TWI676021B - Protein stabilized gold nanocubes, method for making the same, and uses thereof - Google Patents

Abstract

The invention relates to a protein-stabilized gold nano cube, a method for preparing the same and application thereof, and particularly to a protein-stabilized gold nano cube having a specific fluorescence emission wavelength, a method for manufacturing the same, and a use thereof for detecting alkaline phosphatase.

Description

Protein stabilized gold nano cube, its preparation method and application

The invention relates to a protein-stabilized gold nano cube, a method for preparing the same and application thereof, and particularly to a protein-stabilized gold nano cube having a specific fluorescence emission wavelength, a method for manufacturing the same, and a use thereof for detecting alkaline phosphatase.

Alkaline phosphatase (ALP) is a metalloenzyme. Its active site contains two zinc ions (Zn ²⁺ ) and one magnesium (Mg ²⁺ ) ions, which can catalyze nucleosides. Hydrolysis of acids, proteins, alkaloids and other molecules to remove phosphate groups. Because it has the highest activity in an alkaline environment, it is called alkaline phosphatase.

A large amount of alkaline phosphatase exists in human blood, and its concentration can range from about 25 U / L to 100 U / L. However, the increase and decrease of serum alkaline phosphatase concentration may be related to various diseases, such as liver cirrhosis, hepatitis, bone disease, and cancer. Therefore, it is necessary to develop a detection method for alkaline phosphatase with good selectivity and high sensitivity.

The invention provides a protein stabilized gold nanocubes and a manufacturing method thereof. The protein stabilized gold nanocubes can be used for detecting alkaline phosphate Enzyme (alkaline phosphatase).

Therefore, the present invention provides a protein-stabilized nanometer cube, comprising: a plurality of regularly arranged complexes, each of which comprises: a protein; and a gold nanoclusters, which are composed of the protein Around; where the protein-stabilized nanometer cube has an emission wavelength of 468 nm.

The invention further provides a method for manufacturing a protein-stabilized gold nano cube as described above, comprising: (a) mixing a chloroauric acid solution and a protein solution to form a mixed solution; and (b) adjusting the pH value of the mixed solution To about pH 3.7.

The present invention also provides a method for detecting alkaline phosphatase, comprising: (a) providing a sample to be tested, which includes alkaline phosphatase; (b) mixed p-nitrophenyl phosphate, as described above The protein stabilizes the nanometer cube and the test sample to form a test mixture; and (c) Excites the test mixture with light at a wavelength of 405 nm, and detects the emission intensity of the test mixture at a wavelength of 468 nm. .

The schematic diagram of FIG. 1A shows that alkaline phosphatase (ALP) can catalyze the reaction of p-nitrophenyl phosphate to form p-nitrophenol and inorganic phosphate in the presence of water.

The schematic diagram of FIG. 1B shows that in the presence of p-nitrophenyl phosphate in the aqueous solution but no alkaline phosphatase (ALP), the protein-stabilized gold nanocube is excited by incident light with a wavelength of 405 nm and emits fluorescent light.

Figure 1C is a schematic diagram showing p-nitrophenyl phosphate and alkaline phosphatase (ALP) in an aqueous solution. At this time, the reaction generates p-nitrophenol and inorganic phosphate, and p-nitrophenol absorbs incident light at a wavelength of 405 nm, which weakens the emission fluorescence of the protein-stabilized nanometer cube.

FIG. 2 shows a transmission electron microscopy (TEM) image of the protein-stabilized nanometer cube.

Figure 3A shows the fluorescence of p-nitrophenyl phosphate (NPP), alkaline phosphatase (ALP), bovine serum albumin (BSA), p-nitrophenol (pNP), and protein stabilized gold nanocube (PSGNC)- Visible light (UV-vis) absorption spectrum.

FIG. 3B shows fluorescence spectra of bovine serum albumin (BSA), p-nitrophenol (pNP), and protein-stabilized gold nanocube (PSGNC) under the excitation of incident light at a wavelength of 370 nm.

FIG. 4A shows a fluorescence spectrum measured from a test mixed solution (aqueous solution) containing alkaline phosphatase at different concentrations.

