CN113109298B - Method for detecting three-dimensional structure state of sulfhydryl-containing protein - Google Patents
Method for detecting three-dimensional structure state of sulfhydryl-containing protein Download PDFInfo
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Abstract
The invention provides a method for detecting the three-dimensional structural state of protein containing sulfydryl, which realizes the application of Au @ Ag nano particles in the detection of the three-dimensional structural state of the protein containing sulfydryl, can detect the high sensitivity of the structural configuration change of the protein on the scale of a single nano particle, has the correlative relationship between the expansion degree of the protein and the red shift amount of the scattering spectrum of an Au @ Ag nano cube-protein assembly containing sulfydryl, realizes the rapid and high-sensitivity detection of the three-dimensional structural state of the protein, solves the technical problem that the three-dimensional folding state of the protein cannot be monitored in real time in the prior art, and has simple operation.
Description
Technical Field
The invention relates to the field of biosensing, in particular to a method for detecting the three-dimensional structure state of a protein containing sulfydryl based on the construction of a protein plasma assembly by Au @ Ag nano cubic particles.
Background
The biological activity of a protein molecule is determined by a specific amino acid sequence and a specific amino acid space three-dimensional structure thereof, if the three-dimensional structure is changed, the whole protein molecule is changed, even the protein is possibly inactivated, the biological activity function of the protein is closely related to the three-dimensional structure, and therefore, the research of the biological activity of the protein by detecting the structural change of the protein is a very important research means.
Plasmons are an emerging sub-field of nanophotonics, attracting increasing attention because of their potential application in nanoscale control and manipulation of light; surface plasmon resonance is an interaction between the surface of a noble metal particle and incident photoelectrons confined on the surface of the nanoparticle, and the occurrence of plasmon resonance allows the metal nanoparticle to have excellent optical and physical properties including strong absorption and scattering spectra, light stability, and the like. The advent of dark field microscopy has facilitated the study of the effects of nanoparticle plasmons, particularly noble metal size, shape, composition, and local environment, which has further facilitated their use in biomarkers and detection, while enabling their use as sensitive sensors, functional nanoprobes, bioassays, and drug screening based on nanoparticle plasmon resonance properties. And judging the change of the environment of the particles according to the change of the SPR scattering peak signals of the metal nanoparticles. Compared with single metal nanoparticles, the noble metal nano composite material can better control the appearance and uniformity of metal nanoparticles with larger size, and is wider in application than the single metal nanoparticles; most importantly, the noble metal nanoparticles with the composite structure have higher sensitivity, and particularly have great advantages in biological detection and chemical detection.
In vitro protein unfolding and refolding experiments can be achieved by using urea, guanidine hydrochloride (guanidinium salt), heat or acid and base addition, etc., where urea and guanidine hydrochloride denaturation of proteins is a reversible process, and high temperature, etc., is an irreversible process. The protein denaturation is carried out by a denaturant, then the denaturant is removed to refold the protein, and the protein denaturation process is monitored by using a spectrum, which is the prior art means, but the three-dimensional structure state of the protein is detected only by the prior spectrum technology means, so that the problems of low detection accuracy, insensitive detection and the like exist, and real-time monitoring cannot be realized.
Disclosure of Invention
The invention provides a method for detecting the three-dimensional structural state of protein containing sulfydryl, realizes the application of Au @ Ag nano particles in the detection of the three-dimensional structural state of the protein containing sulfydryl, provides a method for quickly and sensitively detecting the three-dimensional structural state of the protein, solves the technical problem that the three-dimensional folding state of the protein cannot be monitored in real time in the prior art, and is simple to operate.
