A modification method and application of NanoSPR biosensor chip
Through the biosensing chip modification method based on NanoSPR technology, combined with ordinary microplate reader or Xlement100 SPR analysis system, the existing SPR technology equipment is solved and efficient and accurate antibody detection is achieved.
Patent Information
- Application Number
- CN202111369871.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-11-18
- Publication Date
- 2025-04-08
- Estimated Expiration
- 2041-11-18
AI Technical Summary
The existing SPR biosensor system requires specially designed equipment and high maintenance costs, limiting its wide application.
The biosensing chip modification method based on nanoplasma resonance (NanoSPR) technology is used to modify the chip through steps such as double distilled water cleaning, reducing agent treatment, antibody protein dropping, blocking and protective liquid treatment, and the detection is combined with a common microplate reader or Xlement100 SPR analysis system.
It realizes the completion of high-throughput sample detection in a short time, simplifies the antibody detection process, reduces equipment and maintenance costs, and has high detection accuracy and relative deviations of no more than 10%.
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Figure CN115144577B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biochemical analysis and detection, and specifically relates to a modification method and application of a NanoSPR biosensor chip. Background Art
[0002] Accurate antibody or protein quantification is essential for selecting engineered cell lines for developing and optimizing antibody production. The current methods for antibody or protein quantification mainly include surface plasmon resonance (SPR) biosensor systems, biomembrane interferometry, etc., which can monitor dynamic biomolecular interactions in real time without labeling and without the influence of a large amount of background. However, these traditional technologies all require special equipment and special personnel to operate. The existing SPR sensor chips are mainly chips from Biacore, which must be used in conjunction with Biacore's supporting instruments. In addition, the maintenance cost of the instruments is also particularly high, which requires a large amount of cost to purchase or use the biosensor system, greatly restricting the widespread application of SPR technology. Summary of the invention
[0003] In view of the shortcomings of the prior art, the object of the present invention is to provide a modification method for a biosensor chip based on nanoplasmon resonance (NanoSPR) technology. The NanoSPR biosensor chip modified by the modification method of the present invention can be combined with an ordinary microplate reader or a standardized Xlement100 SPR analysis system to complete the output of high-throughput sample detection results in a short time, simplify the antibody detection process, and require a small sample volume; it can be used for rapid quantitative detection of human IgG antibodies, Fc fusion proteins or tagged proteins in buffer or cell supernatant.
[0004] To achieve the above object, the present invention provides a method for modifying a NanoSPR biosensor chip, comprising the following steps:
[0005] S1. Use double distilled water to clean the surface of the chip substrate;
[0006] S2. Add a reducing agent solution to the surface of the chip substrate, then add a chloroauric acid aqueous solution, mix well, incubate at 25-45°C for 5-60 minutes, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0007] S3. Add the antibody protein solution to the surface of the nano-gold-modified chip, incubate at 4-37°C for 1.5-24 hours, recover the antibody protein solution after the incubation, and wash the chip coated with the antibody with PBST solution;
[0008] S4. Add blocking solution to the surface of the chip coated with the antibody, incubate at 25-37°C for 1-4 hours, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0009] S5. Add protective solution to the surface of the sealed chip, incubate at 25-37°C for 5-60 min, remove excess protective solution after incubation, and dry to obtain a NanoSPR biosensor chip;
[0010] The chip substrate comprises a base, a nanocup array is printed on the surface of the base, and a metal film is covered on the surface of the nanocup array;
[0011] The reducing agent solution is an aqueous solution of one or more of sodium citrate, sodium borohydride, sodium citrate, mercaptosuccinic acid, hydroxylamine hydrochloride, ascorbic acid, tannic acid, L-cysteine, and white phosphorus;
[0012] The antibody protein solution is an aqueous solution of one of Protein A, Protein G, Protein L, streptavidin or a label antibody;
[0013] The PBST solution is prepared from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, Tween-20, preservative Proclin 300, and double distilled water;
[0014] The blocking solution is prepared by dissolving bovine serum albumin in a CBS solution, and the CBS solution is prepared by potassium carbonate, sodium bicarbonate, a preservative Proclin 300, and purified water;
[0015] The protective solution is prepared by dissolving sugar in PBST solution.
[0016] Preferably, in step S1, the chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, a surface of the nanocup array is covered with a titanium film, a surface of the titanium film is covered with a silver film, and a surface of the silver film is covered with a gold film.
