CN115144577B - A modification method and application of NanoSPR biosensor chip - Google Patents
A modification method and application of NanoSPR biosensor chip Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
- G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
- G01N2021/5903—Transmissivity using surface plasmon resonance [SPR], e.g. extraordinary optical transmission [EOT]
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Abstract
本发明公开了一种基于纳米等离子共振(NanoSPR)技术的生物传感芯片的修饰方法及应用，修饰方法包括以下步骤：清洗芯片基片，修饰纳米金，包被抗体，封闭液封闭，保护液保护。可包被抗体蛋白为Protein A、Protein G、Protein L、链霉亲和素或标签抗体。采用本发明的修饰方法修饰的NanoSPR生物传感芯片联合普通酶标仪或者量准的Xlement100SPR分析系统即可在短时间内完成高通量样本的检测结果输出，简化抗体检测过程，且所需样本体积小；可用于缓冲液或细胞上清中的人IgG抗体、Fc融合蛋白或带标签蛋白的快速定量检测。
The present invention discloses a modification method and application of a biosensor chip based on nanoplasmon resonance (NanoSPR) technology. The modification method comprises the following steps: cleaning the chip substrate, modifying nanogold, coating antibodies, blocking with a blocking solution, and protecting with a protective solution. The antibody protein that can be coated is Protein A, Protein G, Protein L, streptavidin or a labeled antibody. The NanoSPR biosensor chip modified by the modification method of the present invention can be combined with a common microplate reader or a high-precision Xlement100SPR analysis system to complete the output of the detection results of high-throughput samples in a short time, simplify the antibody detection process, and the required sample volume is small; it can be used for rapid quantitative detection of human IgG antibodies, Fc fusion proteins or labeled proteins in buffers or cell supernatants.
Description
Technical Field
The invention belongs to the technical field of biochemical analysis and detection, and particularly relates to a modification method and application of NanoSPR biological sensing chips.
Background
Accurate antibody or protein quantification is critical to the selection of engineered cell lines for development and optimization of antibody production. The current methods for antibody or protein quantification mainly comprise a Surface Plasmon Resonance (SPR) biosensor system, a biological membrane interference technology and the like, and can monitor the interaction of dynamic biological molecules in real time without marking and being influenced by a large amount of background. However, these conventional technologies all require special equipment and special personnel to operate, and the existing SPR sensing chip is mainly a chip of Biacore company, and can be used only by being matched with a matched instrument of Biacore company, and the maintenance cost of the instrument is extremely high, so that a large amount of cost is required for purchasing or using the biosensor system, and the wide application of the SPR technology is greatly restricted.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a modification method of a biosensing chip based on a nano-plasma resonance (NanoSPR) technology, and the NanoSPR biosensing chip modified by the modification method can finish the detection result output of a high-flux sample in a short time by combining a common enzyme-labeled instrument or a standard Xlement100 SPR analysis system, so that the antibody detection process is simplified, the required sample volume is small, and the method can be used for rapid quantitative detection of human IgG antibodies, fc fusion proteins or tagged proteins in buffer solution or cell supernatant.
In order to achieve the above purpose, the present invention provides a method for modifying NanoSPR a bio-sensor chip, comprising the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding a reducing agent solution on the surface of the chip substrate, adding an aqueous chloroauric acid solution, uniformly mixing, incubating at 25-45 ℃ for 5-60 min, removing the solvent after incubation, and cleaning the modified nano gold chip by double distilled water;
S3, adding an antibody protein solution to the surface of the chip modified with the nano gold, incubating for 1.5-24 hours at 4-37 ℃, recovering the antibody protein solution after incubation, and cleaning the chip coated with the antibody by using a PBST solution;
S4, adding a sealing liquid on the surface of the chip coated with the antibody, incubating for 1-4 hours at 25-37 ℃, removing the redundant sealing liquid after incubation, and cleaning the sealed chip by using a PBST solution;
S5, adding a protective solution to the surface of the closed chip, incubating for 5-60 min at 25-37 ℃, removing the redundant protective solution after incubation, and drying to obtain NanoSPR biological sensing chip;
The chip substrate comprises a base, wherein the surface of the base is stamped with a nano cup array, and the surface of the nano cup array is covered with a metal film;
The reducer solution is one or more aqueous solutions 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 tag antibody;
the PBST solution is prepared from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, tween-20, a preservative Proclin300 and double distilled water;
the sealing liquid is prepared by dissolving bovine serum albumin in a CBS solution, wherein the CBS solution is prepared by potassium carbonate, sodium bicarbonate, a preservative Proclin300 and purified water;
the protective solution is prepared by dissolving saccharides in PBST solution.