FIG. 4B shows the relationship between the fluorescence intensity F _{0 (initial)} / F _(final) of each test mixture in FIG. 4A and its ALP concentration.

FIG. 5A shows the fluorescence spectrum measured from a test mixture (serum) containing alkaline phosphatase at different concentrations.

FIG. 5B shows the relationship between the fluorescence intensity F _{0 (initial)} / F _(final) of each test mixture in FIG. 5A and its ALP concentration.

Figure 6 shows the fluorescence of samples containing alkaline phosphatase (ALP), human serum albumin (HSA), bovine serum albumin (BSA), hemoglobin (Hb), thrombin (Th), and lysozyme (Lys), respectively. Intensity F _{0 (initial)} / F _(final) .

The invention provides a protein stabilized gold nanocubes (PSGNC or PSGNCs for short), including: A plurality of regularly arranged complexes, each of which comprises: a protein; and a gold nanoclusters surrounded by the protein; wherein the protein stabilizes the nanometer cubes with an emission wavelength of 468 nm .

The present invention can be more easily understood by referring to the following detailed description of various aspects, examples, and chemical drawings and tables accompanying the description. Before revealing and describing the protein-stabilized gold nano cubes of the present invention, it should be understood that the present invention is not limited to specific preparation methods and applications unless otherwise specifically indicated by the scope of the patent application, which is due to familiarity with the general knowledge of related technologies He knows very well that these things can be changed. It should also be understood that terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the invention.

Ranges are often expressed herein as "about" one particular value and / or to "about" another particular value. When such a range is expressed, it is in a range including one specific value and / or to another specific value. Similarly, when values are expressed as approximations by using the word "about," it should be understood that a particular value can form another aspect. In addition, it should be understood that each endpoint of each range is significant, and one endpoint is related to the other endpoint and independent of each other.

It must be pointed out that, unless the context clearly indicates otherwise, the singular forms "a" and "the" as used in this specification and the scope of the attached patent application include a plurality of the indexed things. Therefore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

The protein-stabilized gold nanocube according to the present invention may be composed of a plurality of regularly arranged complexes. The complex includes the protein and the gold nanoclusters, and the gold nanoclusters are surrounded by the protein. That is, the complex system of the present invention refers to a protein-gold nano-cluster complex, but it is not limited to only consisting of gold nano-cluster and protein. That is, the composites of the present invention may still contain jinami Substances other than clusters and proteins.

The protein described in the present invention is not limited to a specific protein, that is, the protein may have an arbitrary sequence and may have an arbitrary configuration, as long as it can surround the gold nanoclusters. In some embodiments, the protein may be serum albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA), preferably BSA .

The gold nanocluster described in the present invention is composed of gold atoms, and the present invention is not limited in shape. For example, the nano-clusters can be composed of several to hundreds of gold atoms, preferably tens of gold atoms, for example, 10 to 12 gold atoms; the diameter (maximum length) can be 1 nm. To 10nm, or even less than 1nm.

The “gold nanoclusters are surrounded by the protein” in the present invention does not limit whether the gold nanoclusters have a binding relationship with the protein. That is, the protein and the nanocluster may also have binding forces similar to covalent bonds, ionic bonds, coordination chelation, or van der Waals, etc., so that the protein can be located on the nanocluster. In the surroundings, the present invention is not limited. In addition, the present invention does not limit the ratio of the amount of the gold nanoclusters to the protein. For example, a single gold nanocluster may be surrounded by one or more proteins, or a single protein may surround the gold nanoclusters. Without departing from the scope of the present invention.

The PSGNCs of the present invention have an emission wavelength of 468 nm. The emission wavelength in the present invention refers to the wavelength of the emitted light (e.g., fluorescent light) after the PSGNCs are excited by the incident light of the appropriate wavelength. Be restricted. Preferably, the emission wavelength herein may refer to a wavelength (or a maximum emission wavelength) having a maximum emission light intensity, or a position of an emission peak center. However, it should be understood that the PSGNCs of the present invention can still measure emitted light near their maximum emission wavelength.

