CN111781370A - Method for measuring molecular binding capacity by utilizing enzyme-labeling instrument and nano-gold particle-enhanced SPR sensor - Google Patents
Method for measuring molecular binding capacity by utilizing enzyme-labeling instrument and nano-gold particle-enhanced SPR sensor Download PDFInfo
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Abstract
A method for determining molecular binding capacity using a microplate reader and a Surface Plasmon Resonance (SPR) sensor enhanced with gold nanoparticles, comprising: preparing a bottomless pore plate and a specificity detection chip; preparing nano gold particles for amplifying detection signals; covering a specific detection chip with a sample to be detected to capture an antibody; preparing a standard sample of the concentration of a sample to be detected; labeling the gold nanoparticles with a to-be-detected sample labeled antibody, wherein the to-be-detected sample capture antibody and the labeled antibody are paired antibodies; adding a standard sample of the concentration of the sample to be detected into the marked nano gold particles to prepare a mixed solution; detecting the optical density of the specific detection chip at a specific wavelength before adding the mixed solution into the well plate; adding the mixed solution into the pore plate to complete the capture of the complex of the sample to be detected and the marked nano-gold particles; and detecting the optical density of the specificity detection chip under the specific wavelength after the mixed solution is added, and comparing the difference value between the optical density and the optical density obtained in the seventh step to realize the quantitative detection of the sample to be detected.
Description
Technical Field
The embodiment of the invention relates to the technical field of detection, in particular to a method for measuring molecular binding capacity by using a microplate reader and a nano-gold particle enhanced SPR sensor by taking C-type reactive protein (CRP) as an example.
Background
Measurement of molecular interaction kinetics is becoming increasingly important in drug discovery, genetic screening and clinical diagnostics, as dynamic binding information can improve understanding of disease and can provide new ideas for treatment. Surface Plasmon Resonance (SPR) sensors, such as the commercial Biacore SPR biosensor system, can monitor kinetic biomolecular interactions in real time, without labeling, and are not affected by a large background. Since the commercial Biacore SPR biosensor system belongs to the conventional SPR apparatus, it requires a specially-made apparatus, a specially-assigned person for its operation, so that a large amount of money is required to purchase or use the biosensor system.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for measuring the molecular binding capacity by using a microplate reader and a nano-gold particle enhanced SPR sensor by taking CRP as an example to solve the technical problem of high detection cost of a commercial Biacore SPR biosensor system aiming at the defects in the prior art.
According to the invention, the method for determining the molecular binding capacity by using the microplate reader and the SPR sensor enhanced by the nano-gold particles comprises the following steps:
the first step is as follows: preparing a bottomless pore plate and a specificity detection chip;
the second step is as follows: preparing nano gold particles for amplifying detection signals;
the third step: covering a specific detection chip with a sample to be detected to capture an antibody in a bottomless pore plate;
the fourth step: preparing a standard sample of the concentration of a sample to be detected;
the fifth step: labeling the gold nanoparticles with a to-be-detected sample labeled antibody, wherein the to-be-detected sample capture antibody and the labeled antibody are paired antibodies;
a sixth step: adding a standard sample of the concentration of the sample to be detected into the marked nano gold particles to prepare a mixed solution;
a seventh step of: detecting the optical density of the specific detection chip at a specific wavelength before adding the mixed solution into the well plate;
an eighth step: adding the mixed solution into the pore plate to complete the capture of the complex of the sample to be detected and the marked nano-gold particles;
a ninth step: and detecting the optical density of the specificity detection chip under the specific wavelength after the mixed solution is added, and comparing the difference value between the optical density and the optical density obtained in the seventh step to realize the quantitative detection of the sample to be detected.
Preferably, the difference in OD measured before and after addition of the mixed solution is used to characterize the amount of conjugation between CRP and CRP antibody.
Preferably, the sample to be tested is a type C reactive protein.
Preferably, the specific wavelength is 580nm wavelength.
