Bovine serum albumin surface plasma resonance sensor and preparation method thereof

Abstract

The invention belongs to the technical field of surface plasma sensing material preparation and biomedical detection, discloses a bovine serum albumin surface plasma resonance sensor for enhancing sensitivity based on a synthetic hydrotalcite material, and establishes a surface plasma resonance detection method. The preparation method comprises the following steps: firstly, sequentially plating a chromium film and a gold film on a slide by a vacuum coating method, and then covering and synthesizing hydrotalcite (hydrated aluminate magnesium carbonate) particles to modify the surface plasma resonance sensing chip by a spin-coating method. The surface plasmon formant of the SPR sensor modified by 1% hydrotalcite is from 637.9020 nm to 698.1382 nm, the variation amplitude is 60.2362 nm, which is far greater than the deviation amplitude of the formant of 14.98 nm of the currently reported SPR sensor modified by graphene oxide, and the BSA (Bovine Serum Albumin) detection sensitivity is greatly enhanced. The preparation is simple, the cost is low, and the application prospect is relatively strong.

Description

Technical Field

[0001] The invention belongs to the technical field of surface plasma sensor chip preparation and biomedical detection, and particularly relates to a bovine serum albumin surface plasma resonance sensor and a preparation method thereof. Background Art

[0002] Bovine serum albumin (BSA), an important model protein in biomedical research, has a wide range of applications in drug delivery system evaluation, immunoassays, and cell culture optimization. Traditional detection methods, such as UV-visible spectrophotometry (e.g., Bradford and Lowry methods), are simple to use but susceptible to interference from sample impurities (e.g., detergents and reducing agents), resulting in insufficient selectivity. Enzyme-linked immunosorbent assay (ELISA), while highly sensitive, relies on antibody labeling and requires complex procedures, making real-time dynamic monitoring difficult. In recent years, electrochemical sensing technology has improved detection sensitivity through electrode surface modification, but this has been plagued by issues such as easy electrode contamination and complex modification processes.

[0003] Surface plasmon resonance (SPR) technology has been used for protein detection as a label-free, real-time dynamic monitoring method. However, the response signal of traditional SPR sensors to low concentrations of BSA is weak, and the detection limit is difficult to meet the needs of trace analysis. Existing improvement schemes mostly focus on the functional modification of nanomaterials (such as gold nanoparticles and graphene) to enhance the SPR signal, but still face bottlenecks such as complex preparation process, poor material stability or insufficient biocompatibility. For example, a study (ACS Appl. Mater. Interfaces 2018, 10, 34916-34923) used graphene oxide to modify the preparation of SPR sensors. The resonance peak shift amplitude tested in BSA solutions with a concentration range of 0 to 10 mg / mL was 14.98 nm, and the response signal of the sensor to low concentrations of BSA was weak.

[0004] Layered double hydroxides (LDHs), as two-dimensional layered nanomaterials, exhibit significant advantages in the adsorption and immobilization of biomolecules due to their high specific surface area, tunable interlayer channels, and enrichment of surface hydroxyl groups. Previous studies (e.g., ACS Appl. Mater. Interfaces, 2020, 12:123-130) have shown that LDHs can efficiently adsorb proteins through electrostatic interactions. However, their synergistic sensitization mechanism with SPR technology has not been fully explored, especially in the detection of BSA. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention primarily provides a bovine serum albumin surface plasmon resonance sensor chip. Based on this bovine serum albumin surface plasmon resonance sensor chip, a sensor is further developed. The material has a large specific surface area, resulting in high sensitivity in detection applications. Furthermore, the material preparation is simple and low-cost.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a bovine serum albumin surface plasmon resonance sensor chip comprises the following steps: coating a chromium film and a gold film on a substrate glass slide by magnetron sputtering in sequence, wherein the substrate heating temperature is 100°C, the substrate rotation speed is 15 rpm, and the background vacuum is 6*10 -5 Pa; then spin-coating a hydrotalcite solution; the hydrotalcite solution consists of hydrotalcite powder and an ethanol solution; the spin coating is completed and dried.

[0008] Preferably, the spin coating rate is 2000 to 3000 rpm, and the spin time is 90 to 180 seconds.

[0009] Preferably, the amount of the hydrotalcite solution dropped during spin coating is 100 μL to 300 μL.

[0010] Preferably, the thickness of the chromium film is 1 nm to 5 nm; the thickness of the gold film is 35 nm to 55 nm.

[0011] Preferably, the hydrotalcite concentration in the hydrotalcite solution is less than 10%.

