samples to be tested;

[0029] S2. Sample loading: Use a pipette or dropper to accurately add 2-3 drops of the sample to be tested onto the sample pad in the test box to start flowing;

[0030] S3, sample migration: After the sample to be tested passes through the sample pad, it enters the conjugate pad area. At this time, if saxitoxin exists in the sample, the toxin will bind to the colloidal gold-labeled antibody pre-fixed on the conjugate pad to form an antigen-antibody complex, and continue to flow downstream with the sample liquid;

[0031] S4. Detection line reaction: When the sample liquid flows to the detection line, the specific monoclonal antibody in the detection line area will capture the antigen-antibody complex. If saxitoxin is present in the sample, the detection line position will appear red or pink, indicating the presence of the target toxin;

[0032] S5. Verification of control line: The sample liquid continues to flow to the control line area. Regardless of whether the sample contains saxitoxin, the control line will show color. This is because the unbound colloidal gold-labeled antibody on the binding pad binds to the anti-mouse IgG antibody on the control line. The color development of the control line is used to verify the effectiveness of the detection process and ensure that the test card is working properly.

[0033] Preferably, the verification results include three types, namely:

[0034] Positive result: Both the test line and the control line are colored, indicating that the sample contains saxitoxin;

[0035] Negative result: only the control line shows color, while the test line does not, indicating that the sample does not contain saxitoxin;

[0036] Invalid result: If the control line does not develop color, the test is invalid and needs to be retested.

[0037] The present invention provides a saxitoxin colloidal gold rapid detection card, which has the following beneficial effects:

[0038] 1. The present invention significantly improves the detection ability of specific antibodies on the detection line through genetic engineering modification and double labeling strategy, and can achieve accurate detection even in the presence of low concentrations of saxitoxin, effectively solving the problem of insufficient sensitivity and easy occurrence of false positives or false negatives in traditional detection methods.

[0039] 2. The present invention introduces a treatment agent, including a nanofiber membrane and a specific protease, which can reduce the impact of interfering substances in the sample on the test results and ensure that the detection process is stable and reliable. At the same time, the genetic engineering modification of the antibody improves its thermal stability, so that the test card can still maintain good performance under different environmental conditions and extend the service life of the product. These characteristics make the test card more suitable for a wide range of application scenarios, especially on-site rapid screening.

[0040] 3. The test card of the present invention can obtain the test results in a short time through a simple sample dropping step. The components are reasonably arranged and the sample flows smoothly, ensuring the simplicity of operation and the rapid presentation of results, solving the problems of complex operation and long time consumption of traditional detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A perspective view of the present invention;

[0042] Figure 2 It is a schematic diagram of the structure explosion of the present invention;

[0043] Figure 3 It is a schematic diagram of the cross-sectional plan structure of the present invention;

[0044] Figure 4 It is a schematic diagram of the process steps of the present invention.

[0045] Among them, 1. detection box; 2. sample pad; 3. binding pad; 4. detection line; 5. control line; 6. absorption pad. DETAILED DESCRIPTION

[0046] 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.

[0047] Example 1: Preparation and application of basic version of saxitoxin colloidal gold rapid test card

[0048] step:

[0049] Antibody preparation: Based on the known structure of saxitoxin, multiple epitope fragments were designed and conjugated to the key porin (KLH) to form antigens.

[0050] Mice were selected as the immune model, and the immune was carried out according to the strategy of gradually increasing the antigen dose, and the immune cycle was 6-8 weeks. Monoclonal antibodies with high affinity to saxitoxin were obtained by ELISA screening.

[0051] The binding kinetics of the screened antibodies were monitored using surface plasmon resonance (SPR) technology to screen out antibodies with high specificity and affinity.

[0052] Colloidal gold labeling: The prepared monoclonal antibody is combined with colloidal gold particles with a particle size of 20-40nm to form a colloidal gold-labeled antibody.

[0053] Test card assembly: According to the design sequence of the test card, the sample pad, conjugate pad, test line (T line), control line (C line) and absorption pad are assembled into the test box in sequence. The test line and control line are coated with specific antibodies and anti-mouse IgG antibodies respectively.

[0054] Seawater sample extraction (different matrix detection):

[0055] Sample collection: Collect seawater samples to be tested from the sea, usually surface water samples.

[0056] Sample processing: Seawater samples were filtered to remove suspended particulate matter and obtain clear water samples.