[0017] More preferably, the diameter of the nanocup array nanocup is 50-2000nm, the height is 100-800nm, and the spacing is 100-4000nm; the thickness of the titanium film is 2-30nm, the thickness of the silver film is 5-100nm, and the thickness of the gold film is 2-100nm.
[0018] Preferably, in step S2, the concentration of the reducing agent solution is 0.01-100 mM, the concentration of the chloroauric acid aqueous solution is 0.01-100 mM; and the molar ratio of the reducing agent to chloroauric acid is 0.1-1:1.
[0019] Preferably, in step S3, the concentration of the antibody protein solution is 0.5-100 μg / mL.
[0020] Preferably, the PBST solution is prepared from the following raw materials: 29-580 g of disodium hydrogen phosphate dodecahydrate, 2.72-54.4 g of sodium dihydrogen phosphate, 2-40 g of potassium chloride, 20-400 g of sodium chloride, 5-100 mL of Tween-20, 5-100 mL of preservative Proclin 300, and 10 L of double distilled water.
[0021] Preferably, in step S4, the blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared from the following raw materials: 15.9-318 g of potassium carbonate, 29.3-586 g of sodium bicarbonate, 5-100 mL of preservative Proclin 300, and 10 L of purified water.
[0022] Preferably, in step S5, the protective solution is prepared by dissolving 10-200 g of sugar in 1 L of PBST solution, and the sugar is one of dextran, glucose, sucrose and trehalose.
[0023] NanoSPR biosensor chip quantitative detection technology is based on the target antibody binding to the biochip surface to produce a plasma resonance effect with the nanocup chip, causing a change in absorbance at a specific wavelength, and the absorbance at this wavelength is proportional to the content of the antibody in the sample. Therefore, the absorbance change of antibodies of known concentration at a specific wavelength of the nanoplasma chip can be used to establish a standard curve of absorbance change value and concentration value, thereby calculating the antibody content in the sample to be tested. The present invention also provides the use of the NanoSPR biosensor chip modified by the above-mentioned modification method in the rapid quantitative detection of antibodies, Fc fusion proteins or tagged proteins.
[0024] The NanoSPR biosensor chip is assembled with a 96-well microplate to form a 96-well chip plate. The sample is tested using a common microplate reader or a standard Xlement100 SPR analysis system. The test process includes the following steps:
[0025] P1. Use a pipette to add 30-200 μL of washing solution to each well of the 96-well microarray plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0026] P2. Add 30-200 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5-15 min, read the data, and discard the sample solution;
[0027] P3. Add 100-200 μL of washing solution to each well and wash the plate twice, tap the plate, and spin dry;
[0028] P4. Add 30-200 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0029] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0030] After the test, discard the liquid in the well, wash the plate three times with 100 μL elution regeneration solution, and rinse once with 100 μL washing solution, and then it can be reused.
[0031] The elution regeneration solution is a glycine solution (pH<3), and the washing solution is a PBST buffer.
[0032] Compared with the prior art, the present invention has the following beneficial effects:
[0033] (1) The NanoSPR biosensor chip modification method of the present invention modifies the target antibody protein on the surface of the chip substrate with the nanocup array. After the target antibody to be tested is combined with the biochip surface, it will produce a plasma resonance effect with the nanocup chip, causing a change in absorbance at a specific wavelength. The absorbance at this wavelength is proportional to the antibody content in the sample. Therefore, an ordinary microplate reader or a high-precision Xlement100 SPR analysis system can be used to complete the output of high-throughput sample detection results in a short time, simplifying the antibody detection process and requiring a small sample volume.
[0034] (2) The NanoSPR biosensor chip modified by the modification method of the present invention has high sensitivity and good stability and can be reused more than 30 times.
[0035] (3) The NanoSPR biosensor chip modified by the modification method of the present invention is made into a 96-microwell chip plate with high detection throughput. It can complete the simultaneous detection of up to 96 samples in a short time without labeling, which simplifies the antibody detection process. The required sample volume is small and the detection accuracy is high. The lowest detection limit can reach 0.01 μg / mL, the concentration resolution is 0.01 μg / mL, and the relative deviation of the measurement result does not exceed 10%. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is the standard curve of the detection results of Example 1.
