Method for quantitatively analyzing BSA (Bovine Serum Albumin) by SERS (Surface Enhanced Raman Scattering) based on AgNR-coated MIP probe and

Abstract

The invention relates to the technical field of analytical chemistry, in particular to a method for quantitatively analyzing BSA (Bovine Serum Albumin) through SERS (Surface Enhanced Raman Scattering) based on an AgNR-coated MIP probe and a nano-silver sol substrate. Comprising the following steps: (1) preparing a BSA standard working solution system; (2) preparing a blank control solution system; (3) respectively determining the surface-enhanced Raman scattering peak intensity value I at 1621.8 cm <-1 > of the BSA standard working solution system and the blank control solution system in a portable Raman spectrometer, simultaneously determining the surface-enhanced Raman scattering peak intensity value I0 of the blank control solution system, and calculating delta I = I-I0; (4) drawing a working curve according to the relationship between the delta I and the concentration of the BSA; (5) preparing a sample solution according to the method in the step (1), determining the surface enhanced Raman scattering peak intensity value of the sample solution as Isample, and calculating delta Isample = Isample minus I0; and (6) calculating the BSA content of the sample solution according to the working curve. The determination method adopts an SERS (Surface Enhanced Raman Scattering) method in which AgNR-coated MIP is specifically combined with BSA (Bovine Serum Albumin), and is simple, convenient, rapid and high in sensitivity.

Description

Technical Field

[0001] This invention patent relates to the technical field of analytical chemistry, and specifically relates to a method for quantitative analysis of BSA by SERS based on AgNR@MIP probes and a silver sol substrate. Background Art

[0002] Bovine Serum Albumin (BSA) is an important model protein widely used in the fields of biomedicine, food analysis, and environmental monitoring. Changes in its content are often used as important indicators for disease diagnosis, physiological monitoring, and food quality assessment. Therefore, developing a BSA detection method with high sensitivity, high selectivity, simple operation, and applicable to complex matrix samples is of great significance for both scientific research and industry.

[0003] Currently, commonly used BSA detection methods include ultraviolet-visible spectrophotometry (UV-Vis), high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), etc. Although these methods have certain sensitivity and accuracy, they generally have problems such as complex operation procedures, long detection cycles, strong dependence on equipment or reagents, etc., and it is difficult to meet the actual needs of rapid and high-throughput detection. In addition, some methods have high requirements for sample pretreatment and it is difficult to achieve efficient detection of trace BSA.

[0004] In recent years, Surface-Enhanced Raman Scattering (SERS) technology has shown broad prospects in the field of protein detection due to its extremely high signal enhancement ability and molecular specificity. SERS can greatly enhance the Raman signal of target molecules on the surface of metal nanostructures (such as silver and gold), enabling the detection of ultra-low concentration samples. However, SERS platforms still face two main challenges in practical applications: one is the insufficient repeatability and stability of signals, and the other is the lack of selective recognition of target molecules and being easily interfered by complex backgrounds.

[0005] To improve the selectivity and stability of SERS platforms, researchers have begun to combine Molecularly Imprinted Polymers (MIPs) with SERS technology. MIP is a functional material that forms specific recognition sites through template molecule-guided polymerization, and has good structural stability and recognition specificity. When MIP is used in combination with SERS signal enhancement structures, it can simultaneously achieve high-selectivity recognition and high-sensitivity detection of molecules, becoming an important development direction in the field of nanoanalysis in recent years. Summary of the Invention

[0006] To solve the above technical problems, the objective of the present invention is to provide a surface-enhanced Raman scattering (SERS) method based on AgNR@MIP probes and silver nanosol substrates for simple and sensitive quantitative analysis of BSA. This determination method uses the SERS spectroscopy in which AgNR@MIP specifically binds to BSA on the silver nanosol, and the method is simple, rapid, and highly sensitive.