Preferably, in step S1, the chip substrate includes a PET sheet substrate, a nano cup array is stamped on the surface of the PET sheet substrate, a layer of titanium film is covered on the surface of the nano cup array, a layer of silver film is covered on the surface of the titanium film, and a layer of gold film is covered on the surface of the silver film.
More preferably, the diameter of the nano cup array is 50-2000 nm, the height is 100-800 nm, the distance is 100-4000 nm, the thickness of the titanium film is 2-30 nm, the thickness of the silver film is 5-100 nm, and the thickness of the gold film is 2-100 nm.
Preferably, in the 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 the chloroauric acid is 0.1-1:1.
Preferably, in step S3, the concentration of the antibody protein solution is 0.5-100 μg/mL.
Preferably, the PBST solution is prepared from 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, 3005-100 mL of preservative Proclin and 10L of double distilled water.
Preferably, in the step S4, the sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9-318 g of potassium carbonate, 29.3-586 g of sodium bicarbonate, 300-100 mL of preservative Proclin and 10L of purified water.
Preferably, in step S5, the protecting solution is prepared by dissolving 10-200 g of saccharide in 1L of PBST solution, wherein the saccharide is one of dextran, glucose, sucrose and trehalose.
The NanoSPR biological sensing chip quantitative detection technology is based on that after the combination of the target antibody and the surface of the biological chip, a plasma resonance effect is generated with the nano cup chip, the absorbance change is caused at a specific wavelength, and the absorbance at the wavelength is in direct proportion to the content of the antibody in the sample. Therefore, the standard curve of the absorbance change value and the concentration value can be established by utilizing the absorbance change of the antibody with known concentration at the specific wavelength of the nano plasma chip, so that the antibody content in the sample to be detected can be calculated. The invention also provides an application of NanoSPR biological sensing chip modified by the modification method in rapid quantitative detection of antibodies, fc fusion proteins or tagged proteins.
The NanoSPR biological sensing chip and the 96 micropore plate are assembled to form the 96 micropore chip plate, and a common enzyme-labeled instrument or a Xlement SPR analysis system with accurate measurement is adopted for detecting samples, wherein the detection process comprises the following steps:
p1, respectively adding 30-200 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the holes into an enzyme label instrument, recording initial values, and discarding liquid in the holes;
P2, respectively adding 30-200 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5-15 min, reading data, and discarding sample solution;
p3, respectively adding 100-200 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
P4, adding 30-200 mu L of sample diluent into each hole, putting into an enzyme-labeled instrument, and recording an end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
After the detection is finished, the liquid in the hole is discarded, the plate is washed 3 times by 100 mu L of elution regeneration liquid, and then is rinsed 1 time by 100 mu L of washing liquid, so that the plate can be reused.
The eluting regeneration solution is glycine solution (pH < 3), and the washing solution is PBST buffer solution.
Compared with the prior art, the invention has the beneficial effects that:
(1) The NanoSPR biological sensing chip modification method of the invention modifies the target antibody protein on the chip substrate surface with the nano cup array, and after the combination of the target antibody to be detected and the biochip surface, the plasma resonance effect is generated with the nano cup chip, the absorbance change is caused at a specific wavelength, and the absorbance at the wavelength is in direct proportion to the content of the antibody in the sample. Therefore, a common enzyme-labeled instrument or a standard Xlement SPR analysis system can be adopted to finish the output of the detection result of the high-flux sample in a short time, the antibody detection process is simplified, and the required sample volume is small.
(2) The NanoSPR biological sensing chip modified by the modification method has high sensitivity and good stability, and can be repeatedly used for more than 30 times.
(3) The NanoSPR biological sensing chip modified by the modification method is manufactured into a 96-micropore chip board, the detection flux is high, the simultaneous detection of 96 samples at most can be completed in a short time, no mark is needed, the antibody detection process is simplified, the required sample volume is small, the detection precision is high, the lowest detection lower limit can reach 0.01 mug/mL, the concentration resolution is 0.01 mug/mL, and the relative deviation of the measurement result is not more than 10%.
Drawings
FIG. 1 is a standard curve of the test results of example 1.