In a first aspect, the present invention provides a method for detecting the three-dimensional structure state of a thiol-group-containing protein, the method comprising the steps of:
the method comprises the following steps: weighing a certain amount of a sample to be detected, wherein the sample to be detected is protein containing sulfydryl and in an unknown folding state, dissolving the sample to be detected in a phosphoric acid buffer solution, standing for a period of time for incubation, and preparing a protein solution to be detected;
step two: the LSPR biosensor is formed by adopting Au @ Ag nano particles to adsorb and fix on a transparent substrate sheet; diluting the protein solution to be detected obtained in the first step, dripping the diluted protein solution onto the LSPR biosensor, incubating for a period of time, washing and drying to form an Au @ Ag nanoparticle-protein assembly;
step three: and observing the red shift amount of the plasma scattering spectrum of the assembly under a dark field microscope, and directly judging the current folding state of the specific protein to be detected according to a corresponding relation model between the red shift amount of the scattering spectrum and the folding state of the specific protein.
In a second aspect, the present invention provides a method for modeling a correspondence between an amount of red shift of a scattered light spectrum and a folding state of a specific protein, the method comprising the steps of:
selecting high-activity protein in a natural state, preparing the protein into solution, and assembling the solution with Au @ Ag nano particles to form an assembly;
establishing a concentration gradient of a denaturant, and respectively dropwise adding the denaturants with different concentrations onto the Au @ Ag nano cubic particle-protein assembly for reaction to form proteins with different folding degrees; the reaction time is at least half an hour. The denaturant concentration gradient value n is n 1 、n 2 、n 3 The difference values between different gradients are the same, because the relation between the denaturant and the unfolding degree of the protein is a linear change trend, namely when the concentration of the denaturant is lower, the denaturation inducibility of the denaturant to the protein is not strong, and along with the increase of the concentration of the denaturant, the high-concentration denaturant enhances the solubilization of hydrophobic residues in the protein, destroys the hydrophobicity of the surface of the protein, causes the explosion of hydrophobic groups wrapped in the protein, and further gradually induces the protein to be denatured, the higher the concentration of the denaturant is, the higher the unfolding degree of the protein is, and the folding degree P can be marked by 100% -0%; or the folding degree P is divided into a natural folding state, an intermediate state and a complete deformation state, so that the corresponding relation between the corresponding concentration or concentration range of the denaturant and the protein folding degree is formed;
observing the red shift amount of the plasma scattering spectrum of the assembly under a dark field microscope, and carrying out correlation analysis on the red shift amount of the scattering spectrum by using the concentration of the denaturant to obtain a corresponding relation model of the specific concentration n of the denaturant and the red shift amount d of the scattering spectrum, wherein the denaturants with different concentrations correspond to different folding states of the protein, so that a corresponding relation model between the red shift amount d of the scattering spectrum and the folding degree P of the specific protein can be formed.
The method for establishing the model is suitable for all proteins containing the sulfhydryl group, and the model can be used for quickly detecting and judging the three-dimensional structure state of the proteins containing the sulfhydryl group with unknown activity after being established.
In a third aspect, the present invention provides a method for studying the effect of environmental conditions on the three-dimensional structural state of a thiol-group-containing protein, said method comprising the steps of:
selecting high-activity protein in a natural state to prepare a solution, and assembling the solution with Au @ Ag nano particles to form an assembly;
the assembly is placed under different environmental conditions, such as temperature, chemical solution, humidity, radiation and the like, for example, the temperature condition is adopted, and reversible continuous monitoring of the three-dimensional structure state of the protein can be realized within the temperature range of 30-80 ℃;
and observing the red shift amount of the plasma scattering spectrum of the assembly under a dark field microscope, and directly judging the current folding state of the protein according to the corresponding relation model, so that the direct corresponding relation between the corresponding environmental conditions and the folding state of the protein can be obtained.
Further preferably, the Au @ Ag nano particles are Au @ Ag nano cubic particles; compared with a simple spherical structure, the core-shell structure adopting Au @ Ag nano cubic particles has better LSPR performance.
Further, the preparation process of the Au @ Ag nano cubic particles comprises the following steps: preparing gold seeds by adopting a seed growth method, and dropwise adding silver nitrate to react to obtain Au @ Ag nano cubic particle solution; the seed growth method adopts the step-by-step growth from small-size gold spheres to large-size particles, so that the gold sphere nano-particles with uniform appearance and controllable size can be prepared.