[0037] Figure 2 The results of eight replicate wells in Example 1 were obtained by detecting human IgG antibodies in five cell supernatants at the same concentration. DETAILED DESCRIPTION
[0038] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0039] Example 1
[0040] This embodiment provides a method for modifying a NanoSPR biosensor chip with Protein A, comprising the following steps:
[0041] S1. Use double distilled water to clean the surface of the chip substrate;
[0042] S2. Add 50 μL of 1 mM L-cysteine aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0043] S3. Add 50 μL of 10 μg / mL Protein A aqueous solution to the surface of the nano-gold-modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the chip coated with the antibody with PBST solution;
[0044] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0045] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry to obtain the Protein A modified NanoSPR biosensor chip.
[0046] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has an upper diameter of 200nm, a bottom diameter of 150nm, a height of 500nm, and a spacing of 400nm, the surface of the nanocup array is covered with a 2nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 2nm thick gold film.
[0047] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0048] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0049] The protective solution is prepared by dissolving 100 g of sucrose in 1 L of PBST solution.
[0050] The Protein A modified NanoSPR biosensor chip provided in this example is assembled with a 96-microwell plate to prepare a 96-microwell chip plate for detecting human IgG antibodies. The detection method comprises the following steps:
[0051] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0052] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0053] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0054] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0055] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0056] The standard curve of the test results is as follows Figure 1 The results of eight replicate wells testing five human IgG antibodies in the cell supernatant at the same concentration are shown in Figure 2 The calculation results are shown in Table 1. It can be seen that the relative deviation of the measurement results does not exceed 7%.
[0057] Table 1 Detection results of human IgG antibody using Protein A modified NanoSPR biosensor chip
[0058]
[0059] Example 2
[0060] This embodiment provides a method for modifying a NanoSPR biosensor chip with Protein A, comprising the following steps:
[0061] S1. Use double distilled water to clean the surface of the chip substrate;
[0062] S2. Add 50 μL of 1 mM sodium citrate aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0063] S3. Add 50 μL of 10 μg / mL Protein A aqueous solution to the surface of the nano-gold-modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the chip coated with the antibody with PBST solution;
[0064] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0065] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry to obtain the Protein A modified NanoSPR biosensor chip.
[0066] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has an upper diameter of 200nm, a bottom diameter of 150nm, a height of 500nm, and a spacing of 400nm, the surface of the nanocup array is covered with a 9nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 5nm thick gold film.
[0067] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0068] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0069] The protective solution is prepared by dissolving 50 g of sucrose in 1 L of PBST solution.
[0070] The Protein A modified NanoSPR biosensor chip provided in this example is assembled with a 96-microwell plate to prepare a 96-microwell chip plate for detecting Fc fusion protein. The detection method comprises the following steps:
[0071] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0072] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0073] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0074] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0075] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0076] The test results are shown in Table 2. It can be seen that the relative deviation of the measurement results does not exceed 8%.
[0077] Table 2 Detection results of Protein A modified NanoSPR biosensor chip for detecting Fc fusion protein
[0078]
[0079] Example 3
[0080] This embodiment provides a method for modifying a NanoSPR biosensor chip with Protein G, comprising the following steps:
[0081] S1. Use double distilled water to clean the surface of the chip substrate;
[0082] S2. Add 50 μL of 1 mM L-cysteine aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0083] S3. Add 50 μL of 100 μg / mL Protein G aqueous solution to the surface of the nano-gold-modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the chip coated with the antibody with PBST solution;
[0084] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0085] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry to obtain the Protein G-modified NanoSPR biosensor chip.
[0086] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has a diameter of 150nm, a height of 400nm, and a spacing of 300nm, the surface of the nanocup array is covered with a 2nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 2nm thick gold film.
[0087] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0088] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0089] The protective solution is prepared by dissolving 100 g of dextran in 1 L of PBST solution.
[0090] The Protein G modified NanoSPR biosensor chip provided in this example is assembled with a 96-microwell plate to prepare a 96-microwell chip plate for detecting human IgG antibodies. The detection method comprises the following steps:
[0091] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0092] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0093] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0094] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0095] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0096] The test results are shown in Table 3. It can be seen that the relative deviation of the measurement results does not exceed 8%.
[0097] Table 3 Detection results of human IgG antibody by Protein G modified NanoSPR biosensor chip
[0098]
[0099] Example 4
[0100] This embodiment provides a method for modifying a NanoSPR biosensor chip with Protein L, comprising the following steps:
[0101] S1. Use double distilled water to clean the surface of the chip substrate;
[0102] S2. Add 50 μL of 1 mM tannic acid aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0103] S3. Add 50 μL of 10 μg / mL Protein L aqueous solution to the surface of the nano-gold-modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the chip coated with the antibody with PBST solution;
[0104] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0105] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry to obtain the Protein L modified NanoSPR biosensor chip.