FIG. 2 shows the results of example 1 for detection of 8 multiplexed wells of human IgG antibodies in five cell supernatants of the same concentration.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully by reference to the accompanying drawings, in which it is shown, by way of illustration, only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
Example 1
The embodiment provides a modification method of a Protein A modified NanoSPR biological sensing chip, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding 50 mu L of 1mM L-cysteine aqueous solution to the surface of a chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano gold chip by double distilled water;
S3, adding 50 mu L of Protein A aqueous solution of 10 mu g/mL on the surface of the chip modified with the nano gold, incubating for 1.5h at 37 ℃, recovering antibody Protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
s5, adding 150 mu L of protective solution to the surface of the closed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the Protein A modified NanoSPR biological sensing chip.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the upper diameter of each nano cup is 200nm, the bottom diameter of each nano cup is 150nm, the height of each nano cup is 500nm, the distance between each nano cup array and each nano cup array is 400nm, the surface of each nano cup array is covered with a titanium film with the thickness of 2nm, the surface of each titanium film is covered with a silver film with the thickness of 40nm, and the surface of each silver film is covered with a gold film with the thickness of 2 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protective solution is prepared by dissolving 100g of sucrose in 1L of PBST solution.
The Protein A modified NanoSPR biological sensing chip provided by the embodiment is assembled with a 96-micropore plate to form the 96-micropore chip plate for detecting human IgG antibodies, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The standard curve of the detection results is shown in FIG. 1, the results of detecting 8 multiplexed wells of human IgG antibodies in the cell supernatants of five cells of the same concentration are shown in FIG. 2, the calculation results are shown in Table 1, and it can be seen that the relative deviation of the measurement results is not more than 7%.
TABLE 1 Protein A detection results of human IgG antibodies by NanoSPR modified biosensing chip
Example 2
The embodiment provides a modification method of a Protein A modified NanoSPR biological sensing chip, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding 50 mu L of 1mM sodium citrate aqueous solution on the surface of the chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano-gold chip by double distilled water;
S3, adding 50 mu L of Protein A aqueous solution of 10 mu g/mL on the surface of the chip modified with the nano gold, incubating for 1.5h at 37 ℃, recovering antibody Protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
s5, adding 150 mu L of protective solution to the surface of the closed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the Protein A modified NanoSPR biological sensing chip.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the upper diameter of each nano cup is 200nm, the bottom diameter of each nano cup is 150nm, the height of each nano cup array is 500nm, the distance between each nano cup array and each nano cup array is 400nm, the surface of each nano cup array is covered with a titanium film with the thickness of 9nm, the surface of each titanium film is covered with a silver film with the thickness of 40nm, and the surface of each silver film is covered with a gold film with the thickness of 5 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protective solution is prepared by dissolving 50g of sucrose in 1L of PBST solution.
The Protein A modified NanoSPR biological sensing chip provided by the embodiment is assembled with a 96-micropore plate to form the 96-micropore chip plate for detecting the Fc fusion Protein, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The test results are shown in Table 2, and it can be seen that the relative deviation of the measurement results is not more than 8%.
TABLE 2 detection results of Protein A modification NanoSPR biological sensor chip for detecting Fc fusion Protein
Example 3
The embodiment provides a modification method of a Protein G modified NanoSPR biological sensing chip, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding 50 mu L of 1mM L-cysteine aqueous solution to the surface of a chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano gold chip by double distilled water;
S3, adding 50 mu L of Protein G aqueous solution of 100 mu G/mL on the surface of the chip modified with the nano gold, incubating for 1.5h at 37 ℃, recovering antibody Protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
s5, adding 150 mu L of protective solution to the surface of the closed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the Protein G modified NanoSPR biological sensing chip.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the diameter of the nano cup is 150nm, the height of the nano cup array is 400nm, the distance between the nano cup array and the nano cup array is 300nm, the surface of the nano cup array is covered with a titanium film with the thickness of 2nm, the surface of the titanium film is covered with a silver film with the thickness of 40nm, and the surface of the silver film is covered with a gold film with the thickness of 2 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protection solution is prepared by dissolving 100g of glucan in 1L of PBST solution.
The Protein G modified NanoSPR biological sensing chip provided by the embodiment is assembled with a 96-micropore plate to form the 96-micropore chip plate for detecting human IgG antibodies, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The measurement results are shown in Table 3, and it can be seen that the relative deviation of the measurement results is not more than 8%.