In some embodiments, the PSGNCs described herein have an excitation wavelength of 405 nm. The excitation wavelength in the present invention refers to a wavelength of incident light that can excite PSGNCs and cause them to emit light (fluorescence). Preferably, the excitation wavelength herein may refer to the wavelength of the incident light (or the maximum excitation wavelength) that can cause the maximum emitted light intensity, or the position of the center of the excitation peak. However, it should be understood that PSGNCs of the present invention are not only excited by incident light having a wavelength of 405 nm.

Although unwilling to be limited by theory, Xianxin can prevent the gold nanoclusters from further clustering because the protein can surround the gold nanoclusters, thereby maintaining the size of the gold nanoclusters. In addition, the special excitation wavelength of the PSGNCs may be affected by the shape, density, and surface ligands of the gold nanoclusters.

The invention further provides a method for manufacturing a protein-stabilized gold nano cube, which can be used for manufacturing the aforementioned PSGNCs. The method includes: (I) mixing a chloroauric acid solution and a protein solution to form a mixed solution; and (II) adjusting the pH of the mixed solution to about pH 3.7.

The chloroauric acid (HAuCl ₄ ) solution is used to provide a source of gold ions, preferably an aqueous solution of chloroauric acid, and its concentration is preferably about 0.1M or less, and more preferably about 0.1mM to about 10mM. range. For example, the concentration of the chloroauric acid solution may be about 1 mM.

The protein solution may be an aqueous solution of the aforementioned protein, and its concentration is not limited. Preferably, about 0.1 g / ml to about 1 g / ml of protein may be included. For example, the protein solution may be an aqueous BSA solution having a concentration of about 0.5 g / ml. When mixing the chloroauric acid solution and the protein solution, about 0.1 mmol to about 10 mmol of chloroauric acid can be used to mix about 200 g to about 800 g of protein, for example, 1 mmol of chloroauric acid is used to mix about 500 g of protein. In some embodiments, an equal amount of a 1 mM chloroauric acid solution can be used to mix a 0.5 g / ml aqueous BSA solution.

The present invention does not limit the means for mixing the chloroauric acid solution and the protein solution, it is only necessary to make it It can be sufficiently uniformly mixed, for example, it can be mixed by shaking or mechanical stirring. In some embodiments, a magnetic stirrer is used for mixing at a speed of 400 rpm.

After mixing the chloroauric acid solution and the protein solution, a mixed solution can be formed. Generally speaking, the mixed solution is approximately acidic, and its pH value can be lower than pH 3.7. After the mixed solution is formed, the pH of the mixed solution is adjusted to about pH 3.7. For example, an alkaline agent is added to the mixed solution to increase the pH of the mixed solution. For example, the alkaline agent may be sodium hydroxide, sodium citrate, etc., preferably an aqueous solution of sodium hydroxide, such as an aqueous solution of sodium hydroxide having a concentration of about 1 mM. In some embodiments, it is preferable to stir the sodium hydroxide aqueous solution for about 3 hours at a speed of, for example, 600 rpm, so as to fully and uniformly mix it.

Furthermore, in some embodiments, it is preferred that after adjusting the pH of the mixed solution to about pH 3.7, the method of the present invention further comprises (III) leaving the mixed solution to stand for about 48 to about 96 hours. That is, the mixed solution is prevented from being disturbed by shaking or mechanical stirring. Preferably, the mixed solution can be left to stand at a temperature of about 37 ° C., for example, left to stand for about 60 to about 84 hours, and more preferably left to stand for about 72 hours.

The method of the present invention can form the PSGNCs without adding other amino acids, and without stirring / shaking the mixed solution of the chloroauric acid solution and the protein solution for a long time (for example, 12 hours). Compared with other methods for synthesizing gold nanoclusters, the solution is usually adjusted to be alkaline, while the present invention only adjusts the pH of the solution to a range of about pH 3.7. In addition, the method of the present invention does not require long-term stirring, and only needs to stand at 37 ° C (without mechanical stirring).