Preferably, the specificity detection chip is manufactured by a replication forming process, wherein a conical nano-pillar pattern is manufactured on a quartz substrate by utilizing laser interference lithography, then an ultraviolet light curing polymer is uniformly spread on a mold, and the top of the ultraviolet light curing polymer is supported by polyethylene terephthalate; carrying out curing treatment by using ultraviolet light, and then stripping the polyethylene terephthalate substrate and the periodic nano-pore channel pattern from the mold; forming a titanium adhesion layer and a gold layer through electron beam evaporation deposition to realize the formation of a plasmon device; and then, sputtering and depositing a titanium dioxide resonant cavity layer by utilizing radio frequency plasma, and then forming a nano resonant cavity by electron beam evaporation of the titanium adhesion layer and the top gold layer.
Preferably, preparing the gold nanoparticles for amplifying the detection signal includes: and adding a chloroauric acid solution into deionized water, heating to boil, then adding a trisodium citrate solution, continuing heating, stirring by using a magnetic stirrer, changing the color of the solution from black to orange red, heating for a preset time, stopping heating, standing and cooling to room temperature to obtain an Au nanoparticle dispersed solution, namely the final gold nanoparticles.
Preferably, the particle size of the nano gold particles is 13-15 nm.
Preferably, covering the specific detection chip with the sample capture antibody to be tested comprises: placing the specificity detection chip in 11-mercaptoundecanoic acid solution at room temperature, cleaning with ethanol, and drying; adding 1:1 mixed solution of 1-ethyl-3-3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide for incubation; immediately putting the monoclonal antibody CRP capture antibody into a refrigerator for incubation after incubation is finished, and drying the monoclonal antibody CRP capture antibody in the refrigerator after cleaning; adding bovine serum albumin blocking solution for incubation at room temperature, removing the bovine serum albumin blocking solution, adding ethanolamine solution for incubation at room temperature to cover unreacted NHS ester; and cleaning with deionized water, blow-drying and placing in a dry environment.
Preferably, labeling the gold nanoparticles with the sample-labeled antibody to be tested comprises: by K2CO3Adjusting the pH of the nano-gold particle solution to the protein isoelectric point of the CRP labeled antibody by the solution, adding the monoclonal antibody CRP labeled antibody with the final concentration of 6 mug/ml, uniformly mixing, incubating at room temperature, adding bovine serum albumin blocking solution with the final concentration of 1%, and incubating at room temperature; the supernatant was removed by centrifugation using a centrifuge and reconstituted to 1/4 volumes of the original solution.
Preferably, the seventh step includes: taking out the chip pore plate covered with the CRP capture antibody, adding deionized water into the hole to be tested, and detecting the OD value of the hole to be tested under the wavelength of 580nm by using an enzyme-labeling instrument; and after the sample mixed solution is added into the hole and the reaction is finished, absorbing the sample mixed solution, cleaning the test hole by using deionized water, adding the deionized water into the test hole, and detecting the OD value of the test hole at the wavelength of 580 nm.
The invention has at least the following advantages: the quantitative determination between biomolecules is realized by using a common enzyme-labeling instrument and the nano-gold particles, so that the system requirements of special instruments can be reduced to the maximum extent; the invention has the advantages of good selectivity, high sensitivity and the like, and because the gold nanoparticles marked by the specific antibody can be specifically combined with CRP protein, the detection signal can be further amplified without additional detection marking substances, the detection process is simplified, and the detection cost is reduced; in particular, the chip has extremely high sensitivity to changes in the surface refractive index by transmitting light intensity changes in the 580nm band.
Drawings
A more complete understanding of the present invention, and the attendant advantages and features thereof, will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings wherein:
FIG. 1 schematically shows a flow chart of a method for determining the amount of molecular binding using a microplate reader and a gold nanoparticle-enhanced SPR sensor according to a preferred embodiment of the present invention.