[0057] Concentration step (optional): If the toxin concentration is very low, the toxin in the seawater sample can be pre-concentrated by solid phase extraction (SPE) to improve the detection sensitivity.

[0058] Sample testing:

[0059] Use a pipette to take 2-3 drops of the extracted shellfish supernatant or seawater sample and add them to the sample pad of the test card. The sample liquid will flow along the components of the test card.

[0060] During the sample flow process, if saxitoxin is present in the sample, the toxin will bind to the pre-labeled colloidal gold antibody on the conjugate pad to form an antigen-antibody complex and further flow to the detection line area.

[0061] Result interpretation:

[0062] Positive result: If the sample contains saxitoxin, a red or pink line will appear on the test line (T line), indicating a positive test result. The control line (C line) will show color to verify the effectiveness of the test process.

[0063] Negative result: If the sample does not contain saxitoxin, the test line will not show color, but the control line will show color, indicating that the test result is negative.

[0064] Invalid result: If the control line (line C) does not develop color, it means that the test card is invalid and needs to be retested.

[0065] Example 2: Highly sensitive saxitoxin detection card

[0066] step:

[0067] Antibody optimization: Based on Example 1, the affinity maturation technology was optimized for the screened monoclonal antibodies to further improve the binding affinity between the antibodies and saxitoxin.

[0068] Double labeling strategy: Using double labeling technology, monoclonal antibodies are simultaneously labeled with colloidal gold particles and quantum dots (or carbon nanotubes) to improve the sensitivity and visibility of optical detection.

[0069] Detection card assembly: Similar to Example 1, but double-labeled antibodies are used on the conjugate pad to enhance the signal intensity of the detection line (T line).

[0070] Sample detection and result interpretation: The operation is the same as in Example 1, but due to signal enhancement, the detection sensitivity of low concentration toxins is higher.

[0071] Summary: This embodiment significantly improves the detection sensitivity through the double labeling strategy, and is capable of detecting lower concentrations of saxitoxin, which is suitable for occasions requiring high-sensitivity detection.

[0072] Example 3: Enzyme treatment to enhance specificity detection card

[0073] step:

[0074] Antibody preparation: Antibodies were prepared using the same method as in Example 1, but without further optimization.

[0075] Introduction of enzyme treatment agent: A specific protease treatment agent is introduced into the inner cavity of the test line to decompose non-target proteins when the sample flows through the test line and reduce background interference. The treatment agent includes a surface-modified nanofiber membrane to further improve the specific reaction between the enzyme and the target protein.

[0076] Test card assembly: Assemble the sample pad, conjugate pad (containing colloidal gold labeled antibody), test line (containing specific antibody and enzyme treatment agent), control line and absorption pad into the test box.

[0077] Sample detection and result interpretation: The operation was the same as in Example 1. The interference of non-target proteins was reduced and the specificity was enhanced through enzyme treatment.

[0078] Summary: This embodiment greatly reduces background noise and improves detection specificity by introducing an enzyme treatment agent, and is particularly suitable for the detection of complex samples.

[0079] Example 4: Multilayer nanofiber membrane enhanced signal detection card

[0080] step:

[0081] Nanofiber membrane treatment: Multiple layers of surface-modified nanofiber membranes are introduced into the detection line area, which enhance the antibody fixation efficiency and improve the signal transduction performance.

[0082] Antibody preparation and colloidal gold labeling: the same as in Example 1.

[0083] Detection card assembly: The multilayer nanofiber membrane and the specific antibody are fixed together in the detection line area, and the other parts are the same as Example 1.

[0084] Sample detection and result interpretation: The operation is the same as in Example 1. Due to the use of multi-layer nanofiber membranes, the signal intensity is further improved.

[0085] Summary: The introduction of multilayer nanofiber membrane significantly enhanced the signal transduction and antibody fixation effects, and improved the signal strength and accuracy of the detection card.

[0086] Example 5: Environmental adaptability enhanced test card

[0087] step:

[0088] Antibody preparation: Antibodies were prepared using the same method as in Example 1.

[0089] Improved environmental adaptability: Temperature stabilizers and pH buffers are added to the sample pad and conjugate pad to ensure the stability and reliability of the test card under different environmental conditions.