Furthermore, the particle size of the Au @ Ag nano cubic particle is preferably about 55nm, the 55nm particle is selected because the LSPR scattering peak can be controlled in a visible light region, the color of the particle is blue-green under a dark field microscope, the color change span of the particle is long, the change is obvious, and the reaction process can be observed conveniently.
Further, the sample is dissolved in a phosphoric acid buffer solution, is kept stand and incubated for preferably 4-6 hours, and is incubated in a dark room for normal-temperature reaction; proteins purchased directly from the market are generally stored at low temperature, and therefore, proper physiological environment and time are required to be provided for incubation;
further, the volume of the silver nitrate solution is 500-550 mu L, and the concentration is 0.01M; 0.01M of 500-550 mu L of silver nitrate is dripped at a constant speed to stably synthesize Au @ Ag nano cubic particles, too much can cause too large increase of particle size, and too little can etch gold balls into other shapes;
further, the pH value of the phosphoric acid buffer solution is 7.0-7.4.
Further, the transparent substrate sheet is ITO glass, a quartz sheet, organic glass or a mica sheet.
The method of the invention can detect the principle of the protein unfolding and folding process:
the unique size, morphology, components and microenvironment-dependent optical properties of the noble metal nanoparticles enable the plasma to be widely applied to the chemical and biological sensing fields, and the prepared sensor is based on the Local Surface Plasmon Resonance (LSPR) characteristics of the noble metal nanoparticles (such as gold and silver nanoparticles) and takes the movement of the LSPR peak of the particles as a detection signal; the LSPR sensor may be used to measure intermolecular interactions at the surface of metal nanoparticles, and signals obtained from individual metal nanoparticles may provide more detailed information.
According to the invention, the protein containing sulfydryl is assembled on the Au @ Ag nano-particles through an Au-S bond or an Ag-S bond to form an Au @ Ag nano-particle-protein assembly, and the three-dimensional structure state of the protein can be judged through the plasma scattering spectrum red shift amount of the assembly; the judgment is certainly based on the established corresponding relation model between the three-dimensional structure state of the protein and the red shift quantity of the scattering spectrum, and the establishment of the relation model is based on a large number of sample experiments; the model establishment experiment is realized through the condition that the corresponding relation between the concentration of the denaturant and the three-dimensional structure is definite, and the unfolding and refolding processes of the protein can be realized through destroying the hydrophobicity of the surface of the protein or restoring the hydrophobicity of the surface, so that the three-dimensional structure of the protein is changed, the change of the three-dimensional structure of the protein causes the plasma scattering spectrum of the assembly to generate red shift, a one-to-one corresponding relation model of the two is formed, and the model can be directly used for judging the three-dimensional structure state of the protein with unknown activity; because most proteins contain sulfydryl, the method is suitable for most proteins, for example, common bovine serum albumin is the protein containing sulfydryl.
The plasma resonance technology is used for monitoring the three-dimensional structure state of the protein, and the plasma resonance enables the metal nanoparticles to have excellent optical and physical properties, so that the protein configuration change detection can be rapidly and highly sensitively realized, and the method has important significance for researching the three-dimensional structure of the protein.
As shown in figure 1, the scattering spectrum LSPR peak position when Au @ Ag nano particles are not assembled is A, the scattering spectrum LSPR peak position when the assembled protein is in a natural folding state is B, the denaturant with different concentrations can be used for controlling the denaturation degree of the protein, high-concentration guanidine hydrochloride solution is dropwise added to enable the protein to be converted into an intermediate state from the natural folding state, then the protein is in a completely unfolded state, and the scattering spectrum LSPR peak is moved from B to C; because the process of denaturing the protein by taking guanidine hydrochloride, uremia and the like as denaturants is a reversible process, the guanidine hydrochloride on the surface of the denatured protein can be diluted by washing, the concentration of the guanidine hydrochloride on the surface of the denatured protein is reduced, the denatured protein is restored to a natural folded state, and the LSPR peak of a scattering spectrum is restored from C to B'; therefore, under the environment conditions that the protein denaturation is caused but the process is reversible, besides the above denaturants such as guanidine hydrochloride, urea and the like, the continuous and real-time monitoring of the three-dimensional structure state of the protein containing the sulfhydryl group can be realized under the appropriate temperature conditions; the protein folding and unfolding method can also be used in the research field to research the protein folding and unfolding process, and has important significance for researching the biological activity of the protein.