Although unwilling to be limited by theory, Xianxin adjusts the mixed solution to a special pH environment (about pH 3.7), so the problem of excessive aggregation of gold atoms, excessive size, or sedimentation can be avoided without stirring. Due to the long and unstirred process, a single-layered ligand (that is, the protein) can be formed on the surface of the gold nanoclusters. The protein can form a restricted space, so the etching of the edges of the gold nanoclusters can be avoided. (etching) or chelation, so that the nano The shape of the clusters is non-spherical. Therefore, the method of the present invention can make the gold nanoclusters have a specific density, shape, and / or surface ligand type. Compared with the alkaline environment of other methods for synthesizing gold nanoclusters, it can cause the gold nanoclusters of the present case. The fluorescence response of the clusters is blue shifted, so that the obtained PSGNCs have a specific excitation wavelength and / or emission wavelength as described above. For example, the excitation wavelength of the PSGNCs can be lowered to about 405 nm and / or the emission wavelength can be lowered to about 468 nm, so that the PSGNCs can have special applications, for example, can be used to detect alkaline phosphatase.

The invention further relates to the application of the aforementioned PSGNCs, for example, for detecting alkaline phosphatase (ALP). That is, the present invention provides a method for detecting alkaline phosphatase, comprising: (a) providing a sample to be tested, which includes alkaline phosphatase; (b) mixed p-nitrophenyl phosphate (p-nitrophenyl phosphate, abbreviated as pNPP or p-NPP), the aforementioned protein-stabilized gold nano cubes and the sample to be tested to form a test mixture; and (c) exciting the test mixture with light at a wavelength of 405 nm, and detecting the test mixture The emission intensity of the mixed solution at a wavelength of 468 nm was measured.

The invention does not limit the composition and source of the sample to be tested. For example, the sample to be tested may be blood or serum samples of humans or animals, milk, other drugs or reagents. The sample to be tested may be further processed by centrifugation, precipitation, and the like. Preferably, the test sample is a serum sample.

Please refer to FIG. 1A. Alkaline phosphatase can catalyze the hydrolysis of p-NPP in the presence of water to remove phosphate groups from p-NPP to generate p-nitrophenol (p-nitrophenol, pNP or p-NP for short). ) And inorganic phosphate (inorganic phosphate). Therefore, after mixing p-NPP, the aforementioned PSGNCs and the test sample, the ALP in the test sample will catalyze the hydrolysis of p-NPP to generate p- NP, so there will be p-NP in the test mixture formed.

Preferably, the method of the present invention mixes quantitative amounts of p-NPP, the aforementioned PSGNCs, and the test sample, so the amount of p-NP generated can be used to convert the ALP concentration in the test sample.

In some embodiments, the foregoing step (b) may include: (bi) mixing the p-nitrophenyl phosphate with the sample to be tested, so that the alkaline phosphatase reacts with the p-nitrophenyl phosphate; and ( bii) Mix the protein-stabilized nanometer cube and the mixed p-nitrophenyl phosphate with the test sample.

That is, p-NPP and the test sample can be mixed first, and ALP and p-NPP in the test sample can be reacted to generate p-NP, and then the PSGNCs can be mixed therewith. Since alkaline phosphatase is more active in alkaline environment, it is better to make ALP and p-NPP react in alkaline environment, for example, at pH 7 or higher, preferably at about pH 8 to about pH 11, It is more preferable to an environment of about pH 9 to about pH 10. Because the absorption of p-NPP at 405nm is extremely low, but p-NP has obvious absorption at 405nm, it can be used to distinguish between un-hydrolyzed p-NPP and p-NP produced after hydrolysis. .

The method for detecting alkaline phosphatase of the present invention is achieved by an inner filter effect (IFE). IFE is a label-free detection technology, and is suitable for the detection of low-concentration analytes. IFE was proposed by Gabor and Walt in 1991. It includes the participation of two chemical substances, and the absorption bands of these two chemical substances overlap each other. For example, as shown in FIG. 1B, PSGNCs can absorb incident light with a wavelength of 405 nm, and when excited, emit fluorescence with a wavelength of 468 nm, and the excitation of PSGNCs is not affected by the presence of p-NPP. If there is a quantitative amount of PSGNCs in solution, theoretically the fluorescence intensity emitted by the PSGNCs should be a fixed value.

