CN111057537B - Mannose functionalized protein gold nanocluster and preparation method and application thereof - Google Patents

Mannose functionalized protein gold nanocluster and preparation method and application thereof Download PDF

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CN111057537B
CN111057537B CN201911412133.9A CN201911412133A CN111057537B CN 111057537 B CN111057537 B CN 111057537B CN 201911412133 A CN201911412133 A CN 201911412133A CN 111057537 B CN111057537 B CN 111057537B
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protein gold
serum albumin
bovine serum
buffer solution
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CN111057537A (en
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刘欣
沙秋月
胡昭宇
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Huazhong University of Science and Technology
Ezhou Institute of Industrial Technology Huazhong University of Science and Technology
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Ezhou Institute of Industrial Technology Huazhong University of Science and Technology
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Abstract

The invention discloses a mannose functionalized protein gold nanocluster, a preparation method and application thereof, wherein the method comprises the following steps of S1, mixing bovine serum albumin and NH 2 After dissolving mannose in buffer, mixing with EDC, and stirring, purifying and concentrating in sequence to obtain mannose-bovine serum albumin conjugate; the bovine serum albumin and the NH 2 -the mass ratio of mannose to EDC is 9-11:3:1; s2, mixing mannose-bovine serum albumin conjugate with chloroauric acid solution in a buffer solution, regulating the pH value, and stirring and purifying sequentially to obtain the protein gold nanocluster. The preparation method of the protein gold nanocluster disclosed by the invention is convenient, simple, quick and efficient, and the protein gold nanocluster synthesized by the method is nontoxic, good in biocompatibility, good in dispersity, strong in fluorescence and high in detection sensitivity.

Description

Mannose functionalized protein gold nanocluster and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological nano materials, and particularly relates to a mannose functionalized protein gold nanocluster, and a preparation method and application thereof.
Background
The gold nanocluster is a fluorescent nanoparticle which consists of several to tens of Au noble metal atoms, has a size close to the fermi wavelength of electrons, and can emit fluorescence after being excited by energy, wherein the surface of the gold nanocluster is stabilized and protected by ligands such as thiols small molecules, nucleic acid, polymer macromolecules or biological macromolecules. The gold nanocluster has the characteristics of excellent fluorescence performance, no toxicity, high biocompatibility and the like, and is researched and applied in a large number.
The existing gold nanocluster synthesis method generally adopts a core-etching technology, firstly uses a mild reducing agent of tetrakis (hydroxymethyl) phosphonium chloride (THPC) to synthesize gold nanoparticles with the size of about 5nm, and then uses sulfhydryl mannose to etch the gold nanoparticles to obtain gold nanoclusters, wherein the gold nanoclusters have small size and fluorescence performance. However, the gold nanoclusters are modified by using a chemical reagent of sulfhydryl mannose, so that the prepared gold nanoclusters have toxicity and poor biocompatibility, and further application of the gold nanoclusters is limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a mannose functionalized protein gold nanocluster, and a preparation method and application thereof, so as to solve the problems of limited application caused by toxicity and poor biocompatibility of the gold nanocluster due to the fact that the gold nanocluster is synthesized by adopting a chemical reagent in the prior art.
The invention realizes the above purpose through the following technical scheme:
in one aspect, the invention provides a method for preparing mannose-functionalized protein gold nanoclusters, the method comprising,
s1, bovine serum albumin and NH 2 After dissolving mannose in buffer, mixing with EDC, and stirring, purifying and concentrating in sequence to obtain mannose-bovine serum albumin conjugate; the bovine serum albumin and the NH 2 -the mass ratio of mannose to EDC is 9-11:3:1;
s2, mixing mannose-bovine serum albumin conjugate with chloroauric acid solution in a buffer solution, regulating the pH value, and stirring and purifying sequentially to obtain the protein gold nanocluster.
