Preparation method and application of SPR biosensor chip

Abstract

The application relates to a preparation method and application of an SPR biosensing chip. The preparation method of the SPR biosensing chip comprises the following steps: S1. covering a noble metal layer on the surface of a substrate to obtain a substrate-noble metal layer; and S2. sequentially depositing a graphene nanosheet layer and a nanodiamond particle layer on the surface of the substrate-noble metal layer to obtain the SPR biosensing chip. The SPR biosensing chip prepared by the preparation method has high sensitivity and high detection precision, is easy to modify and functionalize, and is relatively easy and simple to operate because the graphene nanosheet is easy to prepare and transfer relative to the large-area layered graphene.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensing, and more particularly to a preparation method and application of an SPR biosensing chip. BACKGROUND

[0002] Surface plasmon resonance is a powerful technique for analyzing biomolecular interactions, which realizes label-free, high-sensitivity real-time optical sensing. Many studies related to biomolecular interactions have been carried out on functionalized plasmonic interfaces, such as diagnosis of human hepatitis B virus (hHBV), detection of prostate specific antigen, detection of vascular endothelial growth factor, etc. One of the common techniques excited by SPR is the Kretschmann configuration, in which we usually choose a gold film layer because the gold film layer has good oxidation resistance and corrosion resistance. However, the immobilization efficiency of biomolecules on the gold film layer is low, which limits the development of the bare gold film layer SPR biosensor. Therefore, researchers have proposed the idea of introducing a sensitization layer to achieve higher biological sensitivity and improve the performance of the sensor. At present, various nanomaterials such as graphene oxide, MoS2, graphene, etc. have been used as sensing platforms for SPR biosensors, such as the Chinese patent entitled "A molybdenum disulfide sensitized surface plasmon resonance sensor and a preparation method thereof" provides a surface plasmon resonance sensor sensitized by molybdenum disulfide.

[0003] Graphene is a high-quality carbon-based material with a two-dimensional honeycomb-like structure of tightly packed carbon atoms linked by sp 2 Hybridly linked carbon atoms. It has many excellent optical, mechanical and electrical properties, exhibits high biomolecule affinity and large specific surface area, which is beneficial to the immobilization of biomolecules, and it is proved to be able to stably adsorb biomolecules with carbon-based ring structure. And graphene has high electron mobility, which can effectively transfer electric charge and increase the electric field strength of the plasmonic interface, thereby increasing the sensitivity of the SPR sensor. However, the large-area layered graphene has high requirements for preparation and transfer, which limits its application. Compared with large-area layered graphene, graphene nanosheets are easy to prepare and transfer, but their sensitization effect on SPR sensors is limited.

[0004] Therefore, it is necessary to solve the problems of high preparation and transfer requirements for SPR biosensor sensitization by large-area layered graphene and low sensitivity of existing SPR biosensors. SUMMARY

[0005] The primary object of the present application is to overcome the problems of high preparation and transfer requirements of the prior art SPR biosensor sensitized by large-area layered graphene and low sensitivity of the prior art SPR biosensor, and to provide a preparation method of an SPR biosensing chip. The SPR biosensing chip obtained by the preparation method has high sensitivity, high detection accuracy, and is easy to modify and functionalize. Moreover, the graphene nanosheet is easy to prepare and transfer relative to the large-area layered graphene, so the operation of the preparation method is relatively easy and simple.

[0006] A further object of the present application is to provide an SPR biosensing chip.

[0007] A further object of the present application is to provide the use of the above-mentioned SPR biosensing chip as a sensor in the field of surface plasmon resonance.

[0008] The above objects of the present application are achieved by the following technical solutions:

[0009] A preparation method of an SPR biosensing chip, comprising the following steps:

[0010] S1. Covering a noble metal layer on the surface of a substrate to obtain a substrate-noble metal layer;

[0011] S2. Depositing a graphene nanosheet layer and a nanodiamond particle layer on the surface of the substrate-noble metal layer in sequence, thereby obtaining the SPR biosensing chip.