[0007] The objective of the present invention can be achieved through the following technical solutions:

[0008] The present invention provides a method for quantitatively analyzing BSA by SERS based on AgNR@MIP probes and silver nanosol substrates, comprising the following steps:

[0009] (1) Prepare a BSA standard working solution system with known concentrations: After mixing the BSA standard solution and the AgNR@MIP solution respectively, let it stand still, then add the AgNP solution (silver nanosol solution) respectively, make up the volume to a fixed volume with ultrapure water respectively, shake well, let it stand still for the first reaction, then add the NaCl solution and the VBB (Victoria Blue B) solution respectively, shake well, and let it stand still for the second reaction to obtain the BSA standard working solution system with different concentrations;

[0010] (2) Prepare a blank control solution system: Use the method in step (1) to prepare the blank control solution system without adding the BSA standard solution;

[0011] (3) Pour the BSA standard working solution system and the blank control solution system prepared according to steps (1) and (2) respectively into quartz cuvettes. On a portable Raman spectrometer, set the instrument parameters, scan to obtain the surface-enhanced Raman scattering spectra of the systems, and measure the intensity value of the surface-enhanced Raman scattering peak of the BSA standard working solution system at 1621.8 cm -1 as I, and at the same time measure the intensity value of the surface-enhanced Raman scattering peak of the blank control solution system at 1621.8 cm -1 as I0, and calculate ΔI = I - I0;

[0012] (4) Make a working curve with the relationship between ΔI and the concentration of the BSA standard working solution system;

[0013] (5) Prepare a sample solution according to the method in step (1), where the added BSA standard solution is replaced with the sample solution, and measure the intensity value of the surface-enhanced Raman scattering peak of the sample solution at 1621.8 cm -1 as I 样品 , and calculate ΔI 样品 = I 样品 - I0;

[0014] (6) According to the working curve in step (4), calculate the content of BSA in the sample solution.

[0015] Further, in step (1), the BSA concentrations in the BSA standard working solution system are 0.1 nmol / L, 0.5 nmol / L, 1 nmol / L, 1.5 nmol / L, 2 nmol / L, 2.5 nmol / L, and 3 nmol / L respectively.

[0016] Further, in step (1), the standing time is 5 - 20 minutes.

[0017] Further, in step (1), the time for the first standing reaction is 5 - 20 minutes.

[0018] Further, in step (1), the time for the second standing reaction is 1 - 10 minutes.

[0019] Further, in step (3), the instrument parameters are power 100 mv, acquisition time 10 ms, and acquisition times 3 times.

[0020] Further, in step (1), the preparation method of the AgNP solution includes the following steps:

[0021] Add a sodium citrate solution and an AgNO3 solution to water. After mixing, slowly add a sodium borohydride solution. When the solution changes from light yellow to dark yellow, continue stirring to obtain an AgNP mixed solution. Wash it with pure water and centrifuge twice, then make up the volume to obtain the AgNP solution, and store it at 4°C.

[0022] Further, in step (1), the preparation method of the AgNR@MIP includes the following steps:

[0023] Step S1: Preparation of Au seeds: Mix a CTAC (cetyltrimethylammonium chloride) solution, a HAuCl4 (tetrachloroauric acid) solution, and a citric acid solution. Under magnetic stirring, add a sodium borohydride solution, let it stand, and heat it in a water bath to induce the formation of a twin crystal surface, and store the seed solution at room temperature.

[0024] Step S2: Preparation of AgNR: First, add the heat-treated seed solution to a growth aqueous solution containing CTAB (cetyltrimethylammonium bromide), HAuCl4, AgNO3, HCl, and AA (L-ascorbic acid), and stir vigorously. Keep the mixture warm, then centrifuge the reaction solution twice to remove excess reagents, then redisperse it in a CTAC solution, dilute it, heat it under magnetic stirring, then add an AgNO3 solution and an AA solution, stir, centrifuge and wash twice and redisperse it in water to obtain an AgNR dispersion.

[0025] Step S3: Preparation of AgNR@MIP: Dissolve the template protein BSA in phosphate buffer solution, add the AgNR dispersion and β-cyclodextrin and stir. Then, add triethylene glycol dimethacrylate, ammonium persulfate and phosphate buffer solution, and ultrasonicate until the solution is dispersed. Stir, wash the obtained product several times until the template protein BSA is not detected, and finally obtain AgNR@MIP after vacuum freeze-drying.