Further, in step S1, the buffer solution is one of a phosphate buffer solution and a 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution, and the pH value of the buffer solution is 6.9-7.2.
Further, in the step S1, the stirring temperature is 20-40 ℃, and the stirring time is 10-14 h.
Further, in the step S2, the molar ratio of the mannose-bovine serum albumin conjugate to the chloroauric acid is 1:0.016-0.022.
Further, in step S2, the buffer solution is one of a phosphate buffer solution and a 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution, and the pH value of the buffer solution is 6.9-7.2.
Further, in step S2, the pH is adjusted to > 10 with the addition of sodium hydroxide solution.
Further, in the step S2, the stirring temperature is 20-40 ℃, and the stirring time is 12-16 h.
On the other hand, the invention also provides the mannose-functionalized protein gold nanocluster prepared by the preparation method of the mannose-functionalized protein gold nanocluster, wherein the excitation wavelength of the protein gold nanocluster is 460-520 nm, the emission wavelength of the protein gold nanocluster is 630-660 nm, and the particle size of the protein gold nanocluster is 1.5-3 nm.
Further, mannose is contained on the surface of the protein gold nanocluster, and the mass concentration of the mannose in the protein gold nanocluster is 760.4-774.8 mg/L.
In still another aspect, the invention further provides application of the mannose-functionalized protein gold nanoclusters to concanavalin detection and breast cancer cell imaging.
The beneficial effects of the invention at least comprise:
the invention provides a mannose functionalized protein gold nanocluster, a preparation method and application thereof, wherein the method comprises the following steps of S1, mixing bovine serum albumin and NH 2 After dissolving mannose in buffer, mixing with EDC, and stirring, purifying and concentrating in sequence to obtain mannose-bovine serum albumin conjugate; the bovine serum albumin and the NH 2 -the mass ratio of mannose to EDC is 9-11:3:1; s2, mannose-bovine serum albumin conjugate and chloroauric acid solutionAnd (3) after mixing in the buffer solution, regulating the pH value, and sequentially stirring and purifying to obtain the protein gold nanocluster. The invention adopts bovine serum albumin and NH 2 Mannose-bovine serum albumin conjugate obtained by the reaction of mannose and two natural biomolecules is used as a reducing agent and a template, and mannose-functionalized protein gold nanoclusters are synthesized with chloroauric acid, and raw materials used for the protein gold nanoclusters are natural and nontoxic, so that the synthesized protein gold nanoclusters are nontoxic, and the raw materials are natural biomolecules, so that the prepared protein gold nanoclusters are good in biocompatibility and high in mannose load. Therefore, the protein gold nanocluster has good comprehensive performance and is widely applied.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a process step diagram of a preparation method of mannose-functionalized protein gold nanoclusters according to an embodiment of the present invention;
FIG. 2 is a fluorescence excitation and emission spectrum of a mannose-functionalized protein gold nanocluster according to an embodiment of the present invention;
FIG. 3 is an ultraviolet-visible absorption spectrum of a mannose-functionalized protein gold nanocluster phenol-sulfuric acid solution pretreated according to an embodiment of the present invention;
FIG. 4 is a fluorescence emission spectrum of a supernatant of a mannose-functionalized protein gold nanocluster mixed with concanavalin of different concentrations according to an embodiment of the present invention;
FIG. 5 is a fluorescence emission spectrum of a supernatant of a mannose-functionalized protein gold nanocluster mixed with concanavalin of different concentrations according to an embodiment of the present invention;
FIG. 6 is a bright field image at excitation wavelength of 488nm of MDA-MB-231 human breast cancer cells incubated with the protein gold nanoclusters of an embodiment of the present invention;
FIG. 7 is a fluorescence field image at excitation wavelength of 488nm of MDA-MB-231 human breast cancer cells incubated with the protein gold nanoclusters of an embodiment of the present invention;
FIG. 8 is an image of superimposed fields at excitation wavelength of 488nm of MDA-MB-231 human breast cancer cells incubated with the protein gold nanoclusters of an embodiment of the present invention.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The technical scheme in the embodiment of the invention aims to solve the technical problems, and the overall thought is as follows:
in one aspect, the embodiment of the invention provides a method for preparing mannose-functionalized protein gold nanoclusters, fig. 1 is a process step diagram of the method for preparing mannose-functionalized protein gold nanoclusters according to the embodiment of the invention, and in combination with fig. 1, the method comprises,
s1, bovine serum albumin and NH 2 After dissolution of mannose in buffer, it is mixed with EDC and stirred, purified and concentrated in sequence to obtain mannose-bovine serum albumin conjugate.
EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and Bovine Serum Albumin (BSA) contains a large amount of carboxyl groups and NH in its amino acid residue 2 Mannose has a large number of amino groups, bovine serum albumin and NH are first added 2 After mixing mannose and adding EDC, the carboxyl group of bovine serum albumin can be activated into a reactive intermediate after adding EDC, and the intermediate can be directly connected with NH 2 -amino groups in mannose react to form a bovine serum albumin-mannose conjugate. If bovine serum albumin is mixed with EDC, NH is added 2 Mannose, EDC, first activates the carboxyl groups in the bovine serum albumin into intermediates, which react with the amino groups of the bovine serum albumin itself, causing self-crosslinking, precipitating the bovine serum albumin and thus affecting the subsequent synthesis process.
Further, the bovine serum albumin and the NH 2 The mass ratio of mannose to EDC is 9-11:3:1.
Bovine serum albumin, NH 2 The ratio of mannose to EDC is controlled in the above-mentioned proper range, and the mannose-bovine serum albumin conjugate can be produced to the maximum without wasting the raw material.
Further, the buffer solution is one of phosphate buffer solution and 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution, and the pH value of the buffer solution is 6.9-7.2.
The pH value of the buffer solution is 6.9-7.2, EDC has the best activity under weak acid environment, and bovine serum albumin has the best activity under 7.4, so that the EDC activity is exerted, the bovine serum albumin is ensured not to be denatured, and the pH value of the buffer solution is 6.9-7.2.
Further, the stirring temperature is 20-40 ℃, and the stirring time is 10-14 h.
Bovine serum albumin is a protein which has an activity temperature of 20 to 40 ℃ and if it is below or above this temperature range, bovine serum albumin is inactivated and a bovine serum albumin-mannose conjugate is not available. By stirring, the reaction process can be accelerated. The bovine serum albumin-mannose conjugate also contains NH 2 Mannose and many small molecular substances, NH can be purified 2 Mannose and a lot of small molecular substances are removed, so that the trapped glycerolThe molecular weight of the conjugate of the sugar syrup and the bovine serum albumin is 10-20 kDa.
S2, after the mannose-bovine serum albumin conjugate and chloroauric acid solution are mixed in a buffer solution, the pH value is regulated, and stirring and purification are sequentially carried out, so that the protein gold nanoclusters (Man-Au NCs) are obtained.
The pH value is regulated to provide optimal reaction conditions for mannose-bovine serum albumin conjugate and chloroauric acid, the reaction can be accelerated by stirring, and impurities and small molecular substances can be removed by purification. The purification process can be carried out in an ultrafiltration tube, the molecular weight of the combination of the bovine serum albumin and the mannose is about 80kDa, and the molecular weight of the small molecular impurities is not more than 1000Da, so that the selection of the ultrafiltration tube with the specification of 10-20 kDa can ensure that almost all the small molecular impurities are removed, and the combination of the bovine serum albumin and the mannose is not lost.
Further, in the step S2, the molar ratio of the mannose-bovine serum albumin conjugate to chloroauric acid is 1:0.016-0.022. The mannose-bovine serum albumin conjugate can be converted into the gold nanoclusters to the greatest extent by controlling the molar ratio of the mannose-bovine serum albumin conjugate to chloroauric acid within the above range.