However, referring to FIG. 1C, since p-NP also absorbs incident light with a wavelength of 405 nm, it does not emit fluorescence with a wavelength of 468 nm, and p-NP has a high extinction coefficient of about 17500M ^-1 cm ^-1 (extinction coeeficient), if ALP and p-NPP are present in the solution at the same time, p-NPP will hydrolyze to produce p-NP, and p-NP will compete to absorb incident light with a wavelength of 405nm, shielding PSGNCs, resulting in excited PSGNCs The decrease in the amount of fluorescein leads to a decrease in the emitted fluorescence intensity at a wavelength of 468 nm. The decrease in the fluorescence intensity can be used to convert the concentration of p-NP in the solution.

The amount of p-NP in the solution was related to the concentration of ALP, and the fluorescence emission intensity of PSGNCs also showed a relative change. Therefore, the method of the present invention excites the mixed solution to be tested with light having a wavelength of 405 nm, and detects the emission intensity of the mixed solution to be tested at a wavelength of 468 nm. The amount of p-NP in the solution can be obtained from the decrease in the emission intensity , And then converted to know the concentration of ALP in the test sample.

For example, in some embodiments, the method for detecting alkaline phosphatase may further include: (d1) providing a plurality of test mixtures, including separately mixing p-nitrophenyl phosphate and the protein-stabilized gold nano cube And alkaline phosphatase of known concentration, wherein the concentrations of alkaline phosphatase in each of the plurality of test mixtures are different; (e1) each of the test mixtures is excited with light at a wavelength of 405 nm, and each of the test solutions is detected Measure the emission intensity of the mixed solution at a wavelength of 468 nm; and (f1) establish a calibration line with the emission intensities of the plurality of test mixtures, and compare the emission intensity of the test mixture in step (c) with the test line , Convert the alkaline phosphatase concentration in the test sample.

In the above step (d1), it is preferable to mix quantitative amounts of p-NPP and PSGNC, and the mixing amount is preferably the same as that of the test solution. In addition, it is preferable that the matrix (solvent) (Liquid conditions and components) are substantially the same as the aforementioned mixed liquid to be tested, so that the detection result can be more accurate. Preferably, the concentration range of alkaline phosphatase in the plurality of test mixtures can cover the concentration of the sample to be tested.

In some other embodiments, the method for detecting alkaline phosphatase may include: (d2) providing a reference mixed solution, comprising mixing the sample to be tested, p-nitrophenyl phosphate, the protein-stabilized nanometer cube, and A known concentration of alkaline phosphatase; (e2) excite the reference mixture with light at a wavelength of 405 nm and measure its emission intensity at a wavelength of 468 nm; and (f2) calculate the emission of the test mixture in step (c) The intensity and the emission intensity difference of the reference mixed solution, and calculate the alkaline phosphatase concentration in the sample to be tested based on the emission intensity difference.

In step (d2), since the alkaline phosphatase of known concentration is directly mixed into the sample to be tested, the influence of the matrix (solution conditions and components) can be avoided, and the detection result can be more accurate. In addition, a plurality of reference mixtures can be prepared, each containing alkaline phosphatase at different concentrations, and the regression curve can be used to calculate the alkaline phosphatase concentration in the test sample to reduce the effect of experimental errors.

The following non-limiting examples are helpful to those having ordinary knowledge in the technical field to which the present invention pertains. These examples should not be seen as unduly limiting the invention. Those with ordinary knowledge in the technical field to which the present invention pertains can make modifications and changes to the embodiments discussed herein without departing from the spirit or scope of the present invention, and still fall within the scope of the present invention.

Example 1: Synthesis of protein-stabilized nanometer cubes

An equal volume of a 1 mM chloroauric acid solution and a 0.5 g / ml BSA solution were mixed and stirred at a speed of 400 rpm until they were uniformly mixed to form a mixed solution. Then, quickly add a 1 mM sodium hydroxide aqueous solution to the mixture to make the pH value about pH 3.7, and stir at 600 rpm. Stir for 3 hours. Then, the mixed solution was left standing (without stirring) at 37 ° C. for 72 hours to form the PSGNCs. The relative quantum yield of the PSGNCs was 28.64%, which was calculated with reference to quinine sulfate (58%). Observing the PSGNCs with a transmission electron microscope, the obtained image is shown in FIG. 2, and the side lengths of the PSGNCs are about 50 nm to 70 nm.