[0026] The beneficial effects that can be produced by this application are as follows:

[0027] Compared with the existing methods, the determination method of the present invention uses the SERS spectroscopy of AgNR@MIP specifically binding to BSA, and the method is simple, fast and highly sensitive. Description of the Drawings

[0028] Figure 1 It is the characterization of AgNR@MIP of the present invention, where a is the particle size diagram of AgNR@MIP; b is the potential diagram of AgNR@MIP.

[0029] Figure 2 It is the XPS full-spectrum characterization diagram and fine diagram of AgNR@MIP and AgNR@MIP-BSA of the present invention, where a is the XPS full-spectrum diagram; b is the fine spectrum of C; c is the fine spectrum of O; d is the fine spectrum of N.

[0030] Figure 3 It is the surface-enhanced Raman scattering spectrum diagram of the bovine serum albumin BSA standard working solution measured by the present invention using AgNP and AgNR@MIP (the corresponding concentrations from bottom to top are 0.1 nmol / L, 0.5 nmol / L, 1 nmol / L, 1.5 nmol / L, 2 nmol / L, 2.5 nmol / L, 3 nmol / L). Detailed Embodiments

[0031] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present application.

[0032] In this embodiment, the 10 nmol / L BSA standard solution is obtained by gradually diluting the 20 mg / ml BSA purchased from Beijing Solarbio Science & Technology Co., Ltd. The template protein BSA is purchased from Beijing Solarbio Science & Technology Co., Ltd. The 0.1 g / L AgNR@MIP solution is obtained by dissolving 10 mg AgNR@MIP in 10 ml ultrapure water and diluting it 10 times. The milk sample is purchased from the convenience store Mengniu pure milk.

[0033] Example

[0034] A method for quantitative analysis of BSA by SERS based on AgNR@MIP probe and silver nanoparticle sol substrate, comprising the following steps:

[0035] (1) Preparation of a BSA standard working solution system with known concentrations:

[0036] Preparation of 0.1 nmol / L BSA standard working solution: In a graduated test tube, add 20 μL of 10 nmol / L BSA standard solution and 60 μL of 0.1 g / L AgNR@MIP solution, let stand for 5 minutes, then add 300 μL of 1 mol / L AgNP solution, make up the volume to 2.0 mL with ultrapure water, shake well, let stand and react for 5 minutes, then add 100 μL of 1 mol / L NaCl solution and 150 μL of 10 μmol / L VBB solution, shake well, let stand and react for 2 minutes;

[0037] Preparation of 0.5 nmol / L BSA standard working solution: In a graduated test tube, add 100 μL of 0.1 μmol / L BSA standard solution and 60 μL of 0.1 g / L AgNR@MIP solution, let stand for 5 minutes, then add 300 μL of 1 mol / L AgNP solution, make up the volume to 2.0 mL with ultrapure water, shake well, let stand and react for 5 minutes, then add 100 μL of 1 mol / L NaCl solution and 150 μL of 10 μmol / L VBB solution, shake well, let stand and react for 2 minutes;

[0038] Preparation of 1 nmol / L BSA standard working solution: In a graduated test tube, add 200 μL of 0.1 μmol / L BSA standard solution and 60 μL of 0.1 g / L AgNR@MIP solution, let stand for 5 minutes, then add 300 μL of 1 mol / L AgNP solution, make up the volume to 2.0 mL with ultrapure water, shake well, let stand and react for 5 minutes, then add 100 μL of 1 mol / L NaCl solution and 150 μL of 10 μmol / L VBB solution, shake well, let stand and react for 2 minutes;

[0039] Preparation of 1.5 nmol / L BSA standard working solution: In a graduated test tube, add 300 μL of 0.1 μmol / L BSA standard solution and 60 μL of 0.1 g / L AgNR@MIP solution, let stand for 5 minutes, then add 300 μL of 1 mol / L AgNP solution, make up the volume to 2.0 mL with ultrapure water, shake well, let stand and react for 5 minutes, then add 100 μL of 1 mol / L NaCl solution and 150 μL of 10 μmol / L VBB solution, shake well, let stand and react for 2 minutes;

