Novel SERS (Surface Enhanced Raman Scattering) material Au-coated PB NPs as well as synthesis method and application thereof in cholesterol detection

Abstract

The invention discloses a novel SERS (Surface Enhanced Raman Scattering) material Au-coated PB NPs and a synthesis method and application thereof in cholesterol detection.The synthesis method comprises the steps that gold nanoparticles are synthesized through a sodium citrate reduction method, then the gold nanoparticles are mixed with potassium ferricyanide to obtain gold nanoparticles coated with cyano groups, then potassium ferrocyanide trihydrate and a ferric trichloride solution are injected in a staggered mode, and the gold nanoparticles coated with cyano groups are obtained; reacting to prepare Prussian blue coated gold nanoparticles (Au coated PB NPs); the material can generate an internal standard signal in a Raman silence zone, an SERS rapid quantitative detection method without adding an additional internal standard substance or pretreatment is realized, and by combining Au-coated PB NPs with a Raman method, the special optical characteristics of the material and the advantages of SERS fingerprint identification are combined, so that the sensitivity of SERS fingerprint identification is greatly improved, and the sensitivity of SERS fingerprint identification is improved. The sensitivity, linear range and detection limit of cholesterol detection are improved, and the operation is simple, convenient and efficient.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and specifically relates to a novel SERS material Au@PB NPs, a synthesis method thereof, and an application thereof in cholesterol detection. Background Art

[0002] Cholesterol is a derivative of cyclopentane polyhydrophenanthrene and plays an important role in basic cell life activities. Cholesterol detection is important in the evaluation and management of various diseases. High cholesterol, especially low-density lipoprotein (LDL) levels, is closely related to the risks of cardiovascular diseases, coronary heart disease, and atherosclerosis. At the same time, it is also an important indicator for diseases such as type 2 diabetes, metabolic syndrome, and fatty liver. Therefore, rapid and accurate detection of cholesterol is of great significance.

[0003] So far, a variety of methods for detecting cholesterol have been developed, such as: electrochemical method, immunoassay, colorimetry, fluorescence method, and chromatography, etc. Electrochemistry offers the advantages of low cost and easy operation. However, it has the disadvantages that the substance must have reduction and oxidation potentials, poor selectivity, and other electroactive substances also affect the measurement accuracy; the immunoassay has low cost and simple operation, but its sensitivity is low; colorimetry can be easily carried out by identifying color changes, but its application may be limited by the interference of the background color of the test sample; fluorescence methods have relatively high sensitivity, but they may suffer from photobleaching or phototoxicity; chromatography can separate, detect, and quantify such metabolites, but it usually involves expensive instruments and cumbersome sample preparation procedures.

[0004] CN 114100586 A discloses a composite material nanozyme and a preparation method thereof. This method synthesizes the composite material NSC / Co 1-xThe hydrogen peroxide produced by the reaction of cholesterol with its oxidase catalyzed by S nanozyme is used to determine the cholesterol content by colorimetry. However, the material synthesis steps of this method are complex and require high-temperature calcination under an inert gas atmosphere, and the reaction conditions are harsh. CN 115753924A discloses a laser-reduced graphene oxide electrochemical electrode and its preparation method. This method uses a three-electrode electrodeposition system and utilizes a laser-reduced graphene oxide device, which can be used as an electrochemical sensor for detecting cholesterol. The detection cost is low and it can be mass-produced, but environmental factors such as temperature and pH may affect the electrochemical signal, and the existing technology has some deficiencies. The Journal of Lipid Research published a research on the method for quantitatively detecting cholesterol by gas chromatography. This method couples gas chromatography with flame ionization detection (GC / FID), and the detection accuracy is high. However, this method requires sample pretreatment and relies on large instruments, which are expensive and the detection time is long. ACS Applied Materials & Interfaces published a research on the on-site colorimetric detection of cholesterol based on polypyrrole nanoparticles. The article couples polypyrrole nanoparticles to induce the reaction of H 2 O 2 and 3,3′,5,5′-tetramethylbenzidine (TMB) to determine the cholesterol content by colorimetry. This method is easily affected by the color of the reagent and has low sensitivity, and there are still deficiencies.