Compared with the prior art, the method has the following technical effects:
according to the first aspect, the method for detecting the three-dimensional structure state of the protein containing the sulfydryl is a new method for detecting the three-dimensional structure of the protein, the protein can be quickly and stably fixed on the surface of Au @ Ag nano cubic particles by utilizing an Au-S bond or an Ag-S bond, pollution-free, quick, green and environment-friendly detection can be realized, and the detection process is simple and quick; compared with the traditional protein configuration change detection means, the method is more convenient and fast, solves the problem that the traditional spectrum detection means can not monitor in real time, and has important significance for researching the three-dimensional structure of the protein space and the biological activity thereof; for example, the activity of homologous proteins that can be synthesized artificially can be detected; provides a research method for treating 'conformation diseases' caused by protein misfolding or inactivation;
in a second aspect, the method for establishing the model of the correspondence between the red shift amount of the scattering spectrum and the folding state of the specific protein is applicable to all proteins containing sulfhydryl groups, and the model can be used for rapidly detecting and judging the three-dimensional structure state of the proteins containing sulfhydryl groups with unknown activity after being established.
In a third aspect, the method for researching the influence of environmental conditions on the three-dimensional structure state of the protein containing the sulfhydryl group can realize real-time cycle detection of protein configuration change on the scale of a single nanoparticle, and can be applied to research on the influence of different environmental conditions on the protein activity according to the relation model between the red shift quantity of the scattering spectrum and the folding state of the specific protein in the second aspect.
Drawings
FIG. 1 is a schematic diagram of the detection of the unfolding and folding process of proteins according to the present invention;
FIG. 2 is a TEM image of Au @ Ag nano cubic particles (about 55nm in particle size) according to example 1 of the present invention;
FIG. 3 is a fluorescence spectrum of BSA prepared in example 1 of the present invention;
FIG. 4 is a graph of the scattering spectra of assemblies formed with different concentrations of BSA found in examples 1-5 of the present invention;
FIG. 5 is a graph of the linear correlation between the scatter spectra of the assemblies synthesized in examples 1-5 of the present invention and the corresponding BSA concentrations;
FIG. 6 is a graph showing the variation of guanidine hydrochloride concentration and the scattering spectrum of the assembly in example 6 of the present invention;
FIG. 7 is a graph of guanidine hydrochloride concentration versus assembled body scattering spectrum variation and a fitting of the red shift amount thereof in example 6 of the present invention;
FIG. 8 is a scattering spectrum of the assembly in example 7 of the present invention;
FIG. 9 is a graph showing the wavelength change of scattering peaks of the assembly in example 7 of the present invention.
Detailed Description
The invention will be better understood from the following examples. It is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.
Taking bovine serum albumin as an example, the selected protein is bovine serum albumin containing free sulfhydryl; preparing 0-9M guanidine hydrochloride solution as a denaturant; dropwise adding guanidine hydrochloride solution to the Au @ Ag nano cubic particle-protein assembly to react for at least half an hour; observing the red shift amount of the plasma scattering spectrum of the assembly under a dark field microscope, and performing related analysis on the red shift amount of the scattering spectrum by using the concentration of guanidine hydrochloride to obtain a related graph of the concentration of guanidine hydrochloride and the red shift amount of the scattering spectrum; washing the assembly after reaction by using a PBS buffer solution to dilute the guanidine hydrochloride solution on the surface of the assembly, and carrying out correlation analysis on the blue shift quantity of the scattering spectrum by using the reaction time to obtain a correlation diagram of the reaction time and the red shift quantity of the scattering spectrum, namely monitoring the renaturation process of the protein.