FIG. 2 schematically shows a graph of the OD difference results for the detection of CRP at different concentrations.
FIG. 3 schematically shows a graph of the rate of change of light intensity results for the detection of CRP at different concentrations.
It is to be noted, however, that the appended drawings illustrate rather than limit the invention. It is noted that the drawings representing structures may not be drawn to scale. Also, in the drawings, the same or similar elements are denoted by the same or similar reference numerals.
Detailed Description
In order that the present disclosure may be more clearly and readily understood, reference will now be made in detail to the present disclosure as illustrated in the accompanying drawings.
The embodiment of the invention provides a method for measuring intermolecular kinetic parameters by using a CRP (CRP) as an example and using a microplate reader.
CPR is an exemplary protein because CRP is an acute phase protein synthesized by hepatocytes when the body is subjected to inflammatory stimuli such as microbial invasion or tissue damage, and CRP is an early blood signal of infection or inflammation and can be used as a biomarker for various diseases. Normal serum CRP levels in healthy subjects were below 5. mu.g/ml, with concentrations significantly increasing after several hours of appearance of foreign antigen, and then even up to 300. mu.g/ml 2 within 48 hours. The blood content of CRP can be detected to have positive effect on disease diagnosis.
Specifically, fig. 1 schematically shows a flowchart of a method for determining the amount of molecular binding using a microplate reader and gold nanoparticles according to a preferred embodiment of the present invention.
As shown in fig. 1, the method for measuring the amount of molecular binding using a microplate reader and a nano-gold particle-enhanced SPR sensor according to a preferred embodiment of the present invention includes:
first step S1: preparing a bottomless pore plate and a specificity detection chip;
for example, the bottomless well plate is a bottomless 96-well plate and can be prepared by printing.
Second step S2: preparing nano gold particles for amplifying detection signals; preferably, the particle size of the nano gold particles is 13-15 nm.
Third step S3: covering a specificity detection chip with a C-type reactive protein capture antibody in a bottomless pore plate;
fourth step S4: preparing a C-type reactive protein concentration standard sample;
fifth step S5: labeling the gold nanoparticles with a C-type reactive protein labeled antibody, wherein the C-type reactive protein capture antibody and the labeled antibody are paired antibodies;
sixth step S6: adding a C-type reactive protein concentration standard sample into the marked gold nanoparticles to prepare a mixed solution;
seventh step S7: detecting the OD (optical density) of the specific detection chip at a specific wavelength (e.g., 580nm wavelength) before adding the mixed solution to the well plate;
eighth step S8: adding the mixed solution into the pore plate to complete the capture of the C-type reactive protein and the labeled nano-gold particle compound;
ninth step S9: and detecting the OD (optical density) of the specific detection chip at a specific wavelength (for example, 580nm wavelength) after the mixed solution is added, and comparing the difference between the OD and the OD obtained in the seventh step to realize the quantitative detection of the C-type reactive protein. Specifically, the difference between OD of the test well before and after loading can be used to characterize the conjugation amount between CRP and CRP antibody, thereby realizing quantitative detection of CRP.
Specific examples of preferred embodiments of the invention are described below.
As a specific example, for example, the specific detection chip employs a replica molding process. Specifically, the method for preparing the specificity detection chip is that a conical nano-pillar pattern is made on a quartz substrate by using laser interference lithography, and then an ultraviolet light curing polymer (for example, NOA-61) is uniformly spread on a mold, and the top of the ultraviolet light curing polymer is supported by polyethylene terephthalate; curing with ultraviolet light, and carefully stripping the polyethylene terephthalate substrate and the periodic nanopore patterns from the mold; and forming a titanium adhesion layer and a gold layer through electron beam evaporation deposition to realize the formation of a plasmon device, then depositing a titanium dioxide resonant cavity layer through radio frequency plasma sputtering, and then forming a nano resonant cavity through the electron beam evaporation of the titanium adhesion layer and the top gold layer. Wherein, the thicknesses of the titanium adhesion layer, the gold layer and the titanium dioxide resonant cavity layer are respectively 16nm, 220nm and 180nm, and the curing time is 3 minutes.
