Ultra-sensitive protein chip for detecting steroid micromolecule testosterone marker and preparation method thereof
By designing a supersensitive protein chip, using dispersed tiled capture molecules and Teflon-coated protein chips, combined with HRP-labeled antibodies and BSA incubation, the problem of insufficient sensitivity of testosterone detection in the prior art is solved, and high sensitivity detection of small molecules of testosterone is achieved, and the accuracy and efficiency of the detection are improved.
Patent Information
- Application Number
- CN202411338963.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-25
- Publication Date
- 2025-05-27
AI Technical Summary
The existing testosterone detection technology is insufficient in sensitivity, making it difficult to accurately detect testosterone concentrations at femtomore level, affecting the clinical diagnosis and treatment effect.
A supersensitive protein chip was designed, using non-overlapping and dispersed and flat capture molecules with a testosterone monoclonal antibody with a density of 2.19x1011 per square millimeter, combined with Teflon coating to prevent cross-contamination, and incubated with HRP-labeled testosterone monoclonal antibody and BSA to reduce false positives, and high sensitivity detection was achieved by capturing luminescent signals through CCD cameras.
Highly sensitive detection of small molecules as low as 10fg/ml testosterone is achieved, providing fast and accurate detection results, reducing detection costs, and improving detection efficiency and accuracy.
Smart Images
Figure CN120044254A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technology of small molecule biomarker detection, and particularly to a super-sensitive protein chip for detecting testosterone and a preparation method thereof. Background Art
[0002] Testosterone (also known as testis hormone, testosterone or androsterone) is a steroid hormone with the molecular formula C19H28O2. It is secreted by the testicles of men or the ovaries of women, and the adrenal glands also secrete a small amount of testosterone. It is a cytoskeleton-associated protein with a relatively small molecular weight and acts by binding to other cytoskeleton proteins. It has important effects on health, including enhancing sexual desire, strength, immune function, and combating osteoporosis.
[0003] Abnormal increases and decreases in testosterone are related to various clinical diseases. Precise and highly sensitive quantitative detection of testosterone is of great significance for clinical diagnosis, treatment process, and prognosis evaluation.
[0004] The present invention is a super-sensitive protein chip technology applied to the detection of testosterone in body fluids such as blood and saliva. This super-sensitive protein chip technology can accurately detect femtomolar (fg / ml) levels of testosterone in the test sample. It will provide a super-sensitive, accurate, economical, and convenient detection method for testosterone detection, contributing to the diagnosis of diseases related to changes in testosterone levels, the determination of disease stages, or the evaluation of the safety and effectiveness of new drugs or new therapies in the target population. Summary of the Invention
[0005] Based on the requirements of the above field, the present invention provides a super-sensitive small molecule testosterone chip and a detection kit, and the technical solutions are as follows:
[0006] A super-sensitive protein chip for detecting small molecule testosterone, characterized in that the protein chip has at least one sub-detection area, each sub-detection area has at least one detection spot, and the detection spot has non-overlapping, dispersed and flatly laid capture molecules with a coating density of 2.19x10 per square millimeter 11 molecules; the capture molecule is a testosterone monoclonal antibody.
[0007] Preferably, the super-sensitive protein chip is characterized in that the diameter of the detection spot is 3 - 5 mm.
[0008] Preferably, the super-sensitive protein chip is characterized in that each sub-detection area is one of the detection spots, which is a regular or irregular figure with an area of 9 - 25 mm, preferably a circle with a diameter of 4 mm.
[0009] Preferably, the super-sensitive protein chip is characterized in that it has 20 - 30 sub-detection areas.
[0010] Preferably, for the ultrasensitive protein chip, it is characterized in that a Teflon coating is provided between adjacent sub-detection regions for separating each sub-detection region to prevent cross-contamination between samples.
[0011] Preferably, for the ultrasensitive protein chip, it is characterized in that between the sub-detection regions, the monoclonal antibodies against testosterone on the detection spots therein are completely identical to each other; or between at least two sub-detection regions, the monoclonal antibodies against testosterone on the detection spots therein are different from each other.
[0012] On the other hand of the present invention, a kit for detecting testosterone is characterized by comprising at least one of the above protein chips, as well as a monoclonal antibody against testosterone labeled with HRP, 2% BSA, a 0.05% PBS-Tween solution, and an HRP luminescent substrate solution.
[0013] On yet another aspect of the present invention, a method for preparing any of the above protein chips is provided, which is characterized by comprising the following steps:
[0014] (1) Obtaining a glass slide having at least one sub-detection region,
[0015] (2) In each sub-detection region, using the monoclonal antibody protein molecules against testosterone to perform uniform spraying and coating to form a coating density of 2.19×10 11 capture molecules per square millimeter that do not overlap with each other and are dispersed and tiled; the capture molecules are small molecule testosterone conjugated with BSA to immunize mice to obtain mouse monoclonal antibodies, and then dried to form the detection spots.