[0005] For the above reasons, the application and popularization of cholesterol detection methods are restricted. Therefore, it is necessary to develop a cholesterol detection method with simple sample treatment, high sensitivity, high accuracy, little environmental influence, and portable instrument. Surface-enhanced Raman scattering (SERS), due to its extensive fingerprint information, has become a powerful tool currently applied to biochemical analysis. It is less sensitive to environmental variables, has high sensitivity, high specificity, the simplicity and practicality of portable instruments, and can provide timely, accurate, and non-destructive information. The local surface plasmon resonance (LSPR) of gold (Au) generates "hot spots". When the analyte molecule approaches the "hot spot" field, the Raman signal will be significantly amplified; Prussian blue (PB) has a unique structure and can generate an internal standard signal in the Raman silent region (1800-2800 cm -1 ) to avoid overlapping with other signals and improve reliability. Au@PB nanocomposites have been used in a variety of biological detections due to their unique optical properties and good biocompatibility. However, there is currently no report on using Au@PB NPs materials combined with Raman spectroscopy technology to detect cholesterol content.

[0006] The present invention provides a novel SERS material and its synthesis method. The obtained gold nanoparticles encapsulated with Prussian blue can catalyze the reaction to proceed and amplify the Raman signal of the sample. Prussian blue, as an endogenous internal standard, has a signal only in the Raman silent region, avoiding the overlapping interference of endogenous biomolecular signals during the detection process, thereby improving the accuracy of quantitative determination of cholesterol. The Raman instrument is portable, simple to operate, fast and sensitive in detection, and the result is not interfered by the color of the sample. A new method for rapid quantitative detection of cholesterol by SERS without adding extra internal standard, without pretreatment, and with a wide linear range is realized. Summary of the Invention

[0007] The object of the present invention is to provide a novel SERS material Au@PB NPs and its synthesis method and application.

[0008] The gold nanoparticles encapsulated with Prussian blue obtained in the present invention can catalyze the reaction of cholesterol with cholesterol oxidase to generate hydrogen peroxide, induce the reaction of hydrogen peroxide with 3,3',5,5'-tetramethylbenzidine (TMB), and obtain oxidized TMB (oxTMB) with Raman signal. This material can also enhance the Raman signal of oxTMB for the detection of cholesterol. This method can perform rapid and accurate SERS quantitative detection without adding extra internal standard and without pretreatment. Using the method of combining Au@PB NPs with Raman to detect cholesterol content has the advantages of high sensitivity, wide linear range, and simple, rapid operation.

[0009] The technical solution of the present invention is as follows:

[0010] A synthesis method of a SERS material Au@PB NPs, comprising the following steps:

[0011] (1) Preparation of gold nanoparticles

[0012] Add chloroauric acid solution to ultrapure water, heat to boiling and incubate at 120 °C for 1 min, add trisodium citrate solution, stir (1000 - 1300 rpm) for 5 min (observing the change of the solution from colorless to wine red), cool to room temperature, and obtain gold nanoparticles (without separation, directly store in the form of solution at 2 - 8 °C in the dark);

[0013] Preferably, the concentration of the chloroauric acid solution is 10 mg / mL; the concentration of the trisodium citrate solution is 10 mg / mL;

[0014] Preferably, the volume ratio of the chloroauric acid solution, ultrapure water, and trisodium citrate solution is 1:60:1;

[0015] (2) Preparation of gold nanoparticles encapsulated with Prussian blue

[0016] Mix the gold nanoparticles obtained in step (1) with K 3[Fe(CN) 6 solution was vortex-mixed for 10 min to obtain Au@CN - NPs (gold nanoparticles coated with cyanide) solution. K 4 [Fe(CN) 6 ·3H 2 O solution and FeCl 3 solution were injected into the Au@CN - NPs solution at the same speed, stirred (500 - 600 rpm) for 3 h, then centrifuged (5000 rpm, 15 min) to discard the supernatant, and dispersed and washed with ultrapure water to obtain Au@PB NPs (gold nanoparticles encapsulated with Prussian blue);