Preferably, the method for preparing the gold nanoparticles comprises the following steps of adding 1mL of 1% chloroauric acid solution into 100mL of deionized water, heating to boil, adding 4mL of 1% trisodium citrate solution, continuing heating, stirring by using a magnetic stirrer, changing the color of the solution from black to orange-red, and stopping heating after the solution is constant in color and is heated for 15 minutes. Standing, and cooling to room temperature to obtain a clear and transparent Au nanoparticle dispersion solution, namely the final gold nanoparticles.
Preferably, covering the specific detection chip with the C-type reactive protein capture antibody comprises: placing the specificity detection chip in a 1mM 11-mercaptoundecanoic acid solution at room temperature for 12 hours, cleaning with 70% ethanol, and drying with nitrogen; adding a 1:1 mixed solution of 400 x 10-3M 1-ethyl-3-3-dimethyl aminopropyl carbodiimide and 100 x 10-3M N-hydroxysuccinimide to incubate for 30 minutes; immediately placing a monoclonal anti-CRP capture antibody of 50 mu g/ml into a refrigerator of 4 ℃ after the incubation is finished, incubating for 5 hours, washing twice with PBS, and drying with nitrogen; adding 30 mu g/ml bovine serum albumin blocking solution, incubating for 30 minutes at room temperature, removing the bovine serum albumin blocking solution, adding 10% ethanolamine solution, and incubating for 30 minutes at room temperature to cover unreacted NHS ester; after being washed twice by deionized water, the mixture is placed in a dry environment after being dried by nitrogen.
Further, preferably, the CRP concentration standard sample is prepared by diluting CRP standard sample solutions (10 ng/ml-10 μ g/ml) prepared into different concentration gradients by using 250 μ g/ml CRP protein solution as a mother solution; the diluted base solution is PBS buffer solution, and is prepared for use.
Preferably, the method for labeling the gold nanoparticles with the CRP-labeled antibody is as follows, using, for example, 0.1M K2CO3The pH of the nano-gold particle solution is adjusted to the protein isoelectric point of the CRP labeled antibody by the solution, the monoclonal antibody CRP labeled antibody with the final concentration of 6 mug/ml is added, the mixture is evenly mixed and incubated for 30 minutes at room temperature, then bovine serum albumin blocking solution with the final concentration of 1% is added, and the incubation is carried out for 10 minutes at room temperature. Setting the rotation speed of the centrifuge at 9000rpm/min, centrifuging for 25 minutes, removing supernatant, and redissolving to 1/4 of the volume of the original solution.
Preferably, the method for preparing the CRP concentration standard sample comprises the steps of taking a CRP protein solution of 250 mug/ml as a mother solution, and diluting CRP standard sample solutions (10 ng/ml-10 mug/ml) prepared into different concentration gradients; the dilution base solution is PBS buffer solution. For example, CRP standard sample solutions prepared as 6 concentration gradients may be diluted with 250 μ g/ml CRP protein solution, 10ng/ml, 50ng/ml, 500ng/ml, 1 μ g/ml, 5 μ g/ml, and 10 μ g/ml, respectively; the dilution base solution is PBS buffer solution.
For the measurement of the standard sample signal for measuring the CRP concentration, for example, the CRP standard sample concentration is measured by using a plasma photon resonance technology and a photosensitive sensing method; adding the CRP standard sample into the complex solution of the gold nanoparticles marked by the CRP marked antibody, uniformly mixing, adding the mixture into a chip pore plate covered with the CRP capture antibody, placing the pore plate into an enzyme-linked immunosorbent assay (ELISA) instrument for oscillation reaction for 30 minutes, recording the OD difference before and after the reaction, further converting the OD difference into a light intensity change value, and calculating a standard curve according to the obtained data.