[0016] The testosterone protein chip of the present invention can quickly and at low cost detect small molecule testosterone as low as 10 fg / ml, providing a powerful detection means for research such as the diagnosis of diseases related to changes in testosterone levels and the determination of disease stages.
[0017] In order to improve the sensitivity, the present invention has precisely adjusted the following items multiple times: strictly and precisely adjusting the pH value of the capture protein according to the properties of the monoclonal antibody against testosterone as the capture protein; strictly calculating the required quantity according to the molecular weight of the capture protein to determine the capture density of the capture molecules; precisely adjusting the required quantity of the detection antibody according to the molecular weight of the detection antibody; adjusting the pH value, temperature, and washing time of the buffer; adjusting the incubation time and temperature; and adjusting various parameters required for signal scanning.
[0018] The capture antibody and the HRP detection antibody of the present invention are small molecule testosterone conjugated with BSA to immunize mice to obtain mouse monoclonal antibodies. This monoclonal antibody can bind to testosterone and can also bind to BSA. The clinical samples will contain BSA components, resulting in false positives.
[0019] To avoid or reduce the generation of false positives, BSA and HRP-labeled detection antibodies are pre-incubated and fully combined to prevent the HRP-labeled detection antibody from binding to BSA in the test sample, effectively avoiding the generation of false positives.
[0020] In the R & D process, it was found that the independent and dispersed arrangement of capture protein molecules plays a decisive role in improving the detection sensitivity. Through intensive analysis and calculation, the present invention obtained that for a detection hole with a diameter of 4 mm, its area is 12.56 square millimeters, and 2.75x10 12 testosterone monoclonal antibodies can be tiled in a single layer. In order to ensure that the capture protein molecules on the test spot are dispersed and spaced from each other, the present invention determines that 2.19x10 2 capture protein molecules are used for coating on a 12.56 mm 11 glass slide, so as to ensure that the capture protein molecules are independent of each other and do not affect each other, enabling a single target protein molecule in the sample to react with a single capture protein molecule on the glass slide. Using the chip of the present invention, the reaction signal between single protein molecules can be captured. Even if the test sample contains only one target protein molecule, the corresponding signal can still be detected.
[0021] For the method of coating and fixing the capture protein of the present invention, a GeSim ultra-micro sample spotting instrument (NanoPlotter NP 2.1 / 2.1E) can be used to uniformly disperse and fix the capture protein molecules on the aldehyde glass slide according to the preset pattern and size. Other instrument devices can also be used to uniformly disperse and coat the capture protein on the glass slide to ensure that the capture protein molecules on the glass slide are evenly dispersed in an array and do not affect each other.
[0022] The present invention claims the coating parameters for dispersedly coating and fixing the capture protein on the glass slide (i.e., the coating density of 2.19x10 11 capture molecules per square millimeter). This dispersed fixing method improves the detection sensitivity. No matter what method is used to disperse and fix the capture protein on the glass slide, the reaction between a single testosterone molecule and the capture antibody can be detected, improving the detection sensitivity. Any capture protein fixing method that reaches the femtogram level is within the scope of the claimed technology.
[0023] In the protein chip provided by the present invention, all testosterone monoclonal antibody molecules in the detection spot are arranged dispersedly and do not affect each other. When detecting, BSA and mouse monoclonal antibody labeled with HRP that are pre-incubated and fully bound are added. For one or several low-concentration testosterone small molecules in the sample to be detected, according to the theory of Poisson distribution, each testosterone monoclonal antibody molecule on the detection spot will capture one testosterone small molecule or not capture it. The captured testosterone molecule binds to the complex of BSA and mouse monoclonal antibody labeled with HRP that are pre-incubated and bound, catalyzes the substrate to generate a luminescence signal, and the detection signal can be captured by a CCD camera. Since one signal unit is generated for each captured testosterone small molecule, the corresponding concentration of testosterone small molecules can be calculated according to the intensity of the luminescence signal on each detection spot.
[0024] The present invention provides a simple, feasible, economical, rapid, and reliable protein chip method for detecting testosterone small molecules. It can detect 24 samples at a time, issue the detection results within 40 minutes, and can qualitatively and quantitatively detect testosterone small molecules in clinical samples with accurate results and high sensitivity.
[0025] The innovation of the present invention lies in the change of the preparation process, that is, the coating method of the capture protein on the chip, which greatly improves the sensitivity. Therefore, it is not limited to being made into a protein chip for detecting testosterone small molecules.