[0012] More preferably, the concentration of hydrotalcite in the hydrotalcite solution is 0.5% to 3%.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The hydrotalcite-modified sensor provided by the present invention achieves excellent detection within a BSA concentration range of 10 mg / mL. In particular, the surface plasmon resonance peak of the SPR sensor modified with 1% hydrotalcite shifts by 60.2362 nm, from 637.9020 nm to 698.1382 nm. This shift is significantly greater than the 14.98 nm resonance peak shift of previously reported graphene oxide-modified SPR sensors. This significantly enhances BSA detection sensitivity and has strong application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of an experimental device in an embodiment of the present invention;

[0016] Figure 2 Schematic diagram of the structure of the sensor of the present invention;

[0017] Figure 3 This is a scanning electron micrograph of the surface morphology of the surface plasmon resonance sensor in the embodiment;

[0018] Figure 4 This is the surface atomic force microscope detection image of the spin-coated surface plasmon resonance sensor when the concentration of the synthesized hydrotalcite dispersion is 1%;

[0019] Figure 5 (a) shows the spectral transmittance distribution of different concentrations of BSA detected by a pure gold film SPR sensor; (b) shows the relationship between the surface plasmon resonance peak of the pure gold film SPR sensor and the change of different concentrations of BSA;

[0020] Figure 6 (a) shows the spectral transmittance distribution of the SPR sensor modified with 0.5% hydrotalcite for detection of different concentrations of BSA; (b) shows the relationship between the surface plasmon resonance peak of the SPR sensor modified with 0.5% hydrotalcite and the change of different concentrations of BSA;

[0021] Figure 7 (a) shows the spectral transmittance distribution of the SPR sensor modified with 1% hydrotalcite for the detection of different concentrations of BSA; (b) shows the relationship between the surface plasmon resonance peak of the SPR sensor modified with 1% hydrotalcite and the change of different concentrations of BSA;

[0022] Figure 8 (a) shows the spectral transmittance distribution of the SPR sensor modified with 2% hydrotalcite for detecting different concentrations of BSA; (b) shows the relationship between the surface plasmon resonance peak of the SPR sensor modified with 2% hydrotalcite and the change of different concentrations of BSA.

[0023] Specific embodiment

[0024] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention and comparative examples. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the implementation examples of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0026] Example 1

[0027] The surface plasmon resonance sensor used in this embodiment was magnetron sputtered onto a custom glass slide (i.e., sized to match the prism) and coated with a 2nm chromium film (Cr) and a 45nm gold film (Au). The Cr film was used to enhance the adsorption capacity of the Au film. A hydrotalcite solution was prepared by mixing a certain mass of synthetic hydrotalcite powder and an ethanol solution (the ethanol volume fraction was not strictly limited, as long as uniform dispersion was achieved). In this embodiment, the hydrotalcite mass concentration was 0.5%. The prepared hydrotalcite solution was then dispersed by oscillation using an ultrasonic oscillator for 10 minutes. A certain amount of the hydrotalcite solution was then spin-coated onto a clean Au wave plate. The solution droplet size was 200 μL, the spin coating rate was 3000 rpm, and the spin time was 180 seconds. After the spin coating was completed, the sample was allowed to dry completely at room temperature before being coupled to the prism using cedar oil.

[0028] The above magnetron sputtering, spin coating and other processes are all performed using existing technologies, and this application will not go into details here.

[0029] Example 2

[0030] The surface plasmon resonance sensor used in this embodiment was coated with a 2nm chromium film (Cr) and a 45nm gold film (Au) on a custom glass slide (i.e., sized to match the prism) by magnetron sputtering. The Cr film is used to enhance the adsorption capacity of the Au film. A hydrotalcite solution was prepared by mixing a certain mass of synthetic hydrotalcite powder and an ethanol solution. In this embodiment, the hydrotalcite concentration was 1% by mass. The prepared hydrotalcite solution was dispersed by ultrasonic oscillation for 10 minutes using an ultrasonic oscillator. A certain amount of the hydrotalcite solution was spin-coated onto a clean Au wave plate using a 200µL droplet of solution at a spin rate of 3000 rpm for 180 seconds. After spin coating, the sample was allowed to dry completely at room temperature before coupling to the prism using cedar oil.

[0031] Example 3

[0032] The surface plasmon resonance sensor used in this embodiment was magnetron sputtered onto a custom glass slide (i.e., sized to match the prism) and coated with a 2nm chromium film (Cr) and a 45nm gold film (Au). The Cr film was used to enhance the adsorption capacity of the Au film. A hydrotalcite solution was prepared by mixing a certain mass of synthetic hydrotalcite powder and an ethanol solution. In this embodiment, the hydrotalcite concentration was 2% by mass. The prepared hydrotalcite solution was dispersed by ultrasonic oscillation using an ultrasonic oscillator for 10 minutes. A certain amount of the hydrotalcite solution was spin-coated onto a clean Au wave plate. The solution droplet size was 200 μL, the spin coating speed was 3000 rpm, and the spin time was 180 seconds. After the spin coating was completed, the sample was allowed to dry completely at room temperature before coupling to the prism using cedar oil.