The OD value of the chip well at a wavelength of 580nm before and after loading is measured, for example, as follows: before sample adding, the chip pore plate covered with the CRP capture antibody is taken out, 100 mu l of deionized water is added into the hole to be tested, and the OD value of the hole to be tested under the wavelength of 580nm is detected by a microplate reader. And after the sample mixed solution is added into the hole and the reaction is finished, absorbing the sample mixed solution, cleaning the test hole for 2 times by using deionized water, adding 100 mu l of deionized water into the test hole, and detecting the OD value of the test hole at the wavelength of 580 nm.
The feasibility of the method is verified through data detection, CRP with different concentrations is detected, an OD difference result graph is shown in figure 3, finally, the light intensity change rate is obtained through an algorithm and is in direct proportion to the concentration of CRP, and the feasibility of the method is proved.
The above-described embodiments of the present invention have at least the following advantages: the quantitative determination between biomolecules is realized by using a common microplate reader and a nano-gold particle enhanced SPR sensor, so that the system requirements of special instruments can be reduced to the maximum extent; the invention has the advantages of good selectivity, high sensitivity and the like, and because the gold nanoparticles marked by the specific antibody can be specifically combined with CRP protein, the detection signal can be further amplified without additional detection marking substances, the detection process is simplified, and the detection cost is reduced; in particular, the chip has extremely high sensitivity to changes in the surface refractive index by transmitting light intensity changes in the 580nm band.
It should be noted that the terms "first", "second", "third", and the like in the description are used for distinguishing various components, elements, steps, and the like in the description, and are not used for indicating a logical relationship or a sequential relationship between the various components, elements, steps, and the like, unless otherwise specified.
It is to be understood that while the present invention has been described in conjunction with the preferred embodiments thereof, it is not intended to limit the invention to those embodiments. It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (10)
1. A method for measuring molecular binding capacity by utilizing a microplate reader and a nano-gold particle-enhanced SPR sensor is characterized by comprising the following steps:
the first step is as follows: preparing a bottomless pore plate and a specificity detection chip;
the second step is as follows: preparing nano gold particles for amplifying detection signals;
the third step: covering a specific detection chip with a sample to be detected to capture an antibody in a bottomless pore plate;
the fourth step: preparing a standard sample of the concentration of a sample to be detected;
the fifth step: labeling the gold nanoparticles with a to-be-detected sample labeled antibody, wherein the to-be-detected sample capture antibody and the labeled antibody are paired antibodies;
a sixth step: adding a standard sample of the concentration of the sample to be detected into the marked nano gold particles to prepare a mixed solution;
a seventh step of: detecting the optical density of the specific detection chip at a specific wavelength before adding the mixed solution into the well plate;
an eighth step: adding the mixed solution into the pore plate to complete the capture of the complex of the sample to be detected and the marked nano-gold particles;
a ninth step: and detecting the optical density of the specificity detection chip under the specific wavelength after the mixed solution is added, and comparing the difference value between the optical density and the optical density obtained in the seventh step to realize the quantitative detection of the sample to be detected.
2. The method for measuring the amount of molecular binding according to claim 1, wherein the OD difference between the measurement before and after the addition of the mixed solution is used to characterize the amount of binding between the sample to be measured and the antibody of the sample to be measured.
3. The method for measuring the amount of molecular binding using the microplate reader and the SPR sensor enhanced with gold nanoparticles according to claim 1 or 2, wherein the sample to be measured is C-type reactive protein.
4. The method for measuring the amount of molecular binding using a microplate reader and a gold nanoparticle-enhanced SPR sensor according to claim 1 or 2, wherein the specific wavelength is 580 nm.