TABLE 3 Protein G detection results of human IgG antibodies by NanoSPR modified biosensing chip
Example 4
The embodiment provides a modification method of a Protein L modified NanoSPR biological sensing chip, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding 50 mu L of 1mM tannic acid aqueous solution on the surface of a chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano-gold chip by double distilled water;
S3, adding 50 mu L of Protein L aqueous solution of 10 mu g/mL on the surface of the chip modified with the nano gold, incubating for 1.5h at 37 ℃, recovering antibody Protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
S5, adding 150 mu L of protective solution to the surface of the closed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the Protein L modified NanoSPR biological sensing chip.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the upper diameter of each nano cup is 200nm, the bottom diameter of each nano cup is 150nm, the height of each nano cup is 500nm, the interval period of each nano cup array is 400nm, the surface of each nano cup array is covered with a titanium film with the thickness of 9nm, the surface of each titanium film is covered with a silver film with the thickness of 40nm, and the surface of each silver film is covered with a gold film with the thickness of 5 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protective solution is prepared by dissolving 50g of sucrose in 1L of PBST solution.
The Protein L modified NanoSPR biological sensing chip provided by the embodiment is assembled with a 96-micropore plate to form a 96-micropore chip plate for detecting Fc fusion proteins, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The measurement results are shown in Table 4, and it can be seen that the relative deviation of the measurement results is not more than 10%.
Table 4 Protein L detection results of Fc fusion protein detection by NanoSPR biological sensor chip
Example 5
The embodiment provides a modification method of a streptavidin modified NanoSPR biological sensing chip, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding 50 mu L of 1mM tannic acid aqueous solution on the surface of a chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano-gold chip by double distilled water;
S3, adding 50 mu L of 10 mu g/mL streptavidin aqueous solution on the surface of the chip modified with the nano gold, incubating for 1.5h at 37 ℃, recovering antibody protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
S5, adding 150 mu L of protective solution on the surface of the closed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the streptavidin-modified NanoSPR biological sensing chip.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the upper diameter of each nano cup is 200nm, the bottom diameter of each nano cup is 150nm, the height of each nano cup is 500nm, the interval period of each nano cup array is 400nm, the surface of each nano cup array is covered with a titanium film with the thickness of 9nm, the surface of each titanium film is covered with a silver film with the thickness of 40nm, and the surface of each silver film is covered with a gold film with the thickness of 5 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protective solution is prepared by dissolving 50g of sucrose in 1L of PBST solution.
The NanoSPR biological sensing chip modified by streptavidin provided by the embodiment is assembled with a 96-micropore plate to prepare the 96-micropore chip plate for detecting the biotinylated antibody, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The measurement results are shown in Table 5, and it can be seen that the relative deviation of the measurement results is not more than 10%.
Table 5 detection results of streptavidin-modified NanoSPR biological sensor chip for detecting biotinylated antibody
Example 6
The embodiment provides a modification method for a biological sensing chip modified NanoSPR by an anti-Flag tag antibody, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding 50 mu L of 1mM L-cysteine aqueous solution to the surface of a chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano gold chip by double distilled water;
S3, adding 50 mu L of 10 mu g/mL of anti-Flag tag antibody aqueous solution on the surface of the chip modified with the nano gold, incubating for 1.5h at 37 ℃, recovering antibody protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
s5, adding 150 mu L of protective solution on the surface of the closed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the NanoSPR biological sensing chip modified by the anti-Flag tag antibody.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the upper diameter of each nano cup is 200nm, the bottom diameter of each nano cup is 150nm, the height of each nano cup is 500nm, the interval period of each nano cup array is 400nm, the surface of each nano cup array is covered with a titanium film with the thickness of 9nm, the surface of each titanium film is covered with a silver film with the thickness of 40nm, and the surface of each silver film is covered with a gold film with the thickness of 5 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protective solution is prepared by dissolving 50g of sucrose in 1L of PBST solution.
The NanoSPR biological sensing chip modified by the anti-Flag tag antibody provided by the embodiment is assembled with a 96-micropore plate to form the 96-micropore chip plate for detecting Flag tag fusion proteins, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The measurement results are shown in Table 6, and it can be seen that the relative deviation of the measurement results is not more than 10%.