[0040] Preparation of 2nmol / L BSA standard working solution: In a graduated test tube, add 400μL 0.1μmol / L BSA standard solution and 60μL 0.1g / L AgNR@MIP solution, let stand for 5 minutes, then add 300μL 1mol / L AgNP solution, dilute to 2.0mL with ultrapure water, shake well, let stand for 5 minutes, then add 100μL 1mol / L NaCl solution and 150μL 10μmol / L VBB solution, shake well, let stand for 2 minutes;

[0041] Preparation of 2.5nmol / L BSA standard working solution: In a graduated test tube, add 500μL 0.1μmol / L BSA standard solution and 60μL 0.1g / L AgNR@MIP solution, let stand for 5 minutes, then add 300μL 1mol / L AgNP solution, dilute to 2.0mL with ultrapure water, shake well, let stand for 5 minutes, then add 100μL 1mol / L NaCl solution and 150μL 10μmol / L VBB solution, shake well, let stand for 2 minutes;

[0042] Preparation of 3nmol / L standard working solution: In a graduated test tube, add 600μL 0.1μmol / L BSA standard solution and 60μL 0.1g / L AgNR@MIP solution, let stand for 5 minutes, then add 300μL 1mol / L AgNP solution, dilute to 2.0mL with ultrapure water, shake well, let stand for 5 minutes, then add 100μL 1mol / L NaCl solution and 150μL 10μmol / L VBB solution, shake well, let stand for 2 minutes;

[0043] The preparation method of the AgNP solution comprises the following steps:

[0044] Add 40 mL of water to a 100 mL conical flask, stir with a magnetic stirrer, add 3.5 mL of 10 g / L trisodium citrate solution and 385 μL of 2.4×10 -2 mol / L AgNO3 solution, after fully mixing, slowly add 4.0mL 0.5mg / mL sodium borohydride solution, the solution changes from light yellow to dark yellow, continue stirring for 10min, and AgNP mixed solution is obtained. The generated AgNP mixed solution is washed with water and centrifuged twice, and the volume is fixed to 20mL to obtain AgNP solution, which is stored at 4℃.

[0045] The preparation method of AgNR@MIP includes the following steps:

[0046] Step S1: Preparation of Au seeds: Add 5 mL of 100 mM CTAC solution, 85 μL of 1% HAuCl4 solution, and 5 mL of 10 mM citric acid solution into a 25 mL beaker. Then add 0.25 mL of 25 mM sodium borohydride solution under magnetic stirring at 500 r / min. After two minutes, transfer the beaker to an 80 °C water bath and heat for 90 minutes to induce the formation of a twin crystal surface, and store the seed solution at room temperature;

[0047] Step S2: Preparation of AgNRs: First, add 2.5 mL of the heat-treated seed solution into the growth aqueous solution containing CTAB (20 mL, 100 mM), HAuCl4 (1 mL, 10 mM), AgNO3 (0.2 mL, 10 mM), HCl (0.4 mL, 1 M), and AA (0.16 mL, 100 mM), and stir vigorously for 5 min. Keep the mixture at 30 °C for 2 hours. Centrifuge the reaction solution twice (8000 rpm, 30 minutes) to remove excess reagents, and then redisperse it in 10 mL of 10 mM CTAC solution. Take 1.25 ml of the above solution and dilute it to 10 ml with 10 mM CTAC solution. Heat it to 60 °C under magnetic stirring at 500 r / min, then add 0.1 ml of 10 mM AgNO3 solution and 0.04 ml of 100 mM AA solution, and stir for 1 hour. Finally, centrifuge and wash the obtained solution 2 times and redisperse it in 10 ml of water to obtain the AgNR dispersion;