Example 1
Specifically, the specific preparation method of Au @ Ag nano cubic particles, protein solution and assemblies in the method is as follows:
(1) Preparation of Au @ Ag nano cubic particles
And (2) reducing sodium borohydride under ice bath to prepare 3nm gold seeds, growing the 3nm gold seeds into 10nm gold balls by using a seed growth method, then dropwise adding growth liquid into the gold balls, growing the gold balls into 30nm, dropwise adding 0.01M silver nitrate solution to obtain 55nm Au @ Ag nano cubic particles (Au @ AgNCs), and measuring the ultraviolet spectrum to be 485nm.
The specific process is as follows:
a. preparation of about 3nm GNPs
A clean and dry 20mL glass bottle which is treated is selected as a reaction bottle, and the cleaned magnetons are put into the reaction bottle to ensure that the whole reaction device is in the middle of stirring. Then, 0.1M CTAB (aqueous solution, aquous: aq) 10mL and 0.01MHAuCL were added to the reaction flask 4 (aq) 0.25mL, and during the loading, the solution was uniformly mixed by keeping the magnetic stirrer stirring. Then quickly adding the prepared NaBH of 0.01M after ice-water bath into the mixed solution 4 (aq) 0.6mL. The color of the solution changes from colorless to dark brown instantly. Putting the obtained solution into a constant-temperature water bath kettle, controlling the temperature at 28 ℃ and reacting for 2 hours to obtain 3nm gold seed solution, namely 3nm GNPs.
b. Preparation of about 10nm GNPs
A clean and dry 20mL glass vial was selected as the reaction vial. The cleaned magnetons are put into a reaction bottle, and the whole reaction device is ensured to be in the middle of stirring. Then, 6mL of 0.1M CTAC (aq) and 5mM HAuCL were added to the reaction flask 4 (aq) 6mL, and during the loading, the solution was uniformly mixed by keeping the magnetic stirrer stirring. Then, 3mL of 0.01M ascorbic acid AA (aq) was added to the mixed solution, followed by 0.3mL of a 5-fold diluted 3nm gold seed solution prepared in (a) above. The solution gradually changed from colorless to wine-red during stirring. Putting the obtained solution into a constant-temperature water bath kettle, controlling the temperature at 38 ℃ and reacting for 2h to obtain 10nm gold seed solution, namely 10nm GNPs.
c. Preparation of about 30nm GNPs
A clean and dry, treated 30mL glass vial was selected as the reaction vial. The cleaned magnetons are put into a reaction bottle, and the whole reaction device is ensured to be in the middle of stirring. Then, 8mL of 0.1M CTAC (aq) and 7mL of 5mM HAuCL4 (aq) were added to the reaction flask, and the solution was uniformly mixed while stirring with a magnetic stirrer. Then, 3mL of 0.01M AA (aq) was added to the mixed solution, followed by 1mL of 10nm gold seed solution. The solution gradually changed from colorless to purple-red during stirring. Putting the obtained solution into a constant-temperature water bath kettle, controlling the temperature at 38 ℃ and reacting for 2h to obtain 30nm gold seed solution, namely 30nm GNPs.
d. Preparation of Au @ Ag nanoparticles of about 55nm
A clean and dry 20mL glass vial was selected as the reaction vial. The cleaned magneton is put into a reaction bottle, and the whole reaction device is ensured to be in the middle of stirring. 7mL of CTAC (aq) of 0.1M, 3mL of AA (aq) of 0.01M and 3mL of prepared 30nm gold ball solution are sequentially added into a reaction flask, and a magnetic stirrer is kept for stirring in the process of sample addition to uniformly mix the solution. Placing in a 60 deg.C constant temperature water bath kettle, and uniformly pumping 0.1M AgNO in 10min by using syringe pump 3 (aq) 0.55mL is added into a reaction bottle, after uniform stirring, the magnetons are taken out and react for more than 3h at the constant temperature of 60 ℃, the color of the solution is changed from light red to orange yellow, and the primarily synthesized gold and silver core-shell nano particles (Au @ AgNCs) are obtained.