[0017] Preferably, the concentration of K 3 [Fe(CN) 6 solution is 500 μM; the concentration of K 4 [Fe(CN) 6 ·3H 2 O solution is 100 μM; the concentration of FeCl 3 solution is 100 μM;

[0018] Preferably, the volume ratio of the gold nanoparticles obtained in step (1), K 3 [Fe(CN) 6 solution, K 4 [Fe(CN) 6 ·3H 2 O solution, and FeCl 3 solution is 15 - 25:5 - 10:1:1;

[0019] Preferably, K 4 [Fe(CN) 6 ·3H 2 O solution and FeCl 3 solution are alternately injected into the vortexed Au@CN - NPs solution at the same speed, and the injection speed is 10 - 20 μL / 10 - 30 s;

[0020] The obtained Au@PB NPs were resuspended in ultrapure water and stored in a 4°C refrigerator protected from light.

[0021] The present invention relates to Au@PB NPs prepared by the above synthesis method.

[0022] The Au@PB NPs of the present invention can be used for the determination of cholesterol content, and the specific method is as follows:

[0023] (1) Prepare a standard working solution

[0024] Disperse cholesterol powder in ethanol and dilute it with PBS buffer (containing 1% Triton X-100) to obtain cholesterol solutions with a series of concentrations; take the cholesterol solution, add cholesterol oxidase and PBS buffer, and incubate at 37 °C for 5-15 min to obtain the standard working solution;

[0025] Preferably, the concentration range of the cholesterol solution is 10-500 μΜ;

[0026] (2) Plot the standard curve

[0027] Sequentially add the standard working solution obtained in step (2), TMB (3,3′,5,5′-tetramethylbenzidine) solution, and Au@PB NPs solution to the HAc-NaAc buffer (pH = 4), incubate at room temperature for 5-15 min, detect the Raman signal, and use the ratio of the SERS signal at 1589 cm -1 to the SERS signal at 2155 cm -1 as the ordinate and the logarithm of the cholesterol concentration as the abscissa to plot the standard curve;

[0028] (3) Detect the cholesterol content

[0029] Take the cholesterol solution to be tested, add cholesterol oxidase and PBS buffer, and incubate at 37 °C for 5-15 min to obtain the sample to be tested. Detect the Raman signal according to the method in step (2), and substitute the signal value into the standard curve plotted in step (2) to obtain the cholesterol content of the sample to be tested.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a novel SERS material Au@PB NPs, its synthesis method and application in cholesterol detection. Au@PB NPs can catalyze the reaction and simultaneously amplify the Raman signal of the product. PB shows a single, strong and unique Raman vibration band at 2155 cm -1 in the Raman silent spectral region, which can be used as an excellent internal standard with low overlap with the analyte fingerprint band, thus reducing inaccuracies when studying complex systems.

[0032] The Raman instrument is portable, easy to operate, fast and sensitive in detection, and the result is not interfered by the color of the sample. Combining the Au@PBNPs material with portable Raman detection can obtain a cholesterol detection method with high sensitivity, high accuracy and convenient detection. The linear range of this method is wider than that of the existing reported methods, which is 10-500 μM; the detection limit in the existing reports is generally in the range of 1×10 -6 -1×10 -12 M, and the detection limit of this method is 0.19×10 -12M is lower than this range. A wider linear range and lower detection limit make the applicability of sample detection broader and the detection results more reliable. Brief Description of the Drawings

[0033] Figure 1 : Scanning electron microscope images of (A) Au NPs and (B) Au@PB NPs.