[0033] The experimental setup of Examples 1 to 3 was carried out based on the following:

[0034] The schematic diagram of the light source test experimental device of the present invention is as follows Figure 1 As shown in the figure, the experimental device schematic diagram consists of a halogen tungsten lamp light source (wavelength range: 360-2500nm); optical fiber; P light polarizer; sensor; fiber optic spectrometer (detection wavelength range: 200-1100nm), and a computer. As shown in Examples 1 to 3, the sensor assembly structure of the present invention is as follows. Figure 2 shown.

[0035] The results of the surface scanning electron microscope test of the sensor according to Example 2 are as follows: Figure 3 As shown in the figure, it can be seen that the hydrotalcite nanoparticles are evenly coated on the substrate, with small particle size and large specific surface area. The above structure has high adsorption characteristics and can greatly improve the SPR sensing performance.

[0036] Figure 4 The figure shows the atomic force microscope test results of the surface of the SPR sensor with 1% hydrotalcite spin-coated in Example 2 of the present invention. According to the test results, the average thickness of the sensor film is calculated to be 218 nm.

[0037] Figures 5 to 8 The spectral transmittance distribution of different SPR sensors for different concentrations of BSA and the relationship between the surface plasmon resonance peak of the SPR sensor and the change of BSA concentration are shown. Figure 5 The preparation of the medium-purity gold film is based on the embodiment, but the spin coating of hydrotalcite is not performed, and the other aspects are the same.

[0038] Combine Figure 1 and Figure 2 , the SPR resonance peak test data of different concentrations of BSA are processed as follows:

[0039] PBS solution was dripped onto the surface of the prepared hydrotalcite SPR sensor to perform SPR spectrum detection. The halogen tungsten lamp light source used in the measurement system emits broadband light of 360-2500nm. The light is incident on the gold film in parallel through a polarizer and a focusing lens. The incident angle is 78°. After the reflected light passes through the prism, it is converged and coupled into a multimode optical fiber through a focusing lens. Finally, the multimode optical fiber light guide input spectrometer detects the signal. The detected data will be further analyzed and processed by a computer to obtain SPR spectrum data. The PBS solution is further removed and BSA solutions of different concentrations (0, 5mg / ml, 10mg / ml, 15mg / ml, 20mg / ml) are dripped into the sensor surface to obtain the corresponding SPR spectrum data according to the above method. Each time a certain concentration of BSA solution is measured, the sensor surface is cleaned with PBS and then the test of the next concentration of BSA solution is performed. Finally, the SPR resonance peak test data of different concentrations of BSA are plotted and linearly fitted. The slope obtained is the average sensitivity within the tested BSA concentration range.

[0040] After testing, the SPR sensor modified with 1% hydrotalcite tested BSA solutions with a concentration range of 0 to 10 mg / mL, and the surface plasmon resonance peak varied from 637.9020 nm to 698.1382 nm, with a variation range of 60.2362 nm and an average sensitivity of 6.024 nm / (mg / mL). This is much larger than the 14.98 nm resonance peak shift amplitude of the graphene oxide-modified SPR sensor reported so far (ACS Appl. Mater. Interfaces 2018, 10, 34916-34923). The average sensitivity of the SPR sensor in the test concentration range of 0 to 10 mg / mL BSA solution was 1.498 nm / (mg / mL).

[0041] The physicochemical indicators in the above embodiments are only some embodiments, but it cannot be determined that the specific embodiments of the present invention are limited to these examples and these application tests. Obviously, the above embodiments of the present invention are only examples made to clearly illustrate the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a bovine serum albumin surface plasmon resonance sensor chip, characterized in that: The method comprises the following steps: coating a chromium film and a gold film on a base glass slide in sequence; and then spin-coating a hydrotalcite solution, wherein the hydrotalcite solution is composed of hydrotalcite powder and an ethanol solution; and drying the solution after the spin-coating is completed.

2. The preparation method according to claim 1, characterized in that The spin coating rate is 2000 to 3000 rpm, and the spin time is 90 to 180 seconds.

3. The preparation method according to claim 1, characterized in that The amount of the hydrotalcite solution dropped during spin coating is 100 μL to 300 μL.

4. The preparation method according to claim 1, characterized in that The thickness of the chromium film is 1nm to 5nm; the thickness of the gold film is 35 to 55nm.

5. The preparation method according to claim 1, characterized in that The hydrotalcite concentration in the hydrotalcite solution is less than 10%.

6. A sensor material obtained by the method for preparing the bovine serum albumin surface plasmon resonance sensor chip according to claim 1.

7. Use of the sensor material according to claim 6 in surface plasmon resonance detection.

8. The use according to claim 7, characterized in that The bovine serum albumin surface plasmon resonance sensor chip is coupled with a prism to obtain a surface plasmon resonance sensor material.

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