S100B protein concentration detection method
By combining field-effect enzymatic detection and immune sensing methods, the existing ELISA method has solved the problems of complex operation, time-consuming and high cost, and achieved rapid, accurate and low-cost S100B protein detection, which is suitable for emergency situations and resource-limited environments.
Patent Information
- Application Number
- CN202510333850.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-20
- Publication Date
- 2025-06-10
AI Technical Summary
The existing ELISA method for S100B protein detection is complex, time-consuming and high detection cost, making it difficult to achieve rapid and accurate detection in emergencies and resource-limited environments.
Field-effect enzymatic detection (FEED) was used to combine with immunosensing methods, and the working electrodes of the electrochemical cell were treated in a structured manner, and the capture antibody was fixed, and the capture antibody-labeled detection antibody was used to form a capture antibody-label-detection antibody complex, and the current signal generated by the complex was measured to detect the S100B protein concentration.
It realizes fast, accurate and cost-effective S100B protein detection, which can detect S100B protein at 10fg/mL level. It is suitable for early diagnosis of mild traumatic brain injury (mTBI), with simple operation and low production cost.
Smart Images
Figure CN120121682A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of protein biomarker detection, and specifically relates to the design of a method for detecting the concentration of S100B protein. Background Art
[0002] The S100B protein is a member of the S100 protein family, and the proteins in this family play an important role in the calcium signal transduction process. The S100B protein is mainly produced by glial cells and has a relatively high concentration in the brain. Therefore, it is often used as a biomarker for nerve injury. A biomarker is a molecular indicator obtained from an organism that can indicate normal biological processes, pathological processes, or the intervention of pharmacological responses to treatment. For the S100B protein, it is usually associated with brain injury. Therefore, detecting its concentration is crucial for the rapid diagnosis and treatment of nervous system injuries.
[0003] ELISA (Enzyme-Linked Immunosorbent Assay) is a commonly used biochemical analysis technique and is currently widely used in clinical detection and quantification of the concentration of antigens (such as S100B protein) in samples. The ELISA method relies on the specific binding of antibodies to antigens and the subsequent detectable signals generated by enzyme-labeled antibodies. However, the ELISA method is complex to operate, time-consuming, and requires professional equipment. Therefore, there is a need for a faster, more convenient, lower-cost, and on-site detectable device or method.
[0004] Microfluidic technology, also known as Lab-on-a-Chip (LOC) technology, is a technology that uses miniaturized fluid channels to process and analyze very small volume samples. In the detection of S100B protein, the application of microfluidic technology can provide multiple advantages, including higher sensitivity, smaller reagent consumption, faster analysis time, and portability, etc.
[0005] A microfluidic device can integrate multiple experimental steps onto a single chip to achieve automated sample processing. In the detection of S100B protein, the sample pretreatment, mixing, reaction, and detection steps can be completed on a single platform, greatly reducing the operation complexity and human error. The small size and high surface area to volume ratio of the microfluidic system make heat and mass transfer more efficient, which can greatly reduce the time required for reaction and analysis. In addition, these systems can be designed to monitor the concentration changes of analytes in real time, which is particularly useful for situations where rapid diagnosis and monitoring of S100B protein levels are required. Microfluidic chips are usually equipped with specially designed detection areas, which can contain immobilized capture antibody areas or other recognition element areas. Due to the high surface area to volume ratio of the microchannel, the interaction between the antibody and the S100B protein is more efficient, thereby improving the sensitivity and specificity of the detection.
[0006] Due to the small size of the microfluidic chip, the required amounts of reagents and samples are correspondingly reduced, which not only reduces costs but is also particularly important when dealing with precious or difficult-to-obtain samples. Due to the miniaturization of microfluidic devices, they are usually easy to carry, which makes it possible to perform S100B protein detection in point-of-care (POC) settings, such as in emergency rooms, stadiums, or battlefields.