[0090] Assembling the test card: Assemble the sample pad and the conjugate pad with improved environmental adaptability into the test card. The other parts are the same as those in Example 1.

[0091] Sample testing and result interpretation: The operation is the same as in Example 1, but the test is conducted under various temperature and pH conditions to ensure that the card can work stably in various environments.

[0092] Summary: Through the improvement of environmental adaptability, this embodiment can still maintain detection performance in a changing environment and is suitable for on-site detection and applications in complex environments.

[0093] Summary of Examples

[0094] These five examples respectively demonstrate the improvement of the saxitoxin colloidal gold rapid test card from various aspects, including basic detection, sensitivity improvement, specificity enhancement, signal enhancement, and environmental adaptability optimization. Each example is improved for specific detection needs, so that the test card has excellent performance in different application scenarios.

[0095] Test experiments and data

[0096] Experimental design:

[0097] Sample concentration gradient test: Different concentrations of saxitoxin (0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL) were used for detection, and the detection period of each example was compared.

[0098] Environmental adaptability test: Expose the test card to different temperatures (4°C, 25°C, 45°C) and different pH conditions (pH4.5, 7.0, 9.5) to observe the stability of the test results.

[0099] Background interference test: non-target proteins (such as bovine serum albumin) were added to the test sample to test the anti-interference ability of each embodiment.

[0100] Experimental data table:

[0101]

[0102]

[0103] Experimental design

[0104] Comparison of detection sensitivity between dual labeling and single labeling:

[0105] By comparing Example 1 with Example 2, the effect of the double labeling strategy on improving detection sensitivity was verified by detecting low-concentration toxin samples.

[0106] Comparison of specificity with and without enzyme treatment:

[0107] Comparing Example 1 with Example 3, the effect of reducing background interference by enzyme treatment was verified by adding interfering substances into the sample.

[0108] Comparison of signal strength between multilayer nanofiber membrane and single-layer nanofiber membrane:

[0109] By comparing Example 1 (not using a multilayer nanofiber membrane) with Example 4 (using a multilayer nanofiber membrane), the same concentration of saxitoxin samples were detected and the difference in signal intensity was observed to verify the enhancement effect of the multilayer nanofiber membrane on signal conduction.

[0110] Experimental data table:

[0111] Experimental data table of detection sensitivity of double labeling and single labeling:

[0112]

[0113] Conclusion: Example 2 uses a dual labeling strategy, which significantly improves the detection sensitivity and can detect lower concentrations of saxitoxin.

[0114] Specific experimental data table with and without enzyme treatment:

[0115]

[0116] Conclusion: After the enzyme treatment was introduced in Example 3, the influence of interfering substances on the detection results was significantly reduced, and the specificity was enhanced, especially in high background samples.

[0117] Experimental data table of signal strength of multilayer nanofiber membrane and single-layer nanofiber membrane:

[0118]

[0119] Conclusion: After using the multilayer nanofiber membrane in Example 4, the detection signal intensity is significantly improved, especially in the detection of low-concentration samples, the signal enhancement effect is particularly obvious.

[0120] Summary

[0121] It can be seen from the above examples and experiments that the innovation and optimization of the saxitoxin colloidal gold rapid test card in various aspects have brought significant performance improvements:

[0122] The basic test card (Example 1) provides basic detection functions and is suitable for toxin detection in routine environments.

[0123] The high-sensitivity detection card (Example 2) greatly improves the detection sensitivity through double labeling technology and is suitable for detecting low-concentration samples.

[0124] The enzyme treatment enhanced specificity detection card (Example 3) reduces background interference, improves the specificity of detection, and is suitable for use in complex samples.

[0125] The multi-layer nanofiber membrane enhanced signal detection card (Example 4) significantly enhances the signal strength and improves the accuracy and visibility of detection.

[0126] The environmental adaptability enhanced detection card (Example 5) performs stably under various environmental conditions and is suitable for a variety of detection scenarios.

[0127] Through experiments, the effectiveness of each embodiment in its design goals is further verified, especially its advantages in sensitivity, specificity, signal strength and environmental adaptability.