Further, the buffer solution is one of phosphate buffer solution and 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution, and the pH value of the buffer solution is 6.9-7.2.
The buffer solution is used as a dissolving solution to ensure the dispersibility of the mannose-bovine serum albumin conjugate.
Further, in step S2, the pH is adjusted to > 10 with the addition of sodium hydroxide solution.
The sodium hydroxide solution is added to adjust the pH value to be more than 10, and the reducibility of the mannose-bovine serum albumin conjugate can be excited under the environment, so that the sodium hydroxide solution can be used as a biological reducer to realize the synthesis of the protein gold nanoclusters.
Further, the stirring temperature is 20-40 ℃, and the stirring time is 12-16 h.
On the other hand, the embodiment of the invention also provides the mannose-functionalized protein gold nanocluster prepared by the preparation method of the mannose-functionalized protein gold nanocluster, wherein the excitation wavelength of the protein gold nanocluster is 460-520 nm, the emission wavelength of the protein gold nanocluster is 630-660 nm, and the particle size of the protein gold nanocluster is 1.5-3 nm.
Under the action of light with the excitation wavelength of 460-520 nm, the emission wavelength of the protein gold nanocluster is 630-660 nm, the emission wavelength is longer, the damage to organisms including cells can not be generated, the application range can be increased, the fluorescence excitation and emission spectrum of the protein gold nanocluster is shown in figure 2, the ultraviolet-visible absorption spectrum of the protein gold nanocluster after phenol-sulfuric acid solution pretreatment is shown in figure 3,
further, mannose is contained on the surface of the protein gold nanocluster, and the mass concentration of the mannose in the protein gold nanocluster is 760.4-774.8 mg/L.
The mannose modified on the protein gold nanoclusters can effectively realize the detection effect of the protein gold nanoclusters on concanavalin (Con A), and the concanavalin is easy to be identified as the mannose is an identification molecule, and the higher the concentration of mannose modified by the protein gold nanoclusters is.
In still another aspect, the embodiment of the invention further provides application of the mannose-functionalized protein gold nanoclusters to concanavalin detection and breast cancer cell imaging.
The mannose modified on the protein gold nanoclusters can react with concanavalin to enable the protein gold nanoclusters to agglomerate and reduce or even not emit light, so that the concanavalin is detected. The fluorescence emission spectra of the supernatants of the gold nanoclusters mixed with concanavalin at different concentrations are shown in FIGS. 4 and 5, wherein the concanavalin concentration is 0-10 nM. The breast cancer cells are provided with mannose receptors, so that the protein gold nanoclusters and the mannose receptors overexpressed on the human breast cancer cells have a specific recognition effect, and therefore, the protein gold nanoclusters can be used for breast cancer cell imaging. The MDA-MB-231 human breast cancer cells are incubated with the protein gold nanoclusters, the confocal is carried out under the excitation wavelength of 488nm, the images of a bright field, a fluorescence field and an superimposed field are respectively shown in fig. 6, 7 and 8, irregular circles or semi-circles with light gray colors in fig. 7 are light emitted by the protein gold nanoclusters under fluorescent irradiation, the light emitted by the protein gold nanoclusters is red under the electron microscope, and the light emitted by the protein gold nanoclusters is red under the superimposed field and the light emitted by the protein gold nanoclusters is round the light gray circles or semi-circles around the periphery of the cells in fig. 8.
The invention provides a mannose functionalized protein gold nanocluster, a preparation method and application thereof, wherein the invention adopts bovine serum albumin and NH 2 Mannose-bovine serum albumin conjugate obtained by the reaction of two natural biomolecules, namely mannose, is taken as a reducing agent and a template, and is synthesized with chloroauric acid to form mannose-functionalized protein gold nanoclusters, and raw materials used by the protein gold nanoclusters are natural and nontoxic, so that the synthesized protein gold nanoclusters are nontoxic, and the raw materials are natural biomolecules, so that the prepared protein gold nanoclusters are good in biocompatibility; the method is convenient, simple, quick and efficient to prepare, and the prepared protein gold nanocluster is good in dispersibility, strong in fluorescence and high in detection sensitivity, can be used for detecting concanavalin, and is low in detection limit of 0.62nmol/L; the mannose has high load and specific recognition effect with mannose receptor over expressed on human breast cancer cells, and can be used for breast cancer cell imaging.