After the gold and silver core-shell nano particles are preliminarily synthesized, purifying, including three times of centrifugation, wherein the centrifugation specifically comprises the following operations:
firstly, the obtained Au @ AgNCs is centrifuged at 990r/min for about 8min to remove large-particle impurities, supernatant is taken after centrifugation is finished, then the Au @ AgNCs is centrifuged at 4800r/min for 8min at high speed to remove redundant surfactant, and 5mL of ultrapure water is added after the lower-layer precipitate is taken. Centrifuging at 5000r/min for 8min to obtain lower layer precipitate and Au @ Ag nano cubic particles with crystal grain size of 55 nm; dissolving and diluting with 0.4mL of ultrapure water, sealing and protecting from light, and storing in a refrigerator at 4 ℃ for later use. Fig. 2 is a TEM image of the prepared gold-silver core-shell nanoparticles, and fig. 2 illustrates that the synthesized gold-silver core-shell nanoparticles have regular and uniform shapes and can provide a better material for the preparation of biosensors.
(2) Preparation of protein solution:
selecting a clean and dry 100mL volumetric flask as a solution storage container, weighing 1g of Bovine Serum Albumin (BSA) as a solute, taking a sodium hydrogen phosphate buffer solution with the pH of 7.0-7.4 as a solvent, standing at normal temperature for 4-6h for incubation, and preparing a 10mg/mL BSA solution, and storing at 4 ℃ for later use; as shown in FIG. 3, the fluorescence spectrum of the BSA solution prepared in this example was analyzed.
(3) Assembled to form Au @ Ag nano cubic particle-bovine serum albumin assembly
Diluting the gold and silver core-shell nanoparticles obtained in the step (1) in a ratio of 1; and (3) diluting the BSA solution obtained in the step (2) according to the proportion of 1.
The more protein is adsorbed on the surface of the Au @ AgNC nano particle, the larger the LSPR scattering peak of the nano particle moves and is linearly related, but the more Ag-S bonds bound on the surface of the particle, the weaker the light scattering of the Au @ AgNCs nano particle under a dark field microscope is caused, so that an appropriate concentration needs to be searched for adsorption assembly.
Thus, the optimal concentration of the Au @ AgNC nanoparticles surface adsorbed protein was obtained in setting examples 2-5.
Example 2 to example 5
Examples 2-5 the same procedure as in example 1 was followed, except that: the dilution ratios of the BSA solutions in the step (3) are different, so that BSA solution concentrations with different concentrations are formed, as shown in FIG. 4, the scattering spectra of assemblies formed by BSA with different concentrations are shown, and FIG. 5 is a graph showing the relationship between the BSA concentration of the assemblies and the red shift amount of the scattering spectra of the assemblies;
the BSA dilution ratios and the diluted concentration values in examples 1-6 are given in the following table:
| example 1 | Example 2 | Example 3 | Example 4 | Example 5 | |
| Dilution ratio of BSA solution | 1:10 | 3:40 | 1:20 | 1:40 | 1:80 |
| BSA solution concentration mg/ |
1 | 0.75 | 0.5 | 0.25 | 0.125 |
The actual observation effect of the dark-field microscope in examples 2 to 5 is the best observation effect in example 3, because Ag has high reactivity and is easily oxidized, and BSA is adsorbed on the surface too much, so that an oxide film is formed on the surface of Ag due to Ag — S bond, and the light scattering brightness is reduced, which is not favorable for visual observation.
Example 6: establishing the corresponding relation between the deformation agent guanidine hydrochloride and the scattering wavelength red shift amount
The Au @ Ag nano cubic particle-bovine serum albumin assembly prepared in the embodiment 3 is used for determining the corresponding relation between the deformation agent guanidine hydrochloride and the scattering wavelength red shift amount, and the specific steps are as follows:
(a) Selecting a guanidine hydrochloride solution as a denaturant, wherein the concentration of the guanidine hydrochloride solution is 1M;
(b) Dripping 200 mu L of guanidine hydrochloride solution obtained in the step (a) on the Au @ Ag nano cubic particle-bovine serum albumin assembly to react for 40min in a dark room at room temperature, so that the bovine serum albumin is denatured and unfolded;
(c) The assembly plasma scattering spectrum red shift amount was observed under a dark field microscope, and the guanidine hydrochloride concentration was subjected to linear correlation analysis on the scattering spectrum red shift amount to obtain a linear correlation chart of the guanidine hydrochloride concentration and the scattering spectrum red shift amount, and the results are shown in fig. 7 and table 1. FIG. 7 is a graph showing the non-linear correlation between the concentrations of guanidine hydrochloride (1M, 3M, 5M, 7M, 9M) and the amount of red shift in the scattering spectrum of the assembly (biosensor) prepared in example 3. From the trend of the change of the red shift amount of the scattering peak, when the concentration of guanidine hydrochloride is increased to 5M, the red shift amount of the LSPR scattering peak of the particle begins to be flat compared with the previous change, and the degeneration effect on BSA tends to be the same after the concentration of guanidine hydrochloride is increased to a certain value.