[0106] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has an upper diameter of 200nm, a bottom diameter of 150nm, a height of 500nm, and an interval period of 400nm, the surface of the nanocup array is covered with a 9nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 5nm thick gold film.
[0107] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0108] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0109] The protective solution is prepared by dissolving 50 g of sucrose in 1 L of PBST solution.
[0110] The Protein L modified NanoSPR biosensor chip provided in this example is assembled with a 96-microwell plate to prepare a 96-microwell chip plate for detecting Fc fusion protein. The detection method comprises the following steps:
[0111] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0112] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0113] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0114] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0115] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0116] The test results are shown in Table 4, and it can be seen that the relative deviation of the measurement results does not exceed 10%.
[0117] Table 4 Detection results of Fc fusion protein by Protein L modified NanoSPR biosensor chip
[0118]
[0119] Example 5
[0120] This embodiment provides a method for modifying a NanoSPR biosensor chip with streptavidin, comprising the following steps:
[0121] S1. Use double distilled water to clean the surface of the chip substrate;
[0122] S2. Add 50 μL of 1 mM tannic acid aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0123] S3. Add 50 μL of 10 μg / mL streptavidin aqueous solution to the surface of the nanogold-modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the chip coated with the antibody with PBST solution;
[0124] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0125] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry the chip to obtain a streptavidin-modified NanoSPR biosensor chip.
[0126] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has an upper diameter of 200nm, a bottom diameter of 150nm, a height of 500nm, and an interval period of 400nm, the surface of the nanocup array is covered with a 9nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 5nm thick gold film.
[0127] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0128] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0129] The protective solution is prepared by dissolving 50 g of sucrose in 1 L of PBST solution.
[0130] The streptavidin-modified NanoSPR biosensor chip provided in this embodiment is assembled with a 96-microwell plate to form a 96-microwell chip plate, which is used to detect biotinylated antibodies. The detection method includes the following steps:
[0131] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0132] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0133] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0134] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0135] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0136] The test results are shown in Table 5, and it can be seen that the relative deviation of the measurement results does not exceed 10%.
[0137] Table 5 Detection results of biotinylated antibodies detected by streptavidin-modified NanoSPR biosensor chip
[0138]
[0139] Example 6
[0140] This embodiment provides a method for modifying a NanoSPR biosensor chip with an anti-Flag tag antibody, comprising the following steps:
[0141] S1. Use double distilled water to clean the surface of the chip substrate;
[0142] S2. Add 50 μL of 1 mM L-cysteine aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0143] S3. Add 50 μL of 10 μg / mL anti-Flag antibody aqueous solution to the surface of the nano-gold modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the chip coated with the antibody with PBST solution;
[0144] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0145] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry the chip to obtain the NanoSPR biosensor chip modified with anti-Flag tag antibody.
[0146] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has an upper diameter of 200nm, a bottom diameter of 150nm, a height of 500nm, and an interval period of 400nm, the surface of the nanocup array is covered with a 9nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 5nm thick gold film.
[0147] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0148] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0149] The protective solution is prepared by dissolving 50 g of sucrose in 1 L of PBST solution.
[0150] The NanoSPR biosensor chip modified with the anti-Flag tag antibody provided in this embodiment is assembled with a 96-microwell plate to prepare a 96-microwell chip plate for detecting Flag tag fusion protein. The detection method comprises the following steps:
[0151] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0152] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0153] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0154] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0155] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0156] The test results are shown in Table 6, and it can be seen that the relative deviation of the measurement results does not exceed 10%.
[0157] Table 6 Detection results of the anti-Flag tag antibody modified NanoSPR biosensor chip for detecting Flag tag fusion protein
[0158]
[0159] Example 7
[0160] This embodiment provides a method for modifying a NanoSPR biosensor chip with an anti-His tag antibody, comprising the following steps:
[0161] S1. Use double distilled water to clean the surface of the chip substrate;
[0162] S2. Add 50 μL of 1 mM ascorbic acid aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0163] S3. Add 50 μL of 10 μg / mL anti-His tag antibody aqueous solution to the surface of the nano-gold modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the antibody-coated chip with PBST solution;
[0164] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0165] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry the chip to obtain the NanoSPR biosensor chip modified with the anti-His tag antibody.