[0128] 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 saxitoxin colloidal gold rapid test card, characterized in that: include: A detection box (1) having a detection slot in the middle thereof for placing other components; A sample pad (2), which is arranged on one side of the interior of the detection box (1) and is used to absorb the sample to be tested; A conjugate pad (3) mounted on one side of the sample pad (2), wherein the conjugate pad (3) comprises a labeled antibody that binds to saxitoxin; A detection line (4) is connected to the binding pad (3) and is located on one side of the inner part of the detection box (1). A specific antibody capable of binding to saxitoxin is fixed on its outer wall and is used to form a visible color line after contacting the target antigen; A control line (5), in contact with the test line (4), is disposed on one side of the interior of the test box (1), and has an antibody that binds to the Fc segment of the labeled antibody fixed on its outer wall, for verifying the effectiveness of the test; An absorption pad (6) is installed on one side of the interior of the detection box (1) and is used to absorb excess liquid.

2. A saxitoxin colloidal gold rapid test card according to claim 1, characterized in that, The labeled antibody is combined with colloidal gold particles, wherein the particle size of the colloidal gold particles is 20-40 nm.

3. A saxitoxin colloidal gold rapid test card according to claim 1, characterized in that: The specific antibody preparation method is: According to the structure of saxitoxin, multiple epitope fragments are designed and conjugated with a carrier protein, wherein the carrier protein is selected from one or more of key pore protein, ovalbumin or bovine serum albumin and is chemically modified; The antigen conjugate is used to immunize an animal model, and a strategy of gradually increasing the antigen dose is adopted, and the immunization process lasts for 6-8 weeks; High-throughput microfluidics technology was used to screen monoclonal antibodies with high affinity and specificity for saxitoxin in the antibody library, and surface plasmon resonance technology was used to monitor the binding kinetics of antibodies and antigens; Finally, the screened monoclonal antibodies are genetically engineered and site-directed mutations are performed on the amino acid sequences of the antibodies.

4. A saxitoxin colloidal gold rapid test card according to claim 3, characterized in that: The specific antibody fixed on the detection line (4) is a mouse monoclonal antibody or a genetically engineered antibody, and is optimized through affinity maturation technology.

5. A saxitoxin colloidal gold rapid test card according to claim 3, characterized in that: The modified antibody is labeled by a double labeling strategy, wherein the colloidal gold label is used for naked eye visual detection, and the nanomaterial label is used to enhance the optical detection signal, and the nanomaterial is a quantum dot or a carbon nanotube.

6. A saxitoxin colloidal gold rapid test card according to claim 1, characterized in that: A treatment agent is introduced into the inner cavity of the detection line (4) before use. The treatment agent includes a nanofiber membrane and an enzyme. The nanofiber membrane is surface-modified.

7. A saxitoxin colloidal gold rapid test card according to claim 6, characterized in that: The enzymes in the treatment agent are specific proteases, which are used to decompose non-target proteins in the sample.

8. A saxitoxin colloidal gold rapid test method, used for a saxitoxin colloidal gold rapid test card according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Prepare samples: collect shellfish extract and environmental water samples to be tested; S2. Sample loading: Use a pipette or dropper to accurately add 2-3 drops of the sample to be tested onto the sample pad (2) in the detection box (1) to start the flow; S3, sample migration: After the sample to be tested passes through the sample pad (2), it enters the binding pad (3) area. At this time, if saxitoxin is present in the sample, the toxin will bind to the colloidal gold-labeled antibody pre-fixed on the binding pad (3) to form an antigen-antibody complex, and continue to flow downstream with the sample liquid; S4, test line (T line) reaction: When the sample liquid flows to the test line (4), the specific monoclonal antibody in the test line (4) area will capture the antigen-antibody complex. If saxitoxin is present in the sample, the test line (4) will appear red or pink, indicating the presence of the target toxin; S5. Verification of the control line (C line): The sample liquid continues to flow to the control line (5) area. Regardless of whether the sample contains saxitoxin, the control line (5) will show color. This is because the unbound colloidal gold-labeled antibody on the binding pad (3) binds to the anti-mouse IgG antibody on the control line (5). The color development of the control line (5) is used to verify the effectiveness of the detection process and ensure that the detection card is working properly.

9. A saxitoxin colloidal gold rapid test method according to claim 8, characterized in that: The verification results include three types: Positive result: Both the test line (4) and the control line (5) are colored, indicating that the sample contains saxitoxin; Negative result: only the control line (5) shows color, while the test line (4) does not show color, indicating that the sample does not contain saxitoxin; Invalid result: If the control line (5) does not develop color, the test is invalid and needs to be retested.

Images