The technical scheme of the present application will be further described below with reference to specific examples.
Example 1
Example 1 provides a method for preparing mannose-functionalized protein gold nanoclusters, comprising reacting 100mg of bovine serum albumin with 30mg of NH 2 Mannose and 10mg of EDC are added into 5 ml of phosphate buffer with pH value of 7 in sequence, stirred for 12 hours at 25 ℃, purified by an ultrafiltration tube with molecular weight cut-off of 10kDa and concentrated to obtain mannose-bovine serum albumin conjugate. The mannose-bovine serum albumin conjugate is redissolved in 2 ml of phosphate buffer, 3 ml of chloroauric acid solution with the concentration of 8 mmol is added, and after stirring for 2 minutes, the mixture is added200 microliters of sodium hydroxide solution with the concentration of 1mol/L is adjusted to 11 in pH value, stirred for 12 hours at 37 ℃, purified by an ultrafiltration tube with the molecular weight cutoff of 10kDa and concentrated to obtain protein gold nanoclusters, and the protein gold nanoclusters are dispersed in 5 milliliters of phosphate buffer.
The excitation wavelength of the obtained protein gold nanocluster is 490nm, and the emission wavelength is 633nm. The particle size of the protein gold nanoclusters is 2nm. After the protein gold nanoclusters are diluted by 10 times and treated by phenol-sulfuric acid, the ultraviolet absorbance on an ultraviolet-visible spectrophotometer is 0.144, and the loading capacity of mannose on the gold nanoclusters is 767.6mg/L by linear equation calculation carried into a standard substance. The detection limit of the concanavalin is 0.62nmol/L.
Example 2
Example 2 provides a method for preparing mannose-functionalized protein gold nanoclusters, comprising reacting 90mg of bovine serum albumin with 30mg of NH 2 Mannose and 10mg of EDC are added to 5 ml of 4-hydroxyethyl piperazine ethane sulfonic acid buffer with pH value of 7 in sequence, stirred for 14 hours at 30 ℃, purified by an ultrafiltration tube with molecular weight cut-off of 10kDa, and concentrated to obtain mannose-bovine serum albumin conjugate. The mannose-bovine serum albumin conjugate is redissolved in 3 ml of 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution, 3 ml of chloroauric acid solution with the concentration of 9 mmol is added, after stirring for 2 minutes, 200 microliters of sodium hydroxide solution with the concentration of 1mol/L is added, the pH value is adjusted to 10, after stirring for 15 hours at 38 ℃, the mixture is purified by an ultrafiltration tube with the molecular weight cutoff of 10kDa, concentration is carried out, and protein gold nanoclusters are obtained, and the protein gold nanoclusters are dispersed in 5 ml of 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution.
The excitation wavelength of the obtained protein gold nanocluster is 500nm, and the emission wavelength is 635nm. The particle size of the protein gold nanoclusters is 2.5nm. After the protein gold nanoclusters are diluted by 10 times and treated by phenol-sulfuric acid, the ultraviolet absorbance on an ultraviolet-visible spectrophotometer is 0.148, and the loading capacity of mannose on the gold nanoclusters is 772.5mg/L by linear equation calculation carried into a standard substance. The detection limit of the concanavalin is 0.62nmol/L.