[0166] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has an upper diameter of 200nm, a bottom diameter of 150nm, a height of 500nm, and an interval period of 400nm, the surface of the nanocup array is covered with a 9nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 5nm thick gold film.
[0167] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0168] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0169] The protective solution is prepared by dissolving 50 g of sucrose in 1 L of PBST solution.
[0170] The NanoSPR biosensor chip modified with the anti-His tag antibody provided in this embodiment is assembled with a 96-microwell plate to prepare a 96-microwell chip plate for detecting the His tag fusion protein. The detection method comprises the following steps:
[0171] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0172] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0173] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0174] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0175] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0176] The test results are shown in Table 7, and it can be seen that the relative deviation of the measurement results does not exceed 10%.
[0177] Table 7 Detection results of His-tag fusion protein detected by anti-His-tag antibody modified NanoSPR biosensor chip
[0178]
[0179] Example 8
[0180] This embodiment provides a method for modifying a NanoSPR biosensor chip with an anti-Fc tag antibody, comprising the following steps:
[0181] S1. Use double distilled water to clean the surface of the chip substrate;
[0182] S2. Add 50 μL of 1 mM ascorbic acid aqueous solution to the surface of the chip substrate, then add 50 μL of 1 mM chloroauric acid aqueous solution, mix well, incubate at 25°C for 60 min, remove the solvent after the incubation, and wash the nano-gold modified chip with double distilled water;
[0183] S3. Add 50 μL of 10 μg / mL anti-Fc tag antibody aqueous solution to the surface of the nano-gold modified chip, incubate at 37°C for 1.5 h, recover the antibody protein solution after the incubation, and wash the antibody-coated chip with PBST solution;
[0184] S4. Add 150 μL of blocking solution to the surface of the chip coated with the antibody, incubate at 37°C for 1 h, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution;
[0185] S5. Add 150 μL of protective solution to the surface of the sealed chip and incubate at 37°C for 5 min. After the incubation, remove the excess protective solution and dry the chip to obtain the NanoSPR biosensor chip modified with anti-Fc tag antibody.
[0186] The chip substrate comprises a PET thin plate substrate, a nanocup array is embossed on the surface of the PET thin plate substrate, the nanocup has an upper diameter of 200nm, a bottom diameter of 150nm, a height of 500nm, and an interval period of 400nm, the surface of the nanocup array is covered with a 9nm thick titanium film, the surface of the titanium film is covered with a 40nm thick silver film, and the surface of the silver film is covered with a 5nm thick gold film.
[0187] The PBST solution is prepared from the following raw materials: 29 g disodium hydrogen phosphate dodecahydrate, 2.72 g sodium dihydrogen phosphate, 2 g potassium chloride, 20 g sodium chloride, 5 mL Tween-20, 5 mL preservative Proclin 300, and 10 L double distilled water.
[0188] The blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared by the following raw materials: 15.9 g of potassium carbonate, 29.3 g of sodium bicarbonate, 5 mL of preservative Proclin 300, and 10 L of purified water.
[0189] The protective solution is prepared by dissolving 50 g of sucrose in 1 L of PBST solution.
[0190] The NanoSPR biosensor chip modified with the anti-Fc tag antibody provided in this embodiment is assembled with a 96-microwell plate to prepare a 96-microwell chip plate for detecting Fc fusion protein. The detection method comprises the following steps:
[0191] P1. Use a pipette to add 50 μL of washing solution to each well of the 96-well microwell chip plate, place it in the microplate reader, record the initial value, and discard the liquid in the well;
[0192] P2. Add 50 μL of standard or diluted sample to each well of the microwell chip plate, shake the plate at 300 rpm for 5 min, read the data, and discard the sample solution;
[0193] P3. Add 100 μL of washing solution to each well and wash the plate twice, tap the plate and spin dry;
[0194] P4. Add 50 μL of sample diluent to each well, place in the microplate reader, and record the endpoint value;
[0195] P5. Data processing, drawing a standard curve, the concentration of the sample to be tested can be read based on the standard curve.
[0196] The test results are shown in Table 8, and it can be seen that the relative deviation of the measurement results does not exceed 8%.