[0026] Other advantages of the protein chip and kit improved in this study:
[0027] It allows simultaneous detection of multiple samples, or repeated detection of the same sample multiple times, or samples taken at different time points to obtain dynamic values, or simultaneous detection of samples from different patients, realizing high-throughput and high-sensitivity detection, and overall reducing the detection cost and improving the detection efficiency. Brief Description of the Drawings
[0028] Figure 1 . Schematic plan view of the ultrasensitive protein chip for testosterone small molecules of the present invention;
[0029] Figure 2 . Schematic diagram of the detection principle of the ultrasensitive protein chip for testosterone small molecules of the present invention;
[0030] Figure 3 . Physical diagram of the ultrasensitive protein chip for testosterone small molecules of the present invention detecting testosterone small molecule standard;
[0031] Figure 4 . Physical diagram of the ultrasensitive protein chip for testosterone small molecules of the present invention detecting testosterone small molecule standard;
[0032] Figure 5 . Physical diagram of the ultrasensitive protein chip for testosterone small molecules of the present invention detecting testosterone in saliva;
[0033] Among them, 1, 2, 3, 4, and 5 are successively 10 times, 100 times, 1000 times, 10000 times, and 100000 times dilution solutions of saliva; the dilution solution is 60% glycerol: 6C: 60% glycerol; 6D: saliva.
[0034] Figure 6 . The ultrasensitive protein chip for detecting testosterone small molecules of the present invention detects saliva dilution samples with known testosterone concentrations. Detailed implementation manners
[0035] The present invention will be further described in detail below in conjunction with the specific implementation manners, but the scope of the present invention is not limited. Unless otherwise specified, the operations used in the following examples are all conventional methods, and the reagents used can be obtained commercially.
[0036] As Figure 1 shown, the present invention provides an ultrasensitive protein chip for detecting testosterone small molecules, which is characterized in that the protein chip has at least one sub-detection area, each sub-detection area has at least one detection spot, and the detection spot has non-overlapping, dispersed and tiled capture molecules with a coating density of 2.19x10 per square millimeter 11 ; the capture molecule is a mouse monoclonal antibody obtained by immunizing mice with testosterone small molecules conjugated to BSA.
[0037] Preferably, for the ultrasensitive protein chip, it is characterized in that the diameter of the detection spot is 3-5 mm.
[0038] Preferably, for the ultrasensitive protein chip, it is characterized in that each sub-detection area is one of the detection spots, which is a regular or irregular figure with an area of 9-25 mm, preferably a circle with a diameter of 4 mm.
[0039] Preferably, for the ultrasensitive protein chip, it is characterized in that it has 20-30 sub-detection areas.
[0040] Preferably, for the ultrasensitive protein chip, it is characterized in that a Teflon coating is provided between adjacent sub-detection areas for separating each detection sub-area to prevent cross-contamination between samples.
[0041] Preferably, for the ultrasensitive protein chip, it is characterized in that the testosterone monoclonal antibodies on the detection spots among the sub-detection areas are completely the same; or among at least two sub-detection areas, the testosterone monoclonal antibodies on the detection spots are different from each other.
[0042] Example 1. Preparation of ultrasensitive testosterone small molecule protein chip
[0043] Reagents used:
[0044] Antibody 1. Product Name: Mouse Monoclonal Antibody IgG1 Kappa against Testosterone. Testosterone Antibody (4E1G2), purchased from Novus Biologicals, catalog number NBP2-45187-0.1mg;
[0045] Testosterone standard, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 86500;
[0046] Antibody 2. Product Name: HRP-labeled Mouse Monoclonal Antibody IgG1 Kappa against Testosterone, Testosterone Antibody (4E1G2) [HRP], purchased from Novus Biologicals, catalog number NBP2-NBP3-11603H;
[0047] Bovine Serum Albumin Fraction V, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 810533
[0048] PBS formulation: Sodium chloride (NaCl) 8g, Potassium chloride (KCl) 0.2g, Disodium hydrogen phosphate (Na 2 HPO 4 ) 1.44g, Potassium dihydrogen phosphate (KH 2 PO 4 ) 0.24g, adjust pH to 7.4, make up to 1L
[0049] PBST formulation: 1L PBS + 1ml Tween-20;
[0050] The chip substrate is a diagnostic glass slide, purchased from Japan. Each chip contains 24 detection round holes (sub-detection areas), with a diameter of 4mm. Each detection round hole is a detection spot. In the sub-detection area, the detection spots evenly cover the detection holes as Figure 1 .
[0051] Chip preparation process: The capture protein used is: Mouse Monoclonal Antibody IgG1 Kappa against Testosterone. Testosterone Antibody (4E1G2). Using a GeSim ultra-micro sample spotting instrument (NanoPlotter NP2.1 / 2.1E), the capture protein molecules are evenly dispersed and fixed on the aldehyde glass slide according to the preset pattern and size. Set the parameters so that the coating density is 2.19x10 per square millimeter 11 capture molecules. Make the detection spots such as Figure 1As shown on the right, the testosterone mouse monoclonal antibody IgG1 Kappa protein molecules are dispersed and non-overlapping.