5. The method for measuring molecular binding amount using microplate reader and SPR sensor enhanced by gold nanoparticles as claimed in claim 1 or 2, wherein the specificity detecting chip is fabricated by a replica molding process in which a tapered nano-pillar pattern is fabricated on a quartz substrate by laser interference lithography, and then an ultraviolet light-curable polymer is uniformly spread on a mold with the top supported by polyethylene terephthalate; carrying out curing treatment by using ultraviolet light, and then stripping the polyethylene terephthalate substrate and the periodic nano-pore channel pattern from the mold; forming a titanium adhesion layer and a gold layer through electron beam evaporation deposition to realize the formation of a plasmon device; and then, sputtering and depositing a titanium dioxide resonant cavity layer by utilizing radio frequency plasma, and then forming a nano resonant cavity by electron beam evaporation of the titanium adhesion layer and the top gold layer.
6. The method for measuring molecular binding amount using microplate reader and the SPR sensor enhanced with gold nanoparticles as claimed in claim 1 or 2, wherein the preparing of the gold nanoparticles for amplifying the detection signal comprises: and adding a chloroauric acid solution into deionized water, heating to boil, then adding a trisodium citrate solution, continuing heating, stirring by using a magnetic stirrer, changing the color of the solution from black to orange red, heating for a preset time, stopping heating, standing and cooling to room temperature to obtain an Au nanoparticle dispersed solution, namely the final gold nanoparticles.
7. The method for measuring molecular binding capacity using the microplate reader and the SPR sensor enhanced with gold nanoparticles as claimed in claim 1 or 2, wherein the particle size of the gold nanoparticles is 10 to 100 nm.
8. The method for measuring the molecular binding capacity by using the microplate reader and the SPR sensor enhanced by gold nanoparticles as claimed in claim 1 or 2, wherein the covering of the specific detection chip with the capture antibody of the sample to be measured comprises: placing the specificity detection chip in 11-mercaptoundecanoic acid solution at room temperature, cleaning with ethanol, and drying; adding 1:1 mixed solution of 1-ethyl-3-3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide for incubation; immediately putting the monoclonal antibody CRP capture antibody into a refrigerator for incubation after incubation is finished, and drying the monoclonal antibody CRP capture antibody in the refrigerator after cleaning; adding bovine serum albumin blocking solution for incubation at room temperature, removing the bovine serum albumin blocking solution, adding ethanolamine solution for incubation at room temperature to cover unreacted NHS ester; and cleaning with deionized water, blow-drying and placing in a dry environment.
9. The method for measuring molecular binding capacity by using the microplate reader and the SPR sensor enhanced by gold nanoparticles as claimed in claim 1 or 2, wherein the labeling of the gold nanoparticles with the sample-to-be-measured labeled antibody comprises: by K2CO3Adjusting the pH of the nano-gold particle solution to the protein isoelectric point of the CRP labeled antibody by the solution, adding the monoclonal antibody CRP labeled antibody with the final concentration of 6 mug/ml, uniformly mixing, incubating at room temperature, adding bovine serum albumin blocking solution with the final concentration of 1%, and incubating at room temperature; the supernatant was removed by centrifugation using a centrifuge and reconstituted to 1/4 volumes of the original solution.
10. The method for measuring the amount of molecular binding using the microplate reader and the SPR sensor enhanced with gold nanoparticles as claimed in claim 1 or 2, wherein the seventh step comprises: taking out the chip pore plate covered with the CRP capture antibody, adding deionized water into the hole to be tested, and detecting the OD value of the hole to be tested under the wavelength of 580nm by using an enzyme-labeling instrument; and after the sample mixed solution is added into the hole and the reaction is finished, absorbing the sample mixed solution, cleaning the test hole by using deionized water, adding the deionized water into the test hole, and detecting the OD value of the test hole at the wavelength of 580 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010433687.3A CN111781370A (en) | 2020-05-20 | 2020-05-20 | Method for measuring molecular binding capacity by utilizing enzyme-labeling instrument and nano-gold particle-enhanced SPR sensor |
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