TABLE 6 detection results of Flag tag fusion proteins by anti-Flag tag antibody-modified NanoSPR biological sensor chip
Example 7
The embodiment provides a modification method of an anti-His tag antibody modified NanoSPR biological sensing chip, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
s2, dropwise adding 50 mu L of 1mM ascorbic acid aqueous solution on the surface of a chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano-gold chip by double distilled water;
S3, adding 50 mu L of 10 mu g/mL of anti-His tag antibody aqueous solution on the surface of the chip modified with the nano gold, incubating for 1.5h at 37 ℃, recovering antibody protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
S5, adding 150 mu L of protective solution to the surface of the sealed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the anti-His tag antibody modified NanoSPR biological sensing chip.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the upper diameter of each nano cup is 200nm, the bottom diameter of each nano cup is 150nm, the height of each nano cup is 500nm, the interval period of each nano cup array is 400nm, the surface of each nano cup array is covered with a titanium film with the thickness of 9nm, the surface of each titanium film is covered with a silver film with the thickness of 40nm, and the surface of each silver film is covered with a gold film with the thickness of 5 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protective solution is prepared by dissolving 50g of sucrose in 1L of PBST solution.
The NanoSPR biological sensing chip modified by the His tag antibody provided by the embodiment is assembled with a 96-micropore plate to prepare a 96-micropore chip plate for detecting His tag fusion proteins, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The measurement results are shown in Table 7, and it can be seen that the relative deviation of the measurement results is not more than 10%.
Table 7 detection results of His tag fusion proteins by anti-His tag antibody modified NanoSPR biological sensor chip
Example 8
The embodiment provides a modification method of an anti-Fc tag antibody modified NanoSPR biological sensing chip, which comprises the following steps:
s1, cleaning the surface of a chip substrate by double distilled water;
s2, dropwise adding 50 mu L of 1mM ascorbic acid aqueous solution on the surface of a chip substrate, adding 50 mu L of 1mM chloroauric acid aqueous solution, uniformly mixing, incubating at 25 ℃ for 60min, removing the solvent after incubation, and washing the modified nano-gold chip by double distilled water;
s3, adding 50 mu L of anti-Fc tag antibody water solution with the concentration of 10 mu g/mL on the surface of the chip modified with the nano gold, incubating for 1.5h at the temperature of 37 ℃, recovering antibody protein solution after incubation, and cleaning the chip coated with the antibody by using PBST solution;
S4, adding 150 mu L of sealing liquid on the surface of the chip coated with the antibody, incubating for 1h at 37 ℃, removing the redundant sealing liquid after the incubation is finished, and cleaning the sealed chip by using a PBST solution;
S5, adding 150 mu L of protective solution on the surface of the closed chip, incubating for 5min at 37 ℃, removing the redundant protective solution after the incubation is finished, and drying to obtain the NanoSPR biological sensing chip modified by the anti-Fc tag antibody.
The chip substrate comprises a PET thin plate substrate, wherein the surface of the PET thin plate substrate is stamped with a nano cup array, the upper diameter of each nano cup is 200nm, the bottom diameter of each nano cup is 150nm, the height of each nano cup is 500nm, the interval period of each nano cup array is 400nm, the surface of each nano cup array is covered with a titanium film with the thickness of 9nm, the surface of each titanium film is covered with a silver film with the thickness of 40nm, and the surface of each silver film is covered with a gold film with the thickness of 5 nm.
The PBST solution is prepared from the following raw materials of 29g of disodium hydrogen phosphate dodecahydrate, 2.72g of sodium dihydrogen phosphate, 2g of potassium chloride, 20g of sodium chloride, 5mL of Tween-20, 300 mL of preservative Proclin and 10L of double distilled water.
The sealing liquid is prepared by dissolving 10g of bovine serum albumin in 1L of CBS solution, wherein the CBS solution is prepared from 15.9g of potassium carbonate, 29.3g of sodium bicarbonate, 300 mL of preservative Proclin and 10L of purified water.
The protective solution is prepared by dissolving 50g of sucrose in 1L of PBST solution.