[0048] Step S3: Preparation of AgNR@MIP: Add 10 mg of the template protein BSA dissolved in 20 mL of phosphate buffer solution (0.02 mol / L, pH = 7.4) into a 50 mL flask, add 5 mL of the AgNR dispersion and 100 mg of β-cyclodextrin, and stir for 1 h. Then, add 2 mmol of triethylene glycol dimethacrylate, 10 mg of ammonium persulfate, and 10 mL of phosphate buffer solution (0.02 mol / L, pH = 7.4), and ultrasonicate until the solution is dispersed. Stir at 60 °C for 12 hours. Wash the obtained product several times with 0.5% sodium dodecyl sulfate until no template protein BSA is detected. Finally, obtain AgNR@MIP after vacuum freeze-drying. The characterization diagrams of AgNR@MIP are as follows Figure 1 shown, where a is the particle size diagram of AgNR@MIP, indicating that the particle size of the synthesized AgNR@MIP is 1.5 μm; b is the potential diagram of AgNR@MIP, and its potential is 6.05 mV, indicating that the synthesized AgNR@MIP has good stability;

[0049] The XPS full-spectrum characterization diagrams and fine diagrams of AgNR@MIP and AgNR@MIP-BSA (i.e., the product obtained by mixing the BSA standard solution and the AgNR@MIP solution in step (1) and allowing it to stand) are as followsFigure 2 As shown in the figure. Where a is the full XPS spectrum, indicating that the N content in AgNR@MIP after binding with BSA has increased significantly; b is the fine spectrum of C, indicating that the binding of BSA to AgNR@MIP introduces oxygen-containing functional groups; c is the fine spectrum of O, further confirming that the interaction between BSA and AgNR@MIP is through oxygen-containing groups; d is the fine spectrum of N, indicating that BSA has successfully bound to AgNR@MIP and brought nitrogen; the XPS change analysis shows that BSA has successfully bound to AgNR@MIP.

[0050] (2) Prepare a blank control solution system: Use the method in step (1) to prepare a blank control solution system without adding BSA standard solution.

[0051] (3) Respectively pour the BSA standard working solution system and the blank control solution system prepared in steps (1) and (2) into a quartz cuvette. On a portable Raman spectrometer, set the instrument parameter power to 100 mv, the acquisition time to 10 ms, and the number of acquisitions to 3 times. Scan to obtain the surface-enhanced Raman scattering spectrum of the system, and measure the intensity value of the surface-enhanced Raman scattering peak at 1621.8 cm -1 as I. At the same time, measure the intensity value of the surface-enhanced Raman scattering peak of the blank control solution system as I0, and calculate ΔI = I - I0; The surface-enhanced Raman scattering spectra of bovine serum albumin BSA standard working solutions measured using AgNP and AgNR@MIP are as Figure 3 shown. Among them, the corresponding concentrations from bottom to top are 0.1 nmol / L, 0.5 nmol / L, 1 nmol / L, 1.5 nmol / L, 2 nmol / L, 2.5 nmol / L, 3 nmol / L, indicating that this measurement method has high sensitivity.

[0052] (4) Make a working curve with the relationship between ΔI and the concentration of BSA.

[0053] (5) Prepare a sample solution according to the method in step (1), where the added BSA standard solution is replaced with the sample solution, and measure the intensity value of the surface-enhanced Raman scattering peak of the sample solution as I 样品 , and calculate ΔI 样品 = I 样品 - I0;

[0054] (6) Calculate the content of BSA in the sample solution according to the working curve in step (4).

[0055] Commercially available milk samples were tested. 1 g of the milk sample was mixed with 5 mL of 5% methanol, and then centrifuged at 5000 rpm for 10 min. The obtained supernatant was passed through a 0.2 μm filter membrane and diluted 100 times with ultrapure water to obtain a milk sample solution. Three portions of the milk sample solution were taken, and a BSA standard solution with a concentration of 1 nmol / L was added respectively to conduct a standard addition recovery experiment. The recovery rates were found to be 96.8%, 98.9%, and 103.3% respectively, and the relative standard deviations were 1.4%, 3.2%, and 2.1% respectively.