[0197] Table 8 Detection results of Fc fusion protein detected by anti-Fc tag antibody modified NanoSPR biosensor chip
[0198]
[0199] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by technicians in this technical field within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention. The protection scope of the present invention shall be based on the protection scope of the claims.
Claims
1. A method for modifying a NanoSPR biosensor chip, characterized in that: The following steps are involved: S1. Clean the chip substrate surface with double distilled water; S2. Add a reducing agent solution to the surface of the chip substrate, then add a chloroauric acid aqueous solution, mix well, and incubate at 25-45°C for 5-60 minutes. After the incubation, remove the solvent and wash the nano-gold modified chip with double distilled water; S3. Add the antibody protein solution to the surface of the nanogold-modified chip and incubate at 4-37°C for 1.5-24 hours. After the incubation, recover the antibody protein solution and wash the chip coated with the antibody with PBST solution. S4. Add blocking solution to the surface of the chip coated with antibodies, incubate at 25-37°C for 1-4 hours, remove excess blocking solution after incubation, and wash the blocked chip with PBST solution; S5. Add protective solution to the surface of the sealed chip, incubate at 25-37°C for 5-60 min, remove excess protective solution after incubation, and dry to obtain the NanoSPR biosensor chip; The reducing agent solution is an aqueous solution of one or more of sodium citrate, sodium borohydride, sodium citrate, mercaptosuccinic acid, hydroxylamine hydrochloride, ascorbic acid, tannic acid, L-cysteine, and white phosphorus; The antibody protein solution is an aqueous solution of one of Protein A, Protein G, Protein L, streptavidin or a label antibody; The PBST solution is prepared from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, Tween-20, preservative Proclin 300, and double distilled water; The blocking solution is prepared by dissolving bovine serum albumin in a CBS solution, and the CBS solution is prepared by potassium carbonate, sodium bicarbonate, a preservative Proclin 300, and purified water; The protective solution is prepared by dissolving sugar in PBST solution; In step S1, the chip substrate comprises a PET thin plate base, a nanocup array is printed on the surface of the PET thin plate base, a surface of the nanocup array is covered with a titanium film, a surface of the titanium film is covered with a silver film, and a surface of the silver film is covered with a gold film.
2. The method for modifying the NanoSPR biosensor chip according to claim 1, characterized in that: The nanocup array nanocup has an upper diameter of 50-2000nm, a bottom diameter of 50-2000nm, a height of 100-800nm, and a spacing of 100-4000nm; the titanium film has a thickness of 2-30nm, the silver film has a thickness of 5-100nm, and the gold film has a thickness of 2-100nm.
3. The method for modifying the NanoSPR biosensor chip according to claim 1, characterized in that: In step S2, the concentration of the reducing agent solution is 0.01-100 mM, the concentration of the chloroauric acid aqueous solution is 0.01-100 mM; and the molar ratio of the reducing agent to chloroauric acid is 0.1-1:
1.
4. The method for modifying the NanoSPR biosensor chip according to claim 1, characterized in that: In step S3, the concentration of the antibody protein solution is 0.5-100 μg / mL.
5. The method for modifying the NanoSPR biosensor chip according to claim 1, characterized in that: The PBST solution is prepared from the following raw materials: 29-580 g of disodium hydrogen phosphate dodecahydrate, 2.72-54.4 g of sodium dihydrogen phosphate, 2-40 g of potassium chloride, 20-400 g of sodium chloride, 5-100 mL of Tween-20, 5-100 mL of preservative Proclin 300, and 10 L of double distilled water.
6. The method for modifying the NanoSPR biosensor chip according to claim 1, characterized in that: In step S4, the blocking solution is prepared by dissolving 10 g of bovine serum albumin in 1 L of CBS solution, and the CBS solution is prepared from the following raw materials: 15.9-318 g of potassium carbonate, 29.3-586 g of sodium bicarbonate, 5-100 mL of preservative Proclin 300, and 10 L of purified water.
7. The method for modifying the NanoSPR biosensor chip according to claim 1, characterized in that: In step S5, the protective solution is prepared by dissolving 10-200 g of sugar in 1 L of PBST solution, and the sugar is one of dextran, glucose, sucrose, and trehalose.
8. A NanoSPR biosensor chip, characterized in that: The NanoSPR biosensor chip is modified by the modification method of any one of claims 1 to 7.
9. Use of the NanoSPR biosensor chip according to claim 8 in rapid quantitative detection of antibodies, Fc fusion proteins or tagged proteins.
Citation Information
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