Table 1 shows the ratio of guanidine hydrochloride concentration and the movement of particle scattering peak
In Table 1, the scattering wavelength of Au @ AgNCs-BSA shows the shift amount of the scattering spectrum after the gold and silver core-shell is connected with BSA, the scattering wavelength is 547nm because the concentration of BSA and Au @ AgNCs is constant, and the second is the shift amount of the scattering spectrum after the reaction of Au @ Ag NCs-BSA and guanidine hydrochloride, and the concentration of guanidine hydrochloride is changed, so the scattering spectrum shifts.
As can be seen from Table 1, the Au @ Ag nano cubic particles-bovine serum albumin groups obtained in the embodiment of the invention have consistent structures, the scattering spectrum of the assembly body can generate obvious red shift after guanidine hydrochloride is added for reaction, the red shift amount can obviously show the change of the particle color under a dark field microscope, and the guanidine hydrochloride concentration and the scattering spectrum red shift amount are in a relevant relationship, so that the protein configuration change detection which can be detected by naked eyes is realized.
FIG. 6 is a graph showing that Au @ Ag NCs-BSA constructed has the identification property for guanidine hydrochloride with different concentrations, and the scattering spectrum shift amount is different according to the concentrations, and FIG. 7 is a fitting graph made according to different concentrations and corresponding to different spectrum red shift amounts, and shows that the concentration of guanidine hydrochloride and the spectrum red shift amount have a correlation relationship.
As can be seen from Table 1 and FIG. 7, the biosensor prepared according to the present invention can effectively monitor the protein development process, and has the advantages of high sensitivity, high specificity, real-time performance, rapidness, etc.
Example 7: protein three-dimensional state reversible experiment of deforming agent guanidine hydrochloride
Dropwise adding a 5M guanidine hydrochloride solution to the assembly synthesized in the embodiment 3, reacting for 40min, and observing the movement condition of a plasma scattering spectrum; on the basis, the guanidine hydrochloride concentration on the surface of the denatured protein is washed and diluted, the reaction is carried out for 20min, and the movement condition of the plasma scattering spectrum is observed.
The LSPR scattering peak of the assembly prepared in example 3 in the pristine state was 547nm; dropwise adding 5M guanidine hydrochloride solution on the Au @ AgNCs-BSA assembly for reaction for 40min, determining that the LSPR scattering peak of the Au @ AgNCs is 585nm, the LSPR scattering spectrum of the particles is red-shifted, and the movement amount is 38nm after the reaction is finished; then washing and diluting the guanidine hydrochloride concentration on the surface of the denatured protein on the basis, reacting for 20min, and determining that the LSPR scattering peak of Au @ AgNCs is 557nm, the LSPR scattering spectrum of the particles is blue-shifted, and the movement amount is 28nm; the results are shown in fig. 8, fig. 9 and table 2.