[0012] The present application has found through repeated research that the graphene nanosheet layer can improve the sensitivity of the SPR biosensing to a certain extent, and on this basis, the deposition of the nanodiamond particle layer can further improve the sensitivity of the SPR biosensing (relative to the case where no graphene nanosheet layer and nanodiamond particle layer are deposited, the sensitivity is improved by 12.6% to 32.8%). Specifically, the graphene nanosheet layer and the nanodiamond particle layer are deposited on the surface of the substrate-noble metal layer in sequence, and the graphene nanosheet layer and the nanodiamond particle layer are used as a sensitizing layer. The nanodiamond particles of the nanodiamond particle layer have irregular shapes and contain oxygen-containing functional groups. On the one hand, they can provide a large specific surface area to provide sufficient binding sites for biomolecules; on the other hand, the nanodiamond particles can be effectively connected to the graphene nanosheets of the graphene nanosheet layer to improve the electron mobility, thereby making the SPR biosensing chip have high sensitivity and high detection accuracy. In addition, the graphene nanosheet is easy to prepare and transfer relative to the large-area layered graphene, so the preparation of the SPR biosensing chip is relatively easy and simple. Moreover, the nanodiamond particle layer also makes the SPR biosensing chip easier to modify and functionalize.

[0013] The SPR biosensor chip prepared by the preparation method has high sensitivity, high detection precision, and is easy to modify and functionalize.

[0014] If only the graphene nanosheet layer is deposited, the nano-diamond particle layer is not deposited, and the sensitizing layer is only the graphene nanosheet layer, the sensitizing effect of the sensitizing layer on the SPR biosensor chip is poor, and the sensitivity of the SPR biosensor chip is low; if the graphene nanosheet-nano-diamond particle mixed layer is deposited, and the sensitizing layer is the graphene nanosheet-nano-diamond particle mixed layer (i.e., the sensitizing layer is not a two-layer structure including the graphene nanosheet layer and the nano-diamond particle layer, but a one-layer structure including only the graphene nanosheet and nano-diamond particle mixed layer), the sensitizing layer is difficult to be fixed on the SPR biosensor, and the sensitizing layer constantly falls off during the test process, so that the sensitivity of the SPR biosensor cannot be improved.

[0015] Preferably, the substrate in step S1 is a prism.

[0016] Preferably, the noble metal layer in step S1 is a gold layer or a silver layer.

[0017] More preferably, the noble metal layer is a gold layer.

[0018] More preferably, the thickness of the noble metal layer is 49-51 nm.

[0019] Preferably, the covering process in step S1 is to plate a noble metal layer on the surface of the substrate by vacuum evaporation.

[0020] Preferably, the graphene nanosheet in the graphene nanosheet layer in step S2 has a sheet diameter of 50-500 nm.

[0021] Preferably, the nano-diamond particle in the nano-diamond particle layer in step S2 has a particle diameter of 25-35 nm.

[0022] Preferably, the deposition process in step S2 is to deposit the graphene nanosheet layer by using the graphene nanosheet dispersion liquid, and then to deposit the nano-diamond particle layer by using the nano-diamond particle dispersion liquid.

[0023] More preferably, the deposition process in step S2 is to transfer the graphene nanosheet dispersion liquid to the surface of the substrate-noble metal layer, dry, and deposit to form the graphene nanosheet layer; and then to transfer the nano-diamond particle dispersion liquid to the graphene nanosheet layer, dry, and deposit to form the nano-diamond particle layer.

[0024] More preferably, the concentration of the graphene nanosheet dispersion liquid is 0.08-0.12 mg / mL; and the concentration of the nanodiamond particle dispersion liquid is 0.08-0.12 mg / mL.

[0025] Preferably, the number of times of the deposition in step S2 is 1-2 times.

[0026] The deposition step of step S2 can be performed for multiple times, for example, when the number of times of the deposition is 2 times, the specific process is to first deposit a graphene nanosheet layer and a nanodiamond particle layer, and then deposit a graphene nanosheet layer and a nanodiamond particle layer.

[0027] An SPR biosensor chip is prepared by the preparation method.

[0028] The SPR biosensor chip is used as a sensor in the field of surface plasmon resonance, for example, the SPR biosensor chip is prepared into an SPR sensing detection system comprising the SPR biosensor chip.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The SPR biosensor chip prepared by the preparation method has high sensitivity and high detection precision, is easy to modify and functionalize, and is relatively easy and simple to operate due to the easy preparation and transfer of the relatively large-area layered graphene of the graphene nanosheet. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 SEM image of the surface of the SPR biosensor chip of Example 1.

[0032] Figure 2 Structure diagram of a sensing detection system for refractive index sensing property test of the present application.