[0056] The above is a specific description of the preferred embodiment of the present invention, but the present invention is not limited to the described embodiment. Those skilled in the art can make various equivalent deformations or substitutions without departing from the spirit of the present invention, and these equivalent deformations or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for SERS quantitative analysis of BSA based on AgNR@MIP probe and silver sol substrate, characterized in that, It includes the following steps: (1) Prepare a BSA standard working solution system with known concentrations: After mixing the BSA standard solution and the AgNR@MIP solution respectively, let it stand, then add the AgNP solution respectively, make up the volume to a fixed value with ultrapure water respectively, shake well, let it react for the first time while standing, then add the NaCl solution and the VBB solution respectively, shake well, and let it react for the second time while standing to obtain the BSA standard working solution system with different concentrations; (2) Prepare a blank control solution system: Use the method in step (1) to prepare the blank control solution system without adding the BSA standard solution; (3) Pour the BSA standard working solution system and the blank control solution system prepared in steps (1) and (2) respectively into quartz cuvettes. On a portable Raman spectrometer, set the instrument parameters and scan to obtain the surface-enhanced Raman scattering spectra of the systems. Measure the surface-enhanced Raman scattering peak intensity value I of the BSA standard working solution system at 1621.8 cm -1 At the same time, measure the surface-enhanced Raman scattering peak intensity value I0 of the blank control solution system at 1621.8 cm -1 At, and calculate ΔI = I - I0; (4) Make a working curve based on the concentration relationship between ΔI and the BSA standard working solution system; (5) Prepare the sample solution according to the method in step (1), where the added BSA standard solution is replaced with the sample solution, and measure the surface-enhanced Raman scattering peak intensity value I of the sample solution at 1621.8 cm -1 as I 样品 , and calculate ΔI 样品 = I 样品 - I0; (6) Calculate the content of BSA in the sample solution according to the working curve in step (4).

2. The method for quantitatively analyzing BSA by SERS based on the AgNR@MIP probe and the silver nanosol substrate according to claim 1, wherein, In step (1), the BSA concentrations of the BSA standard working solution system are 0.1 nmol / L, 0.5 nmol / L, 1 nmol / L, 1.5 nmol / L, 2 nmol / L, 2.5 nmol / L, and 3 nmol / L respectively.

3. The method for SERS quantitative analysis of BSA based on AgNR@MIP probe and silver sol substrate according to claim 1, characterized in that, In step (1), the standing time is 5 - 20 minutes.

4. The method for quantitatively analyzing BSA by SERS based on an AgNR@MIP probe and a silver sol substrate according to claim 1, wherein In step (1), the reaction time for the first standing reaction is 5 - 20 minutes.

5. The method for quantitatively analyzing BSA by SERS based on an AgNR@MIP probe and a silver sol substrate according to claim 1, wherein In step (1), the reaction time for the second standing reaction is 1 - 10 minutes.

6. The method for quantitatively analyzing BSA by SERS based on an AgNR@MIP probe and a silver sol substrate according to claim 1, wherein In step (3), the instrument parameters are power 100 mv, acquisition time 10 ms, and acquisition times 3 times.

7. The method for quantitatively analyzing BSA by SERS based on an AgNR@MIP probe and a silver sol substrate according to claim 1, wherein In step (1), the preparation method of the AgNR@MIP includes the following steps: Step S1: Preparation of Au seeds: Mix the CTAC solution, the HAuCl4 solution and the citric acid solution, add the sodium borohydride solution under magnetic stirring, let it stand, heat it in a water bath to induce the formation of a twin crystal surface, and store the seed solution at room temperature; Step S2: Preparation of AgNR: First add the heat-treated seed solution to the growth aqueous solution containing CTAB, HAuCl4, AgNO3, HCl and AA, and stir vigorously, keep the mixture warm, then centrifuge the reaction solution twice to remove the excess reagents, then redisperse it in the CTAC solution, dilute it, heat it under magnetic stirring, then add the AgNO3 solution and the AA solution, stir, centrifuge and wash 2 times and redisperse it in water to obtain the AgNR dispersion; Step S3: Preparation of AgNR@MIP: Dissolve the template protein BSA in the phosphate buffer solution, add the AgNR dispersion and β-cyclodextrin and stir, then add triethylene glycol dimethacrylate, ammonium persulfate and the phosphate buffer solution, ultrasonicate until the solution is dispersed, stir, wash the obtained product several times until the template protein BSA is not detected, and finally obtain AgNR@MIP after vacuum freeze-drying.

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