Example 3
Example 3 provides a method of preparing mannose-functionalized protein gold nanoclusters, comprising reacting 110mg of bovine serum albumin, 30mg of NH 2 Mannose and 10mg of EDC are added into 5 ml of phosphate buffer with pH value of 7 in sequence, stirred for 11 hours at 35 ℃, purified by an ultrafiltration tube with molecular weight cut-off of 10kDa and concentrated to obtain mannose-bovine serum albumin conjugate. The mannose-bovine serum albumin conjugate is redissolved in 2 ml of phosphate buffer, 3 ml of chloroauric acid solution with the concentration of 10 mmol is added, after stirring for 2 minutes, 200 microliters of sodium hydroxide solution with the concentration of 1mol/L is added, the pH value is adjusted to 10, after stirring for 14 hours at 30 ℃, the mixture is purified by an ultrafiltration tube with the molecular weight cut-off of 10kDa, concentration is carried out, and protein gold nanoclusters are obtained and dispersed in 5 ml of phosphate buffer.
The excitation wavelength of the obtained protein gold nanocluster is 470nm, and the emission wavelength is 653nm. The particle size of the protein gold nanoclusters is 1.8nm. After the protein gold nanoclusters are diluted by 10 times and treated by phenol-sulfuric acid, the ultraviolet absorbance on an ultraviolet-visible spectrophotometer is 0.144, and the loading capacity of mannose on the gold nanoclusters is 763.5mg/L by linear equation calculation carried into a standard substance. The detection limit of the concanavalin is 0.62nmol/L.
Example 4
Example 4 provides a method of preparing mannose-functionalized protein gold nanoclusters, comprising reacting 105mg of bovine serum albumin, 30mg of NH 2 Mannose and 10mg of EDC are added to 5 ml of 4-hydroxyethyl piperazine ethane sulfonic acid buffer with pH value of 6.9 in sequence, stirred for 11 hours at 35 ℃, purified by an ultrafiltration tube with molecular weight cut-off of 10kDa, and concentrated to obtain mannose-bovine serum albumin conjugate. The mannose-bovine serum albumin conjugate was redissolved in 2 ml of 4-hydroxyethylpiperazine ethanesulfonic acid buffer at pH 6.93 ml of chloroauric acid solution with the concentration of 11 mmol is added, after stirring for 2 minutes, 200 microliters of sodium hydroxide solution with the concentration of 1mol/L is added, the pH value is adjusted to 10, after stirring for 14 hours at 30 ℃, the solution is purified by an ultrafiltration tube with the molecular weight cutoff of 10kDa and then concentrated, so that protein gold nanoclusters are obtained, and the protein gold nanoclusters are dispersed in 5 ml of phosphate buffer.
The excitation wavelength of the obtained protein gold nanocluster is 510nm, and the emission wavelength is 653nm. The particle size of the protein gold nanoclusters is 1.8nm. After the protein gold nanoclusters are diluted by 10 times and treated by phenol-sulfuric acid, the ultraviolet absorbance on an ultraviolet-visible spectrophotometer is 0.144, and the loading capacity of mannose on the gold nanoclusters is 771.5mg/L by linear equation calculation carried into a standard substance. The detection limit of the concanavalin is 0.62nmol/L.
Example 5
Example 5 provides a method of preparing mannose-functionalized protein gold nanoclusters, comprising reacting 105mg of bovine serum albumin, 30mg of NH 2 Mannose and 10mg of EDC are added sequentially to 5 ml of phosphate buffer with pH value of 7.2, stirred at 35 ℃ for 11 hours, purified by an ultrafiltration tube with molecular weight cut-off of 10kDa and concentrated to obtain mannose-bovine serum albumin conjugate. The mannose-bovine serum albumin conjugate is redissolved in 2 ml of phosphoric acid buffer solution with the pH value of 7.2, 3 ml of chloroauric acid solution with the concentration of 10 millimoles is added, after stirring for 2 minutes, 200 microlitres of sodium hydroxide solution with the concentration of 1mol/L is added, the pH value is adjusted to 10, after stirring for 14 hours at 30 ℃, the mixture is purified by an ultrafiltration tube with the molecular weight cutoff of 10kDa, concentration is carried out, and protein gold nanoclusters are obtained and dispersed in 5 ml of phosphoric acid buffer solution.