[0033] Figure 3 Graph of the refractive index sensing property test result of the prism-gold layer obtained in the first step of Example 1 as an SPR biosensor.

[0034] Figure 4 Graph of the test result of the refractive index sensing property test of the SPR biosensor chip of Example 1 as an SPR biosensor.

[0035] Figure 5 Graph of the test result of the refractive index sensing property test of the SPR biosensor chip of Example 2 as an SPR biosensor.

[0036] Figure 6 Schematic diagram of the working process of the SPR biosensor chip of the embodiment of the present application.

[0037] Figure 7 The test result graph of the refractive index sensing property test of the prism-gold layer of the comparative example 1 and the comparative SPR biosensor chip as the SPR biosensor respectively.

[0038] Figure 8 The test result graph of the refractive index sensing property test of the prism-gold layer of the comparative example 2 and the comparative SPR biosensor chip as the SPR biosensor respectively. DETAILED DESCRIPTION

[0039] In order to more clearly and completely describe the technical solutions of the present application, the present application is further described in detail below through specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application, and various changes can be made within the scope of the present application.

[0040] Example 1

[0041] The present embodiment provides a preparation method of an SPR biosensor chip, comprising the following steps:

[0042] 1. A gold layer with a thickness of 50 nm is plated on the surface of the prism by vacuum evaporation method, and then 95% alcohol is used for cleaning to obtain a prism-gold layer 1#. The vacuum degree of the vacuum chamber is 7x10 -3 Pa, and the temperature is 60℃.

[0043] 2. 100 μL of 0.1 mg / mL graphene nanosheet dispersion liquid is dropped on the surface of the gold layer of the prism-gold layer 1#, and is left to stand for 24 h and is naturally dried. The graphene nanosheets are deposited on the surface of the gold layer and form a graphene nanosheet layer. The graphene nanosheets in the graphene nanosheet dispersion liquid are single-layer graphene with a sheet diameter of 100-300 nm, and the dispersing agent of the graphene nanosheet dispersion liquid is a mixed liquid of deionized water and anhydrous ethanol with a volume ratio of 2:1.

[0044] 3. 100 μL of 0.1 mg / mL nanodiamond particle dispersion liquid is dropped on the graphene nanosheet layer formed in the second step, and is left to stand for 24 h and is naturally dried. The nanodiamond particles are deposited on the graphene nanosheet layer and form a nanodiamond particle layer to obtain an SPR biosensor chip 1#. The structure of the SPR biosensor chip 1# is that the prism, the gold layer and one sensitizing layer are sequentially stacked from bottom to top, and the sensitizing layer comprises the graphene nanosheet layer and the nanodiamond particle layer from bottom to top. The particle size of the nanodiamond particles in the nanodiamond particle dispersion liquid is 30 nm, and the dispersing agent of the nanodiamond particle dispersion liquid is deionized water.

[0045] The surface of the SPR biosensor chip 1# is scanned by an electron microscope to obtain a SEM graph as shown in FIG. 1. Figure 1 ​

[0046] Example 2

[0047] The present example provides a preparation method of an SPR biosensor chip, comprising the following steps:

[0048] Take the SPR biosensor chip 1# of Example 1, drop 100 μL of 0.1 mg / mL graphene nanosheet dispersion liquid on the surface of the SPR biosensor chip 1#, stand for 24 h and dry naturally; then drop 100 μL of 0.1 mg / mL nanodiamond particle dispersion liquid, stand for 24 h and dry naturally, to obtain the SPR biosensor chip 2#. The structure of the SPR biosensor chip 2# is that the prism, the gold layer and the two layers of the sensitizing layer are sequentially stacked from bottom to top, and each layer of the sensitizing layer comprises the graphene nanosheet layer and the nanodiamond particle layer from bottom to top.

[0049] Example 3

[0050] The present example provides a preparation method of an SPR biosensor chip, which is different from the SPR biosensor chip of Example 1 in that the gold layer is replaced by a silver layer. The structure of the obtained SPR biosensor chip is that the prism, the silver layer and the one layer of the sensitizing layer are sequentially stacked from bottom to top, and the sensitizing layer comprises the graphene nanosheet layer and the nanodiamond particle layer from bottom to top.