Example 2: Properties of protein-stabilized nanometer cubes

The protein-stabilized gold nano cubes (PSGNC) obtained above, as well as p-nitrophenyl phosphate (pNPP), alkaline phosphatase (ALP), bovine serum albumin (BSA), and p-nitrophenol (pNP) were fluoresced. The results of light-visible (UV-vis) absorption spectrum detection are shown in FIG. 3A.

As shown in FIG. 3A, PSGNCs and p-NP also have a hump absorption at a wavelength of 300 nm. The absorption of PSGNCs here may be related to quantum sized homomoleptic cluster formation (Au _10-12 ), and it shows that it has an aspheric structure.

In addition, the above-obtained protein-stabilized gold nano cubes (PSGNC), bovine serum albumin (BSA), and p-nitrophenol (pNP) were detected by emission spectrum, excited at a wavelength of 370 nm, and the emission spectrum was recorded as shown in FIG. 3B As shown.

It can be seen from FIGS. 3A and 3B that the PSGNCs and pNP both have obvious absorption at 405 nm, and the effects of the other components can be eliminated. In addition, the PSGNCs have an excitation wavelength of 405 nm and an emission wavelength of 468 nm, and have a maximum fluorescence intensity at an emission wavelength of 468 nm.

Example 3: Detection of alkaline phosphatase in aqueous solution

Dissolve 20U of ALP in 10ml of reaction solution (the reaction solution contains 100M Tris-HCl (pH 9.5), 50mM magnesium chloride, 10mM sodium chloride, 0.2% Tween 20) for later use; prepare p-NPP as a 100mM solution for later use ; In addition, the PSGNCs obtained in the foregoing Example 1 were diluted 10 times for use.

The above 20U ALP solution was diluted with deionized water to a concentration of 1U, 0.5U, 0.25U, 0.125U, 0.0625U, 0.312U, respectively, and mixed with p-NPP (final concentration is 1mM) to perform a reaction. The reaction volume The reaction temperature was 500 μl, the reaction temperature was 37 ° C., and the reaction time was 30 minutes.

After the reaction was completed, 500 μl of the above PSGNCs solution was added to form a calibration mixture, and the samples were excited with light at a wavelength of 405, and the fluorescence spectrum emitted was detected. The results are shown in FIG. 4A. The higher the concentration of ALP in the test mixture, the lower the fluorescence intensity emitted by PSGNCs.

In addition, the relative fluorescence intensity F _{0 (initial)} / F _{(final) of} each test mixture at the emission wavelength of 468 nm was calculated, and the ALP concentration was plotted as shown in FIG. 4B. Calculate the linear regression determination coefficient R ² = 0.9873, showing that F _{0 (initial)} / F _(final) has a linear response to ALP concentration, and calculate the limit of detection (LOD) to 1.616U. / L (S / N = 3).

Example 4: Detection of alkaline phosphatase in serum

Serum was isolated from a human blood sample as a serum sample, and ALP was added to a concentration of 1U, 0.5U, 0.25U, 0.125U, 0.0625U, 0.312U to 5 μl of the serum sample, and mixed with p-NPP (final concentration of 1mM) to The reaction was performed with a reaction volume of 500 μl, a reaction temperature of 37 ° C., and a reaction time of 30 minutes.

After the reaction was completed, 500 μl of the above PSGNCs solution was added to form a calibration mixture, and the samples were excited with light at a wavelength of 405, and the fluorescence spectrum emitted was detected. The results are shown in FIG. 5A. The results measured in the serum samples were similar to those in FIG. 4A.

In addition, the relative fluorescence intensity F _{0 (initial)} / F _{(final) of} each test mixture at the emission wavelength of 468 nm will be calculated, and the ALP concentration is plotted as shown in Figure 5B. Calculate the linear regression determination coefficient R ² = 0.9808, which is also shown in the serum sample. F _{0 (initial)} / F _(final) has a linear relationship with the concentration of ALP, and the detection limit is calculated as 9.441 U / L (S / N = 3).

Example 5: Selectivity of alkaline phosphatase detection method

Thrombin (Th), hemoglobin (Hb), and lysozyme (Lys) are metalloenzymes that belong to the same metal group as ALP, and BSA and HSA are present in large amounts in serum. This method is used to test the selectivity of the alkaline phosphatase detection method of the present invention.