[0052] Example 2. Method for using the ultrasensitive testosterone small molecule protein chip
[0053] The operation process and principle of the protein chip of the present invention are as follows:
[0054] 1. Pre-incubate HRP-labeled testosterone mouse monoclonal antibody IgG1 Kappa and 2% BSA; obtain the HRP detection mixture;
[0055] 2. On the chip prepared in Example 1, add one detection sample (8 ul) to each sub-detection area, store at room temperature for 20 minutes, so that the testosterone small molecules that may be contained in the sample bind to the testosterone mouse monoclonal antibody IgG1 Kappa on the chip;
[0056] 3. Wash the chip with 0.05% PBS-Tween, 5 s each time, wash 5 times, to remove non-specific binding;
[0057] 4. Add the HRP detection mixture to form a complex of testosterone mouse monoclonal antibody IgG1 Kappa - testosterone - HRP-labeled testosterone mouse monoclonal antibody IgG1 Kappa and 2% BSA on the chip, as Figure 2 shown.
[0058] 4. Wash the chip with 0.05% PBS-Tween, 5 s each time, wash 5 times, to remove non-specific binding;
[0059] 5. Add the horseradish peroxidase luminescent substrate.
[0060] Expected result: If the sample contains testosterone small molecules, the detection spot shows a luminescent signal; if the sample does not contain testosterone small molecules, the detection spot has no signal.
[0061] The actual detection results are as Figure 3 and Figure 4 shown. For the detection results using the testosterone standard product, with the chip prepared according to Example 1, its detection sensitivity for testosterone reaches 10 fg / ml.
[0062] Sensitivity verification
[0063] Use the same method as in Example 2.
[0064] The samples to be tested are saliva dilutions and controls, as Figure 5: 1 - 5 are 10 times, 100 times, 1000 times, 10000 times, and 100000 times dilutions of saliva respectively; the diluent is 60% glycerol; 6C: 60% glycerol; 6D: undiluted saliva; it shows that testosterone in saliva diluted 10000 times can be detected.
[0065] Figure 6 The detection is carried out with the saliva diluent whose testosterone content has been determined as the sample, and the control is water; the detection method is the same as that in Example 2, showing that the detection sensitivity of the chip of the present invention to testosterone is 10 fg / ml.
Claims
1. An ultrasensitive protein chip for detecting testosterone small molecules, characterized in that: The protein chip has at least one sub-detection area, each sub-detection area has at least one detection spot, and the detection spots are non-overlapping and dispersed, with a coating density of 2.19x per square millimeter. 11 The capture molecules are obtained by combining testosterone small molecules with BSA to immunize mice and obtain mouse monoclonal antibodies.
2. The ultrasensitive protein chip according to claim 1, characterized in that: The diameter of the detection spot is 3-5 mm.
3. The ultrasensitive protein chip according to claim 1, characterized in that: Each sub-detection area is a detection spot, which is a regular or irregular pattern with an area of 9-25 mm.
4. The ultrasensitive protein chip according to claim 1, characterized in that: Each sub-detection area is a circle with a diameter of 4 mm.
5. The ultrasensitive protein chip according to claim 1, characterized in that: There are 20-30 sub-detection areas.
6. The ultrasensitive protein chip according to claim 1, characterized in that: There is a Teflon coating between adjacent sub-detection areas to separate the sub-detection areas and prevent cross contamination between samples.
7. The ultrasensitive protein chip according to claim 1, characterized in that: Between the sub-detection areas, the testosterone mouse monoclonal antibodies on the detection spots therein are completely identical to each other.
8. The ultrasensitive protein chip according to claim 1, characterized in that: There are at least two sub-detection zones between which the testosterone mouse monoclonal antibodies on the detection spots are different from each other.
9. A kit for detecting testosterone small molecules, characterized in that: The invention comprises at least one ultrasensitive protein chip according to any one of claims 1 to 8, as well as HRP-labeled mouse monoclonal antibody, 0.05% BSA PBS-Tween solution and HRP luminescent substrate solution.
10. The method for preparing the ultrasensitive protein chip according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) obtaining a slide having at least one sub-detection area, (2) In each sub-detection area, the testosterone mouse monoclonal antibody protein molecules are evenly dispersed and sprayed to form a non-overlapping, dispersed and flat layer with a density of 2×10 per square millimeter. 11 The coating density of the capture molecules is 1 capture molecule; the capture molecules are testosterone small molecules combined with BSA to immunize mice, obtain mouse monoclonal antibody protein molecules and then dry to form the detection spots.