[0034] Figure 2 : EDS images of (A) Au element in Au NPs, and (B) Au, (C) C, (D) N, (E) Fe elements in Au@PB NPs.

[0035] Figure 3 : UV-visible absorption spectra of PB, Au NPs, and Au@PB NPs.

[0036] Figure 4 : SERS spectra of different substances.

[0037] Figure 5 : (A) Raman spectra of H 2 O 2 at different concentrations and (B) linear relationship between the logarithm of H 2 O 2 concentration and I 1589 / I 2155

[0038] Figure 6 : (A) Detection of the SERS intensity of Au@PB NPs within 20 days and (B) detection of the SERS signal intensity of 10 batches of Au@PB NPs.

[0039] Figure 7 : (A) Raman spectra of cholesterol at different concentrations and (B) linear relationship between the logarithm of cholesterol concentration and I 1589 / I 2155 Detailed Description of the Embodiments

[0040] To make the objectives, technical solutions, and advantages of the present invention clearer, the following further elaborates on the present invention in detail with reference to the accompanying drawings and specific embodiments. The described embodiments are only partial embodiments of the present invention, rather than all embodiments. All other implementation manners obtained by those of ordinary skill in the art without making creative improvements fall within the scope protected by the present invention.

[0041] Example 1

[0042] Preparation of gold nanoparticles (Au NPs). Specifically:

[0043] Take 1000 μL (10 mg / mL) of chloroauric acid solution, add it to 60 mL of ultrapure water, then heat the solution to boiling and incubate at 120 °C for 1 minute. Quickly add 1 mL of trisodium citrate solution (1%) and stir the reaction at 1200 rpm for 5 minutes. A change from a colorless solution to a wine-red color is observed. After the reaction is completed, stir the solution to cool it to room temperature and store it in the refrigerator at 2 - 8 °C in the dark.

[0044] Example 2

[0045] Preparation of Prussian blue-coated gold nanoparticles (Au@PB NPs). Specifically:

[0046] Vortex mix 10 mL of Au NPs (prepared in Example 1) with 3 mL of potassium ferricyanide K 3 [Fe(CN) 6 (500 μM) solution for 10 min to obtain Au NPs coated with CN - . Then, inject potassium ferrocyanide trihydrate K 4 [Fe(CN) 6 ·3H 2 O (100 μM) solution and FeCl 3 (100 μM) solution into the Au@CN - NPs solution at the same speed and stir the reaction for 3 h. After the reaction is completed, centrifuge at 5000 revolutions per minute for 15 min, discard the supernatant, redisperse and wash it 3 times in ultrapure water, resuspend it in 1 mL of ultrapure water, and store it in the refrigerator at 4 °C in the dark.

[0047] Perform corresponding scanning electron microscopy, EDS, ultraviolet, and SERS characterizations on each layer of the "Au@PB NPs" constructed. The results are shown in Figure 1 , Figure 2 , Figure 3 , Figure 4 .

[0048] Figure 1 In, (A) Scanning electron microscopy image of Au NPs at 150,000 times magnification, (B) Scanning electron microscopy images of Au@PB NPs at 150,000 times and 300,000 times magnification. The synthesized Au NPs and Au@PB NPs show regular spherical particles, evenly arranged. The particle size of Au nanoparticles is about 30 nm, and the particle size of Au@PB nanoparticles is about 35 nm.

[0049] Figure 2Energy-dispersive spectroscopy (EDS) was used to detect Au NPs and Au@PB NPs. EDS spectra can effectively analyze the elements contained in a sample. Among them, (A) shows that Au elements are mainly distributed on the surface of Au NPs, and (B-E) show that Au, C, N, and Fe elements are mainly distributed on the surface of Au@PB nanoparticles, indicating that PB was successfully coated on the surface of Au.

[0050] Figure 3 The UV-visible absorption spectra of PB, Au NPs, PB NPs, and Au@PB NPs are shown. The two absorption peaks of gold nanoparticles (Au NPs) and Prussian blue nanoparticles (PB NPs) are located at 530 nm and 740 nm respectively. For Au@PB NPs, these two absorption peaks appear simultaneously, which further proves the successful synthesis of Au@PB NPs.