The excitation wavelength of the obtained protein gold nanocluster is 483nm, and the emission wavelength is 643nm. The particle size of the protein gold nanoclusters is 1.8nm. After the protein gold nanoclusters are diluted by 10 times and treated by phenol-sulfuric acid, the ultraviolet absorbance on an ultraviolet-visible spectrophotometer is 0.144, and the loading capacity of mannose on the gold nanoclusters is 765.5mg/L by linear equation calculation carried into a standard substance. The detection limit of the concanavalin is 0.62nmol/L.
TABLE 1
Figure BDA0002350249580000081
Table 1 shows that the mannose loading amount of the protein gold nanoclusters and the detection lower limit of concanavalin provided by examples 1 to 5 are 771.5-767.6 mg/L, the mannose loading amount is high, the detection lower limit of concanavalin is 0.62nmol/L, and the detection lower limit is low.
The linear equation of the standard is NH with a purity of 95% or more on the market 2 Mannose as standard substance, which is dispersed in buffer solutions with different volumes to obtain NH with different concentrations 2 Mannose solution, absorbance of which is detected, NH is obtained from concentration and absorbance data 2 -a linear regression equation between the concentration of mannose and the absorbance, which is the linear regression equation for the standard.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (9)

1. A method for preparing mannose functionalized protein gold nanoclusters is characterized in that the method comprises the following steps of,
s1, bovine serum albumin and NH 2 After dissolving mannose in buffer, mixing with EDC, and stirring, purifying and concentrating in sequence to obtain mannose-bovine serum albumin conjugate; the bovine serum albumin and the NH 2 -the mass ratio of mannose to EDC is 9-11:3:1;
s2, mixing mannose-bovine serum albumin conjugate with chloroauric acid solution in a buffer solution, regulating the pH value, and stirring and purifying sequentially to obtain the protein gold nanocluster.
2. The method for preparing mannose functionalized protein gold nanoclusters according to claim 1, wherein in the step S1, the buffer solution is one of a phosphate buffer solution and a 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution, and the pH value of the buffer solution is 6.9-7.2.
3. The method for preparing mannose-functionalized protein gold nanoclusters according to claim 1, wherein in the step S1, the stirring temperature is 20 to 40 ℃, and the stirring time is 10 to 14 hours.
4. The method for preparing mannose-functionalized protein gold nanoclusters according to claim 1, wherein in step S2, the molar ratio of the mannose-bovine serum albumin conjugate to the chloroauric acid is 1:0.016-0.022.
5. The method for preparing mannose functionalized protein gold nanoclusters according to claim 1, wherein in the step S2, the buffer solution is one of a phosphate buffer solution and a 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution, and the pH value of the buffer solution is 6.9-7.2.
6. The method for preparing mannose-functionalized protein gold nanoclusters according to claim 1, wherein in step S2, the pH value is adjusted to > 10 by adding sodium hydroxide solution.
7. The method for preparing mannose-functionalized protein gold nanoclusters according to claim 1, wherein in the step S2, the stirring temperature is 20 to 40 ℃, and the stirring time is 12 to 16 hours.
8. The mannose-functionalized protein gold nanocluster prepared by the method for preparing mannose-functionalized protein gold nanoclusters according to any one of claims 1 to 7, wherein the excitation wavelength of the protein gold nanocluster is 460 to 520nm, the emission wavelength of the protein gold nanocluster is 630 to 660nm, and the particle size of the protein gold nanocluster is 1.5 to 3nm.
9. The mannose functionalized protein gold nanocluster according to claim 8, wherein the surface of the protein gold nanocluster contains mannose, and the mass concentration of the mannose in the protein gold nanocluster is 760.4-774.8 mg/L.
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