[0051] Comparative Example 1

[0052] The present comparative example provides a preparation method of a comparative SPR biosensor chip, comprising the following steps:

[0053] 1. A gold layer with a thickness of 50 nm is plated on the surface of the prism by vacuum evaporation method, and then cleaned with 95% alcohol to obtain the prism-gold layer 2#. The vacuum degree of the vacuum chamber is 7 x 10 -3 Pa, and the temperature is 60°C.

[0054] 2. Mix 0.1 mg / mL graphene nanosheet dispersion liquid and 0.1 mg / mL nanodiamond particle dispersion liquid at a ratio of 1:1 to obtain a mixed dispersion liquid. Then drop 100 μL of the mixed dispersion liquid on the surface of the gold layer of the prism-gold layer 2#, stand for 24 h and dry naturally, and the graphene nanosheet and the nanodiamond particle are deposited on the surface of the gold layer and form a graphene nanosheet-nanodiamond particle mixed layer to obtain the comparative SPR biosensor chip 1#. The structure of the comparative SPR biosensor chip 1# is that the prism, the gold layer and the one layer of the sensitizing layer are sequentially stacked from bottom to top, and the sensitizing layer is the graphene nanosheet-nanodiamond particle mixed layer. The graphene nanosheet dispersion liquid is the same as that of Example 1, and the nanodiamond particle dispersion liquid is the same as that of Example 1.

[0055] 3. Take the comparative SPR biosensor chip 1# of step 2, and drop 100 μL of the same mixed dispersion liquid as in step 2 on the surface thereof, and let stand for 24 h and dry naturally to obtain a comparative SPR biosensor chip 2#.

[0056] The difference between the comparative SPR biosensor chip 1# and the comparative SPR biosensor chip 2# is that the comparative SPR biosensor chip 1# has one graphene nanosheet-nanodiamond particle mixed layer deposited thereon, and the comparative SPR biosensor chip 2# has two graphene nanosheet-nanodiamond particle mixed layers deposited thereon.

[0057] Comparative Example 2

[0058] The present comparative example provides a method for preparing a comparative SPR biosensor chip, comprising the following steps:

[0059] 1. A gold layer with a thickness of 50 nm is vacuum-deposited on the surface of a prism, and then the prism-gold layer 3# is obtained by cleaning with 95% alcohol. The vacuum degree of the vacuum chamber is 7 x 10 -3 Pa, and the temperature is 60°C.

[0060] 2. 100 μL of a graphene nanosheet dispersion liquid with a concentration of 0.1 mg / mL is dropped on the surface of the gold layer of the prism-gold layer 3#, and let stand for 24 h and dry naturally. The graphene nanosheet is deposited on the surface of the gold layer and forms a graphene nanosheet layer to obtain a comparative SPR biosensor chip 3#. The structure of the comparative SPR biosensor chip 3# is that the prism, the gold layer and one sensitized layer are sequentially stacked from bottom to top, and the sensitized layer is the graphene nanosheet layer.

[0061] 2. Take the comparative SPR biosensor chip 3# of step 2, and drop 100 μL of the same graphene nanosheet dispersion liquid as in step 2 on the surface thereof, and let stand for 24 h and dry naturally to obtain a comparative SPR biosensor chip 4#.

[0062] The difference between the comparative SPR biosensor chip 3# and the comparative SPR biosensor chip 4# is that the comparative SPR biosensor chip 3# has one graphene nanosheet layer deposited thereon, and the comparative SPR biosensor chip 4# has two graphene nanosheet layers deposited thereon.

[0063] Performance test

[0064] The prism-gold layer 1# of Example 1, the SPR biosensor chip 1#, the SPR biosensor chip 2# of Example 2, the prism-gold layer 2# of Comparative Example 1, the comparative SPR biosensor chip 1# and the comparative SPR biosensor chip 2#, the prism-gold layer 3# of Comparative Example 2, the comparative SPR biosensor chip 3# and the comparative SPR biosensor chip 4# were used as the SPR biosensor to perform the refractive index sensing property test. Taking the SPR biosensor chip 1# as an example, the schematic diagram of the sensing detection system is shown in Figure 2 The light source used by the sensing detection system was a halogen tungsten lamp light source (AvaLight-HAL-(S)-Mini, Beijing Aiwantis Technology Co., Ltd., wavelength range: 360-2600 nm), and the spectrometer used was a YOKOGAWA spectrometer (YOKOGAWA AQ6370D, YOKOGAWA Measurement Technology Co., Ltd., wavelength detection range: 600-1800 nm). The test process was as follows: under normal temperature environment, the refractive index liquids with refractive indexes of 1.331, 1.334, 1.337, 1.340, 1.343 RIU were added to each SPR biosensor in turn, and after the addition of each refractive index liquid, the spectrum test / recording was performed, and after the test, the alcohol was used for cleaning to prevent the residual of molecules, and then the addition of the next refractive index liquid and the spectrum test / recording were performed.