[0051] Figure 4 The SERS spectra of different substances are shown. Compared with the Au NPs spectrum, in the Raman spectrum of Au@PB NPs, a strong PB Raman vibration band feature is shown at 2155 cm -1 −1. And different from many other internal standard substances, this PB spectral band is located in the Raman silent region, thus greatly reducing signal overlap and interfering with the signal of the analyte, and is suitable for use as an internal standard for SERS quantitative analysis in complex systems. In the system of Au@PB NPs and TMB, weak characteristic peaks appear at 497 cm -1 −1, 553 cm -1 −1, 1178 cm -1 −1 and 1589 cm -1 −1. These characteristic peaks are attributed to C-N bending, skeletal ring deformation, and C-N stretching respectively. In the TMB-H 2 2 2 O mixture, TMB is oxidized to ox TMB, and the typical characteristic peaks at 553 cm -1 −1, 1178 cm -1 −1, 1319 cm -1 −1 and 1589 cm -1 −1 are enhanced.

[0052] Example 3

[0053] Au@PB NPs were used to detect different concentrations of H 2 2 2 . Specifically:

[0054] 13 μL of TMB (0.5 mM) solution and 175 μL of Au@PB NPs solution (prepared in Example 2) were added to 940 μL of HAc-NaAc buffer solution with a pH value of 4. Different concentrations of H 2 2 2(10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M and 10 -12 M) was taken, 25 μL was added to the above mixed solution, and incubated at room temperature for 10 min. The Raman signal was detected and the characteristic peaks were quantitatively analyzed.

[0055] Figure 5 Among them, (A) the SERS signals at 553 cm -1 , 1178 cm -1 , 1319 cm -1 and 1589 cm -1 increased with the increase of H 2 O 2 concentration. It shows that H 2 O 2 effectively converted TMB into ox TMB and immobilized ox TMB on the surface of Au@PB NPs. A strong characteristic of the PB Raman vibration band was shown at 2155 cm -1 , and the repeatability was good. Through the fitting process of the data, it was found that taking PB as the internal standard, after standardizing the peak at 1589 cm -1 , there was an approximate linear relationship y = -2.2015x + 29.343 with the logarithm of H 2 O 2 concentration, where x is the logarithm of H 2 O 2 concentration, and y is the ratio (R -1 ) of the SERS signal at 1589 cm -1 to the SERS signal at 2155 cm 2 (R Figure 5 = 0.9914), as shown in 2 O 2 The detection limit of H -12 was calculated to be 0.19×10

[0056] Example 4

[0057] Stability and repeatability tests of Au@PB NPs. Specifically:

[0058] Ten batches of Au@PB NPs materials were synthesized according to the above method. The stability of one batch of materials was analyzed for 20 days, and the calculated RSD was 5.2%, as shown in Figure 6 (A); at the same time, ten batches of materials were detected with H of the same concentration. 2 O 2 It can be seen from the figure that after normalizing the Raman signal at 1589 cm -1 , the degree of data dispersion decreased, and the RSD was 4.87%, as shown in Figure 6 (B). In summary, the material has good stability and repeatability.

[0059] Example 5

[0060] Quantitative analysis of cholesterol with PB as the internal standard. Specifically:

[0061] 0.01 g of cholesterol powder was dispersed in 1 mL of ethanol and diluted to different concentrations of cholesterol (10, 20, 40, 60, 80, 100, 200, 300, 400, 500 μM) with 9 mL of PBS (containing 1% Triton X-100). 10 μL of the cholesterol solution was taken and added to 5 μL of cholesterol oxidase (1 U / mL) and 45 μL of PBS buffer, and incubated at 37 °C for 10 min. 25 μL of the above reaction solution was taken and added to a cuvette containing Au@PB NPs, TMB and HAc-NaAc buffer to detect the Raman signal. Cholesterol oxidase was added to cholesterol for the detection of cholesterol concentration. When the cholesterol concentration was 10 - 500 μM, I 1589 / I 2155 showed a good linear relationship with the logarithmic concentration of cholesterol Y = 1.6049X - 0.4811, R 2 = 0.9926, as shown in Figure 7 .