[0007] In practical applications, microfluidic technology can be combined with optical detection (such as fluorescence or absorbance measurement), electrochemical detection, or other sensing technologies to achieve highly sensitive and specific detection of S100B protein. The progress of these technologies continuously promotes the application prospects of microfluidics in biomedical diagnosis, especially in the field of biomarker detection. Therefore, creating a method that can quickly and accurately determine S100B protein levels on-site is of great significance for the timely diagnosis of brain injury, assessment of disease status, and monitoring of treatment effects. Summary of the Invention
[0008] The purpose of the present invention is to solve the problems of the existing ELISA method for S100B protein detection, which is complex in operation, time-consuming, and high in detection cost. A method for detecting S100B protein concentration is proposed, which is applicable to real-time monitoring of S100B protein in emergency situations and resource-limited environments to facilitate timely clinical decision-making and patient management.
[0009] The technical solution of the present invention is as follows: A method for detecting S100B protein concentration includes the following steps:
[0010] S1. Structure the working electrode area of the electrochemical cell to obtain a structured working electrode.
[0011] S2. Fix the capture antibody against S100B protein on the structured working electrode area to obtain a functionalized working electrode.
[0012] S3. Incubate the sample to be tested with the functionalized working electrode at 4°C to promote the binding of the capture antibody to the S100B protein in the sample to be tested.
[0013] S4. Remove the unbound capture antibody and other components of the sample to be tested.
[0014] S5. Add the HRP-labeled detection antibody against S100B protein to the functionalized working electrode to form a capture antibody-labeled-detection antibody complex on the functionalized working electrode.
[0015] S6. Connect the working electrode interface, counter electrode interface, and reference electrode interface of the electrochemical cell to an electrochemical workstation respectively, and measure the current signal generated by the capture antibody-label-detection antibody complex.
[0016] S7. Plot the S100B biosensing detection standard curve based on the known S100B protein concentrations at different levels and their corresponding current signal intensities. Obtain the S100B protein concentration of the test sample from the position of the current signal intensity generated by the test sample in the S100B biosensing detection standard curve.
[0017] Further, step S1 includes the following sub-steps:
[0018] S11. Deposit the mixture of single-walled carbon nanotubes, Nafion solution, and glutaraldehyde solution on the working electrode of the electrochemical cell and let it stand for 10 minutes.
[0019] S12. Wash the working electrode with deionized water to remove impurities and obtain a structured working electrode.
[0020] Further, in step S11, the material ratio of single-walled carbon nanotubes, Nafion solution, and glutaraldehyde solution is 1:2:1.
[0021] Further, in step S11, the solvent of the Nafion solution is a mixture of ethanol and water, and its concentration is 0.5 wt%.
[0022] Further, in step S11, the concentration of the glutaraldehyde solution is 3%.
[0023] Further, step S3 includes the following sub-steps:
[0024] S31. Place the test sample on the functionalized working electrode and incubate them together in a 4°C refrigerator for 1 hour to promote the binding of the capture antibody to the S100B protein in the test sample.
[0025] S32. Apply bovine serum albumin as a blocking agent to the functionalized working electrode and let it stand for 10 minutes.
[0026] Further, step S4 is specifically: Wash the functionalized working electrode with deionized water multiple times to remove the unbound capture antibody and other components of the test sample.
[0027] Further, step S5 includes the following sub-steps:
[0028] S51. Add horseradish peroxidase and a detection antibody against the S100B protein to the enzyme solution at a molar ratio of 3:1, so that the horseradish peroxidase attaches to the detection antibody, forming an HRP-labeled detection antibody solution against the S100B protein, with a concentration of 0.2 μg / mL.
[0029] S52. Add the HRP-labeled detection antibody solution against the S100B protein to the functionalized working electrode and incubate for 40 minutes.
[0030] S53. Wash the functionalized working electrode with deionized water to form a capture antibody - label - detection antibody complex on the functionalized working electrode.