The NanoSPR biological sensing chip modified by the anti-Fc tag antibody provided by the embodiment is assembled with a 96-micropore plate to prepare a 96-micropore chip plate for detecting the Fc fusion protein, and the detection method comprises the following steps:
p1, respectively adding 50 mu L of washing liquid into each hole of a 96-micropore chip plate by using a liquid transfer device, putting the mixture into an enzyme labeling instrument, recording an initial value, and discarding liquid in the hole;
P2, respectively adding 50 mu L of standard substance or diluted sample into each hole of the microporous chip plate, vibrating the plate at a rotating speed of 300rpm for reacting for 5min, reading data, and discarding sample solution;
P3, respectively adding 100 mu L of washing liquid into each hole to wash the plate for 2 times, beating the plate, and spin-drying;
p4. adding 50 μl of sample diluent into each well, placing into an enzyme-labeled instrument, and recording end point value;
P5., data processing, drawing a standard curve, and reading the concentration of the sample to be measured according to the standard curve.
The measurement results are shown in Table 8, and it can be seen that the relative deviation of the measurement results is not more than 8%.
Table 8 detection results of Fc fusion protein detected by anti-Fc tag antibody modified NanoSPR biological sensor chip
The foregoing is merely specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are intended to be covered by the scope of the present invention, which is defined by the claims.
Claims (9)
1. A method for modifying NanoSPR a biosensing chip, comprising the steps of:
s1, cleaning the surface of a chip substrate by double distilled water;
S2, dropwise adding a reducing agent solution on the surface of the chip substrate, adding chloroauric acid aqueous solution, uniformly mixing, incubating at 25-45 ℃ for 5-60 min, removing the solvent after incubation, and cleaning the modified nano gold chip by double distilled water;
s3, adding an antibody protein solution to the surface of the chip modified with the nano gold, incubating for 1.5-24 hours at 4-37 ℃, recovering the antibody protein solution after incubation, and cleaning the chip coated with the antibody by using a PBST solution;
s4, adding a sealing liquid on the surface of the chip coated with the antibody, incubating for 1-4 hours at 25-37 ℃, removing the redundant sealing liquid after incubation, and cleaning the sealed chip by using a PBST solution;
S5, adding a protective solution to the surface of the closed chip, incubating for 5-60 min at 25-37 ℃, removing the redundant protective solution after incubation, and drying to obtain NanoSPR biological sensing chip;
The reducer solution is one or more aqueous solutions 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 tag antibody;
the PBST solution is prepared from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, tween-20, a preservative Proclin300 and double distilled water;
the sealing liquid is prepared by dissolving bovine serum albumin in a CBS solution, wherein the CBS solution is prepared by potassium carbonate, sodium bicarbonate, a preservative Proclin300 and purified water;
the protective solution is prepared by dissolving saccharides in PBST solution;
in the step S1, the chip substrate includes a PET sheet substrate, a nano cup array is stamped on the surface of the PET sheet substrate, a layer of titanium film is covered on the surface of the nano cup array, a layer of silver film is covered on the surface of the titanium film, and a layer of gold film is covered on the surface of the silver film.
2. The method for modifying NanoSPR biological sensing chips according to claim 1, wherein the upper diameter of the nano-cup array nano-cup is 50-2000nm, the bottom diameter is 50-2000nm, the height is 100-800 nm, the distance is 100-4000 nm, the thickness of the titanium film is 2-30 nm, the thickness of the silver film is 5-100 nm, and the thickness of the gold film is 2-100 nm.
3. The method for modifying a NanoSPR biosensor chip according to claim 1, wherein in the 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 the chloroauric acid is 0.1-1:1.
4. The method for modifying a NanoSPR-biosensing chip according to claim 1, wherein in step S3, the concentration of the antibody protein solution is 0.5-100 μg/mL.
5. The NanoSPR biological sensing chip modification method according to claim 1 is characterized in that the PBST solution is prepared from 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, 300-100 mL of preservative Proclin and 10L of double distilled water.
6. The method for modifying NanoSPR biological sensing chip according to claim 1, wherein in the step S4, the sealing liquid is prepared by dissolving 10g bovine serum albumin in 1L of CBS solution, and the CBS solution is prepared from 15.9-318 g potassium carbonate, 29.3-586 g sodium bicarbonate, 300-100 mL preservative Proclin and 10L purified water.
7. The method for modifying NanoSPR biological sensor chip according to claim 1, wherein in step S5, the protective solution is prepared by dissolving 10-200 g of saccharide in 1L of PBST solution, and the saccharide is one of dextran, glucose, sucrose and trehalose.
8. A NanoSPR biosensing chip, characterized in that it is modified by the modification method of NanoSPR biosensing chip according to any one of claims 1-7.
9. Use of NanoSPR of the biosensing chip of claim 8 in rapid quantitative detection of antibodies, fc fusion proteins or tagged proteins.
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