[0062] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing Au@PB NPs, a SERS material, characterized in that: The following steps are involved: (1) Preparation of gold nanoparticles The chloroauric acid solution was added to ultrapure water, heated to boiling and incubated at 120°C for 1 min, trisodium citrate solution was added, stirred for reaction for 5 min, and cooled to room temperature to obtain gold nanoparticles; (2) Preparation of Prussian blue-coated gold nanoparticles The gold nanoparticles obtained in step (1) were vortex-mixed with the K3[Fe(CN)6] solution for 10 min to obtain Au@CN - NPs solution, K4[Fe(CN)6]·3H2O solution and FeCl3 solution were injected into Au@CN at the same speed. - The NPs solution was stirred for reaction for 3 h, then centrifuged and the supernatant was discarded, and the solution was dispersed and washed with ultrapure water to obtain Au@PB NPs.

2. The method for synthesizing the SERS material Au@PB NPs according to claim 1, characterized in that: In step (1), the concentration of the chloroauric acid solution is 10 mg / mL; the concentration of the trisodium citrate solution is 10 mg / mL.

3. The method for synthesizing the SERS material Au@PB NPs according to claim 1, characterized in that: In step (1), the volume ratio of chloroauric acid solution, ultrapure water and trisodium citrate solution is 1:60:

1.

4. The method for synthesizing the SERS material Au@PB NPs according to claim 1, characterized in that: In step (2), the concentration of K3[Fe(CN)6] solution is 500 μM; the concentration of K4[Fe(CN)6]·3H2O solution is 100 μM; and the concentration of FeCl3 solution is 100 μM.

5. The method for synthesizing the SERS material Au@PB NPs according to claim 1, characterized in that: In step (2), the volume ratio of gold nanoparticles, K3[Fe(CN)6] solution, K4[Fe(CN)6]·3H2O solution, and FeCl3 solution is 15-25:5-10:1:

1.

6. The method for synthesizing the SERS material Au@PB NPs according to claim 1, characterized in that: In step (2), K4[Fe(CN)6]·3H2O solution and FeCl3 solution are injected into the Au@CN in the vortex at the same speed. - In the NPs solution, the injection rate is 10-20 μL / 10-30 s.

7. Au@PB NPs prepared by the synthesis method according to any one of claims 1 to 6.

8. Use of Au@PB NPs as claimed in claim 7 in the determination of cholesterol content.

9. The use according to claim 8, characterized in that Here’s how: (1) Prepare standard working solution Disperse cholesterol powder in ethanol and dilute with PBS buffer to obtain cholesterol solutions of a series of concentrations; add cholesterol oxidase and PBS buffer to the cholesterol solution and incubate at 37°C for 5 to 15 minutes to obtain a standard working solution; The concentration of cholesterol solution ranges from 10 to 500 μM; (2) Draw a standard curve The standard working solution of step (2), TMB solution, and Au@PB NPs solution were added to the HAc-NaAc buffer in sequence and incubated at room temperature for 5 to 15 min. The Raman signal was detected at 1589 cm -1 The SERS signal at 2155 cm -1 The ratio of the SERS signals at the position is taken as the ordinate, and the logarithm of the cholesterol concentration is taken as the abscissa to draw a standard curve; (3) Detection of cholesterol content Take the cholesterol solution to be tested, add cholesterol oxidase and PBS buffer, and incubate at 37°C for 5 to 15 minutes to obtain the sample to be tested. Detect the Raman signal according to the method in step (2), and substitute the signal value into the standard curve drawn in step (2) to obtain the cholesterol content of the sample to be tested.

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