[0031] Further, the enzyme solution in step S51 is a mixed solution of phosphate buffer and a cross - linker. The pH value of the phosphate buffer is 7.4 and its concentration is 0.01 M. The cross - linker is glutaraldehyde or NHS - ester. When the cross - linker is glutaraldehyde, its concentration is 0.2% w / v, and the volume ratio of the phosphate buffer to glutaraldehyde is 100:3; when the cross - linker is NHS - ester, its concentration is 5 mM, and the volume ratio of the phosphate buffer to NHS - ester is 100:4.
[0032] Further, the concentration of the enzyme solution in step S51 is 0.3 mg / mL.
[0033] The beneficial effects of the present invention are as follows: The present invention combines field - effect enzymatic detection (FEED) with an immunosensing method, providing a rapid, accurate, user - friendly and cost - effective detection method for the S100B protein. When the capture antibody - label - detection antibody complex is fixed on the working electrode, it is ready for measurement; when the sample to be tested contains the S100B protein, a current path is formed between the working electrode and the counter electrode, and the concentration information of S100B is obtained by detecting the generated current signal. The present invention can detect the S100B protein at a level of 10 fg / mL. Using the method of the present invention to detect low - concentration human serum and the biomarker of mild traumatic brain injury (mTBI) that may be in its body fluid makes the present invention an early diagnosis technology for mTBI, and the operation of the present invention is simple and the production cost is low. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 Shown is a flowchart of a method for detecting the concentration of S100B protein provided by an embodiment of the present invention.
[0035] Figure 2 Shown is a schematic diagram of the structural processing of the working electrode provided by an embodiment of the present invention.
[0036] Figure 3The figure shows a schematic diagram of an HRP-labeled detection antibody provided by an embodiment of the present invention.
[0037] Figure 4 The figure shows a schematic diagram of the generation of a capture antibody-label-detection antibody complex provided by an embodiment of the present invention.
[0038] Figure 5 The figure shows a schematic diagram of the details of a capture antibody-label-detection antibody complex provided by an embodiment of the present invention. Detailed implementation manners
[0039] Now, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the embodiments shown and described in the drawings are merely exemplary, intended to explain the principles and spirit of the present invention, and not to limit the scope of the present invention.
[0040] An embodiment of the present invention provides a method for detecting the concentration of S100B protein, as Figure 1 shown, including the following steps S1 to S7:
[0041] S1. Perform structuring on the working electrode area of the electrochemical cell to obtain a structured working electrode.
[0042] In an embodiment of the present invention, a traditional electrochemical cell is used, which includes a three-electrode system: a working electrode, a reference electrode, and a counter electrode.
[0043] Step S1 includes the following sub-steps S11 to S12:
[0044] S11. As Figure 2 shown, deposit a mixture of single-walled carbon nanotubes, Nafion solution, and glutaraldehyde solution on the working electrode of the electrochemical cell and let it stand for 10 minutes.
[0045] In an embodiment of the present invention, the material ratio of single-walled carbon nanotubes, Nafion solution, and glutaraldehyde solution is 1:2:1. The solvent of the Nafion solution is a mixture of ethanol and water, and its concentration is 0.5 wt%, and the concentration of the glutaraldehyde solution is 3%.
[0046] S12. As Figure 2 shown, use deionized water to clean the working electrode to remove impurities and obtain a structured working electrode.
[0047] S2. As Figure 4 shown, fix the capture antibody against the S100B protein in the structured working electrode area to obtain a functionalized working electrode.
[0048] In an embodiment of the present invention, the capture antibody molecules are fixed on the structured working electrode by a certain covalent or physical adsorption method.
[0049] S3. Incubate the sample to be tested with the functionalized working electrode at 4°C to promote the binding of the capture antibody to the S100B protein in the sample to be tested.
[0050] Step S3 includes the following sub-steps S31 to S32:
[0051] S31. As Figure 4 shown, place the sample to be tested on the functionalized working electrode and incubate them together in a 4°C refrigerator for 1 hour to promote the binding of the capture antibody to the S100B protein in the sample to be tested.
[0052] S32. As Figure 4 shown, apply bovine serum albumin as a blocking agent to the functionalized working electrode and let it stand for 10 minutes.