TABLE 2 measurement of the amount of movement of the assembly scatter spectra during unfolding and refolding
| Guanidine hydrochloride concentration (M) | Peak value (nm) | Amount of movement (nm) |
| 5 | 585 | 38 |
| Diluting guanidine hydrochloride: washing and soaking in PBS buffer solution for 40min | 557 | 28 |
The experiment shows that after Au @ Ag NCs-bovine serum albumin reacts with guanidine hydrochloride, the protein configuration is unfolded, namely, the dispersion spectrum generates red shift to indicate that guanidine hydrochloride solution realizes the denaturation control of protein, meanwhile, the dispersion spectrum generates red shift to indicate that the protein is successfully fixed and synthesized into the assembly biosensor, then the concentration of a denaturant on the surface of the denatured protein is diluted, the protein is recovered to a folded state, namely, the dispersion spectrum generates blue shift, and the cyclic detection of the change of the protein configuration is realized. The biosensor prepared by the invention not only has the advantages of real-time, rapidness, high sensitivity, high specificity identification and the like, but also can realize the cyclic monitoring of the protein configuration, and has important significance for researching the protein structure and the biological activity thereof.
Claims (9)
1. A method for detecting the state of the three-dimensional structure of a thiol-group-containing protein, said method comprising the steps of: weighing a certain amount of sulfhydryl-containing protein in an unknown folded state as a sample to be detected, standing and incubating to form a protein solution to be detected; forming an LSPR biosensor by adopting Au @ Ag nano particles; diluting the obtained protein solution to be detected, then dropwise adding the diluted protein solution to the LSPR biosensor, incubating for a period of time, washing and drying to form an Au @ Ag nanoparticle-protein assembly; observing the red shift amount of the plasma scattering spectrum of the assembly under a dark-field microscope, and directly judging the current folding state of the specific protein to be detected according to a corresponding relation model between the red shift amount of the scattering spectrum and the folding state of the specific protein;
the method for establishing the corresponding relation model between the red shift quantity of the scattering spectrum and the folding state of the specific protein comprises the following steps: selecting high-activity protein in a natural state, standing and incubating to prepare a solution, and then assembling the solution with Au @ Ag nano particles to form an assembly; establishing a concentration gradient of a denaturant, and respectively dropwise adding the denaturants with different concentrations onto the Au @ Ag nano cubic particle-protein assembly for reaction to form proteins with different folding degrees, wherein the reaction time is at least half an hour; observing the red shift amount of the plasma scattering spectrum of the assembly under a dark field microscope, and performing correlation analysis on the red shift amount of the scattering spectrum by using the concentration of the denaturant to obtain a corresponding relation model of the specific concentration of the denaturant and the red shift amount of the scattering spectrum, wherein the denaturants with different concentrations correspond to different folding states of the protein, so that a corresponding relation model between the red shift amount of the scattering spectrum and the folding degree of the specific protein is formed.
2. The method according to claim 1, wherein the step of establishing a concentration gradient of the denaturant is specifically as follows: setting denaturant concentration gradient value n, wherein the difference values between different gradient values n are the same, different folding states of the protein can be represented by folding degrees P, and the folding degrees P are divided into a natural folding state, an intermediate state and a complete deformation state.
3. The method according to claim 1, wherein the denaturing agent is guanidine hydrochloride.
4. A method for studying the effect of environmental conditions on the three-dimensional structural state of a thiol-group-containing protein, said method comprising the steps of: selecting high-activity protein in a natural state, standing and incubating to prepare a solution, and then assembling the solution with Au @ Ag nano particles to form an assembly; placing the assembly under different environmental conditions including temperature, chemical solution, humidity, radiation conditions; observing the red shift amount of the plasma scattering spectrum of the assembly under a dark field microscope, and directly judging the current folding state of the protein according to the corresponding relation model in claim 1, namely obtaining the direct corresponding relation between the corresponding environmental conditions and the folding state of the protein.
5. The method according to any one of claims 1 to 4, wherein the Au @ Ag nanoparticles are Au @ Ag nano cubic particles.
6. The method of claim 5, wherein the Au @ Ag nano cubic particles have a particle size of about 55 nm.
7. The method according to any one of claims 1 to 4, wherein the thiol-group-containing protein is bovine serum albumin at a concentration of 0.125mg/mL to 1mg/mL.
8. The method of claim 7, wherein the concentration of bovine serum albumin is 0.5mg/mL.
9. The method according to any one of claims 1 to 4, wherein the protein static incubation is configured to be performed in a solution, specifically, in a phosphoric acid buffer solution with pH of 7.0 to 7.4, for 4 to 6 hours at normal temperature in a dark room.
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