The samples contained Th (1U / ml), Hb (0.5μg / ml), Lys (0.02U / ml), BSA (25mg / ml), and HSA (25mg / ml). The above concentrations are all common in serum Existing concentration. In the same steps as above, the samples were separately mixed with p-NPP (final concentration of 1 mM) for reaction. The reaction volume was 500 μl, the reaction temperature was 37 ° C., and the reaction time was 30 minutes.

After the reaction was completed, 500 μl of the above PSGNCs solution were separately added and excited with light at a wavelength of 405, and the fluorescence intensity at an emission wavelength of 468 nm was detected. The calculation of F _{0 (initial)} / F _(final) is shown in FIG. 6. Because only ALP can convert p-NPP to p-NP, which significantly reduces the fluorescence intensity; on the other hand, none of the other proteins can convert p-NPP to p-NP, so it does not significantly reduce the fluorescence intensity. The method of the present invention has good selectivity for ALP.

The above embodiments are only for explaining the principle of the present invention and its effects, but not for limiting the present invention. Modifications and changes made by those skilled in the art to the above embodiments still do not violate the spirit of the present invention. The scope of rights of the present invention should be listed in the scope of patent application described later.

Claims (8)

A protein stabilized gold nanocubes, comprising: a plurality of complexes, each of which comprises: a protein; and a gold nanoclusters surrounded by the protein; The emission wavelength of the protein-stabilized nanometer cube is 468nm, and the protein-stabilized nanometer cube is prepared by the following method: (I) mixing an equal amount of a 1mM chloroauric acid solution and a 0.5g / ml beef A serum albumin (BSA) solution to form a mixed solution; and (II) adjust the pH of the mixed solution to about pH 3.7, and leave the mixed solution for about 72 hours. For example, the protein-stabilized nanometer cube of claim 1 has a side length of about 50 nm to about 70 nm. A method for manufacturing a protein-stabilized gold nano cube as claimed in claim 1 or 2, comprising: (I) mixing an equal amount of a 1 mM chloroauric acid solution and a 0.5 g / ml bovine serum albumin solution to form a mixture And (II) adjust the pH of the mixed solution to about pH 3.7, and continue to stand for about 72 hours. A method for detecting alkaline phosphatase, comprising: (a) providing a test sample containing alkaline phosphatase; (b) mixing p-nitrophenyl phosphate, as requested The protein-stabilized nanometer cube of item 1 or 2 and the sample to be tested to form a mixed solution to be tested; and (c) Exciting the mixed solution to be tested with light having a wavelength of 405 nm, and detecting that the mixed solution to be tested is at a wavelength Emission intensity at 468 nm. The method of claim 4, wherein step (b) comprises: (bi) mixing the p-nitrophenyl phosphate with the sample to be tested so that the alkaline phosphatase reacts with the p-nitrophenyl phosphate; and ( bii) Mix the protein-stabilized nanometer cube and the mixed p-nitrophenyl phosphate with the test sample. The method according to claim 4, further comprising: (d1) providing a plurality of test mixtures, including separately mixing p-nitrophenyl phosphate, the protein-stabilized gold nano cube, and a known concentration of alkaline phosphatase, each of which The concentrations of alkaline phosphatase in the plurality of test mixtures are different; (e1) each of the test mixtures is excited with light at a wavelength of 405 nm, and the emission intensity of each of the test mixtures at a wavelength of 468 nm is detected; and ( f1) Establish a calibration curve based on the emission intensities of the plurality of test mixtures, and compare the emission intensity of the test mixture in step (c) with the calibration curve to convert the alkaline phosphatase in the test sample. concentration. The method of claim 4, further comprising: (d2) providing a reference mixed solution, comprising mixing the test sample, p-nitrophenyl phosphate, the protein-stabilized gold nano cube, and alkaline phosphatase of known concentration; (e2) Excite the reference mixture with light at a wavelength of 405 nm and measure its emission intensity at a wavelength of 468 nm; and (f2) Calculate the emission intensity of the test mixture in step (c) and the emission intensity of the reference mixture Difference, and calculate the alkaline phosphatase concentration in the test sample based on the difference in emission intensity. The method according to any one of claims 4 to 7, wherein the test sample is a serum sample.