[0053] In the embodiment of the present invention, bovine serum albumin is used to reduce non-specific binding.
[0054] S4. Remove the unbound capture antibody and other components of the sample to be tested.
[0055] In the embodiment of the present invention, as Figure 4 shown, use deionized water to wash the functionalized working electrode multiple times to remove the unbound capture antibody and other components of the sample to be tested. Other components of the sample to be tested refer to components other than the S100B protein.
[0056] At the same time, washing with deionized water multiple times can also ensure that bovine serum albumin as a blocking agent does not interfere with subsequent steps and meets the detection conditions.
[0057] S5. Add an HRP-labeled detection antibody against the S100B protein to the functionalized working electrode to form a capture antibody-label - detection antibody complex on the functionalized working electrode.
[0058] Step S5 includes the following sub-steps S51 to S53:
[0059] S51. As Figure 3 shown, add horseradish peroxidase (HRP) and a detection antibody against the S100B protein to the enzyme solution at a molar ratio of 3:1, so that horseradish peroxidase attaches to the detection antibody to form an HRP-labeled detection antibody solution against the S100B protein, and its concentration is 0.2 μg / mL.
[0060] In the embodiments of the present invention, the enzyme solution is a mixed solution of phosphate buffer solution (PBS) and a crosslinking agent. The pH value of the phosphate buffer solution is 7.4, and its concentration is 0.01 M (mol / L). The crosslinking agent is glutaraldehyde or NHS-ester (N-hydroxysuccinimide ester). When the crosslinking agent is glutaraldehyde, its concentration is 0.2% w / v, and the volume ratio of the phosphate buffer solution to glutaraldehyde is 100:3. When the crosslinking agent is NHS-ester, its concentration is 5 mM (mmol / L), and the volume ratio of the phosphate buffer solution to NHS-ester is 100:4.
[0061] In the embodiments of the present invention, the concentration of the enzyme solution in step S51 is 0.3 mg / mL.
[0062] S52, as Figure 4 shown, add the HRP-labeled detection antibody solution against the S100B protein to the functionalized working electrode and incubate for 40 minutes.
[0063] S53. Wash the functionalized working electrode with deionized water to form a capture antibody-label - detection antibody complex on the functionalized working electrode.
[0064] S6. Connect the working electrode interface, counter electrode interface, and reference electrode interface of the electrochemical cell to the electrochemical workstation respectively, and measure the current signal generated by the capture antibody-label - detection antibody complex.
[0065] In the embodiments of the present invention, as Figure 5 shown, when the test sample contains the S100B protein, the substrate of HRP undergoes a catalytic reaction to generate a quantifiable chemical signal, that is, Figure 5 the current signal I in , forming a current path between the working electrode and the counter electrode of the electrochemical cell. The current intensity is positively correlated with the concentration of the S100B protein in the test sample. Therefore, by measuring the intensity of the generated current signal, the concentration of the S100B protein can be indirectly reflected. The reduced peak current of HRP is the detection signal, and the gating voltage VG of the detection system will amplify the detection signal.
[0066] S7. Draw the S100B biosensing detection standard curve according to the known S100B protein with different concentrations and the corresponding current signal intensities, and obtain the concentration of the S100B protein in the test sample through the position of the current signal intensity generated by the test sample in the S100B biosensing detection standard curve.
[0067] The present invention can detect S100B at the level of 10 fg / mL, and the reportable range is from fg / ml to ng / ml, indicating that the method of the present invention for detecting extremely low mTBI markers in human serum is feasible.
[0068] Those of ordinary skill in the art will realize that the embodiments described herein are provided to assist the reader in understanding the principles of the present invention, and it should be understood that the scope of protection of the present invention is not limited to such specific statements and embodiments. Those of ordinary skill in the art can make various other specific deformations and combinations that do not depart from the essence of the present invention based on these technical revelations disclosed in the present invention, and these deformations and combinations are still within the scope of protection of the present invention.