[0065] The refractive index sensing property test results of the prism-gold layer 1# of Example 1 as the SPR biosensor are shown in Figure 3 , wherein Figure 3 (a) is the spectrum diagram, Figure 3 (b) is the linear fitting diagram of the resonance wavelength and the refractive index. It can be known from Figure 3 that as the refractive index of the refractive index liquid increases, the resonance wavelength drifts to the long wavelength; the linear fitting of the resonance wavelength and the refractive index is performed, the slope of the fitting straight line is the sensitivity of the sensor, and the sensitivity of the SPR biosensor chip with the prism-gold layer without sensitization is 11400 nm / RIU, and the linear correlation coefficient is 0.9898.

[0066] The refractive index sensing property test results of the SPR biosensor chip 1# of Example 1 as the SPR biosensor are shown in Figure 4 , wherein Figure 4 (a) is the spectrum diagram, Figure 4 (b) is the linear fitting diagram of the resonance wavelength and the refractive index. It can be known from Figure 4 that the slope of the fitting straight line is the sensitivity of the sensor, and the sensitivity of the SPR biosensor chip 1# is 12840 nm / RIU, which is increased by 12.6% compared with the prism-gold layer 1#.

[0067] The refractive index sensing characteristics test results of SPR biosensor chip 2# in Example 2 as an SPR biosensor are as follows: Figure 5 As shown, where Figure 5 (a) is the spectrum. Figure 5 (b) is a linear fit plot of the resonant wavelength and refractive index. From Figure 5 It can be seen that the slope of the fitted line is the sensitivity of the sensor chip. The sensitivity of SPR biosensor chip 2# is 15140nm / RIU, which is 32.8% higher than that of prism-gold layer 1#.

[0068] from Figure 4 (a) and Figure 5 The spectrum in (a) also shows that as the number of repeating sensitizing layers increases, the initial resonance wavelength (the spectral resonance wavelength when the refractive index of the liquid is 1.331 RIU) shifts to a longer wavelength, and the degree of distortion of the resonance valley of the spectrum increases accordingly. This indicates that the increase in the number of sensitizing layers has a more significant effect on the regulation of the plasma interface, resulting in an increase in sensitivity; however, it also enhances the scattering and dissipation of surface plasma waves, and the resonance valley becomes wider and shallower, that is, the degree of distortion increases.

[0069] The working process of the SPR biosensor chip in the embodiments of the present invention is as follows: Figure 6 As shown, the SPR effect, which occurs between incident light and a noble metal film at a specific wavelength λ, creates a resonance trough centered on λ in the transmission spectrum. When the amount of biomolecules attached to the SPR biosensor chip changes, the refractive index around the chip also changes, causing a change in the resonance conditions. This change manifests as a shift in the SPR resonance trough in the transmission spectrum. Therefore, sensing of the analyte can be achieved by monitoring the shift in λ.

[0070] The spectra and linear fitting plots of refractive index sensing characteristics obtained by comparing Prism-Gold Layer 2#, SPR Biosensor Chip 1#, and SPR Biosensor Chip 2# as SPR biosensors in Comparative Example 1 are shown below. Figure 7 As shown, where, Figure 7 (a) is the spectrum of prism-gold layer 2#. Figure 7 (b) is a linear fitting diagram of the resonant wavelength and refractive index of prism-gold layer 2#. Figure 7 (c) For comparison, the spectrum of SPR biosensor chip 1# is shown. Figure 7 (d) is a linear fitting graph comparing the resonant wavelength and refractive index of SPR biosensor chip 1#. Figure 7 (e) For comparison, the spectrum of SPR biosensor chip 2# is shown. Figure 7 (f) is a linear fitting graph comparing the resonant wavelength and refractive index of the SPR biosensor chip 2#. From Figure 7It can be seen that the slope of the fitting straight line is the sensitivity of the sensor, the sensitivity of the prism-gold layer 2# is 11760 nm / RIU, the sensitivity of the comparative SPR biosensor chip 1# is 11580 nm / RIU, and the sensitivity of the comparative SPR biosensor chip 2# is 11340 nm / RIU. With the increase of the deposition times of the mixed layer, the sensitivity of the SPR biosensor chip gradually decreases, and the resonance wavelength of the initial resonance spectrum (RI = 1.331) drifts to the right with the increase of the deposition times. This is because in the experimental process, the sensitization layer continuously falls off, the drift amount continuously decreases, and the slope of the linear fitting continuously decreases, that is, the sensitivity continuously decreases.

[0071] The spectrum and the linear fitting graph of the resonance wavelength and the refractive index of the prism-gold layer 3# of Comparative Example 2, the comparative SPR biosensor chip 3# and the comparative SPR biosensor chip 4# respectively as the SPR biosensor for the refractive index sensing property test are shown in Figure 8 , wherein, Figure 8 (a) is the spectrum of the prism-gold layer 3#, Figure 8 (b) is the linear fitting graph of the resonance wavelength and the refractive index of the prism-gold layer 3#, Figure 8 (c) is the spectrum of the comparative SPR biosensor chip 3#, Figure 8 (d) is the linear fitting graph of the resonance wavelength and the refractive index of the comparative SPR biosensor chip 3#, Figure 8 (e) is the spectrum of the comparative SPR biosensor chip 4#, Figure 8 (f) is the linear fitting graph of the resonance wavelength and the refractive index of the comparative SPR biosensor chip 4#. From Figure 8 It can be seen that the slope of the fitting straight line is the sensitivity of the sensor, the sensitivity of the prism-gold layer 3# is 11580 nm / RIU, the sensitivity of the comparative SPR biosensor chip 3# is 12000 nm / RIU, which is 3.6% higher than that of the prism-gold layer 3#; the sensitivity of the comparative SPR biosensor chip 4# is 12480 nm / RIU, which is 7.8% higher than that of the prism-gold layer 3#. It can be seen that under the same deposition times, the sensitization effect of the graphene nanosheet layer is not as good as that of the graphene nanosheet layer and the nanodiamond particle layer as a sensitization layer together, which shows that the graphene nanosheet layer and the nanodiamond particle layer as a sensitization layer together have better sensitization effect.

[0072] Obviously, the above embodiments of the present application are merely exemplary but not intended to limit the embodiments of the present application. Based on the above description, any other variations or changes can be made by those skilled in the art without departing from the spirit and principles of the present application. It is not necessary to list all the embodiments here. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application shall fall within the scope of the claims of the present application.

Claims

1. A method for fabricating an SPR biosensor chip, characterized in that, Includes the following steps: S1. A noble metal layer is coated on the surface of the substrate to obtain a substrate-noble metal layer; S2. Graphene nanosheets and nanodiamond particles are sequentially deposited on the surface of the substrate-noble metal layer to obtain the SPR biosensor chip. The deposition process described in step S2 is as follows: the graphene nanosheet dispersion is directly transferred to the surface of the substrate-noble metal layer, dried, and deposited to form a graphene nanosheet layer; then the nanodiamond particle dispersion is transferred to the graphene nanosheet layer, dried, and deposited to form a nanodiamond particle layer.

2. The preparation method according to claim 1, characterized in that, The precious metal layer mentioned in step S1 is a gold layer or a silver layer.

3. The preparation method according to claim 1, characterized in that, The covering process described in step S1 is as follows: a noble metal layer is deposited on the surface of the substrate by vacuum evaporation.

4. The preparation method according to claim 1, characterized in that, The graphene nanosheets in step S2 have a diameter of 50~500 nm.

5. The preparation method according to claim 1, characterized in that, The nanodiamond particles in the nanodiamond particle layer described in step S2 have a particle size of 25~35 nm.

6. The preparation method according to claim 1, characterized in that, The concentration of the graphene nanosheet dispersion is 0.08~0.12 mg / mL; the concentration of the nanodiamond particle dispersion is 0.08~0.12 mg / mL.

7. The preparation method according to claim 1, characterized in that, The deposition process in step S2 is repeated 1 to 2 times.

8. An SPR biosensor chip, characterized in that, It is prepared by any of the preparation methods described in claims 1 to 7.

9. The application of the SPR biosensor chip of claim 8 as a sensor in the field of surface plasmon resonance.

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