Claims
1. A method for detecting S100B protein concentration, characterized in that: The following steps are involved: S1. Structuring the working electrode region of the electrochemical cell to obtain a structured working electrode; S2, immobilizing the capture antibody against S100B protein on the structured working electrode area to obtain a functionalized working electrode; S3, incubating the sample to be tested with the functionalized working electrode at 4°C to promote the binding of the capture antibody to the S100B protein in the sample to be tested; S4, removing unbound capture antibodies and other components of the sample to be tested; S5, adding an HRP-labeled detection antibody targeting the S100B protein to the functionalized working electrode to form a capture antibody-label-detection antibody complex on the functionalized working electrode; S6, connecting the working electrode interface, the counter electrode interface and the reference electrode interface of the electrochemical cell to the electrochemical workstation respectively, and measuring the current signal generated by the capture antibody-marker-detection antibody complex; S7. Draw a S100B biosensor detection standard curve based on known different concentrations of S100B protein and corresponding current signal intensities, and obtain the S100B protein concentration of the sample to be tested by the position of the current signal intensity generated by the sample to be tested in the S100B biosensor detection standard curve.
2. The method for detecting the concentration of S100B protein according to claim 1, characterized in that: The step S1 comprises the following sub-steps: S11, depositing a mixture of single-walled carbon nanotubes, Nafion solution and glutaraldehyde solution on a working electrode of an electrochemical cell and leaving it to stand for 10 minutes; S12. Clean the working electrode with deionized water to remove impurities and obtain a structured working electrode.
3. The method for detecting the concentration of S100B protein according to claim 2, characterized in that: In the step S11, the material-liquid ratio of the single-walled carbon nanotubes, the Nafion solution and the glutaraldehyde solution is 1:2:
1.
4. The method for detecting the concentration of S100B protein according to claim 2, characterized in that: The solvent of the Nafion solution in step S11 is a mixture of ethanol and water, and the concentration thereof is 0.5 wt %.
5. The method for detecting the concentration of S100B protein according to claim 2, characterized in that: The concentration of the glutaraldehyde solution in step S11 is 3%.
6. The method for detecting the concentration of S100B protein according to claim 1, characterized in that: The step S3 comprises the following sub-steps: S31, placing the sample to be tested on the functionalized working electrode and incubating them in a refrigerator at 4°C for 1 hour to promote the binding of the capture antibody to the S100B protein in the sample to be tested; S32. Apply bovine serum albumin as a blocking agent to the functionalized working electrode and let it stand for 10 minutes.
7. The method for detecting the concentration of S100B protein according to claim 1, characterized in that: The step S4 specifically includes: using deionized water to rinse the functionalized working electrode multiple times to remove unbound capture antibodies and other components of the sample to be tested.
8. The method for detecting the concentration of S100B protein according to claim 1, characterized in that: The step S5 comprises the following sub-steps: S51, adding horseradish peroxidase and a detection antibody for S100B protein into the enzyme solution at a molar ratio of 3:1, so that the horseradish peroxidase is attached to the detection antibody to form an HRP-labeled detection antibody solution for S100B protein, the concentration of which is 0.2 μg / mL; S52, adding a detection antibody solution labeled with HRP against S100B protein to the functionalized working electrode and incubating for 40 minutes; S53. Clean the functionalized working electrode with deionized water to form a capture antibody-label-detection antibody complex on the functionalized working electrode.
9. The method for detecting the concentration of S100B protein according to claim 8, characterized in that: The enzyme solution in step S51 is a mixed solution of phosphate buffer and a cross-linking agent, the pH value of the phosphate buffer is 7.4, and its concentration is 0.01M. The cross-linking agent is glutaraldehyde or NHS-ester. When the cross-linking agent is glutaraldehyde, its concentration is 0.2% w / v, and the volume ratio of phosphate buffer to glutaraldehyde is 100:3; when the cross-linking agent is NHS-ester, its concentration is 5mM, and the volume ratio of phosphate buffer to NHS-ester is 100:
4.
10. The method for detecting the concentration of S100B protein according to claim 8, characterized in that: The concentration of the enzyme solution in step S51 is 0.3 mg / mL.