Surface plasma resonance sensing chip and application thereof in detection of malachite green

Abstract

The invention discloses a surface plasma resonance sensing chip and application thereof in detection of malachite green. The invention finds that the refractive index sensitivity of the SPR sensing chip can be effectively improved by regulating and controlling the deposition thickness of the ZIF-67 on the noble metal film, so that the refractive index sensitivity of the SPR sensing chip can be up to 2570.94 nm / RIU, which is improved by 42.30% compared with that of a pure noble metal film. In addition, the SPR sensing chip disclosed by the invention can be used for specifically detecting malachite green, and the surface refractive index of the SPR sensing chip is remarkably changed due to the interaction between ZIF-67 and malachite green. The preparation method of the SPR sensing chip is simple and easy to operate, and the prepared SPR sensing chip is high in sensitivity and good in linear correlation degree and has wide application prospects in the field of biological environment detection.

Description

Technical Field

[0001] The present invention relates to the technical field of SPR biosensors, and in particular to a surface plasmon resonance sensor chip and application thereof in detecting malachite green. Background Art

[0002] Surface plasmon resonance (SPR) technology is a highly sensitive, real-time, label-free detection method based on optical sensing. Its core principle is that when incident light strikes the interface between a metal film (such as gold or silver) and a dielectric medium at a specific angle, if the frequency of the light matches the oscillation frequency of the metal's surface plasmon, it triggers resonance in the metal's free electrons. This causes the electrons to absorb light energy, significantly reducing the intensity of the reflected light within a certain angle. The angle at which the reflected light intensity is minimized is called the SPR angle. When incident light excites free electrons on the metal surface at a specific angle, slight changes in the refractive index of the interface cause a significant shift in the resonance angle or light intensity, enabling label-free dynamic monitoring of biomolecular interactions. Due to its label-free, real-time, and highly sensitive nature, this technology is widely used in drug development (such as antibody-antigen binding kinetics analysis), life science research, environmental toxin detection, and clinical diagnostics (such as cancer marker screening). In recent years, with the convergence of nanophotonics, fiber optic sensing, and artificial intelligence technologies, SPR is developing towards portability, high throughput, and multi-parameter integration, providing a valuable tool for precision medicine and point-of-care testing.

[0003] Malachite green (Malachite Green) is a synthetic triphenylmethane basic dye that typically appears as dark green crystals or bluish-green powder with a metallic luster. Originally used as an industrial dye in the textile, leather, and paper industries, it later gained widespread application in aquaculture due to its antibacterial and antiparasitic properties to prevent fungal and parasitic infections in fish. However, studies have shown that malachite green and its metabolite, leucomalachite green, are highly toxic, carcinogenic, and teratogenic in vivo, accumulating readily through the food chain and posing a health threat. Consequently, most countries, including China, the European Union, and the United States, have banned its use in aquaculture. However, due to its low cost and significant efficacy, its illegal misuse continues to occur, making it a key target of food safety regulation. Therefore, developing a detection method for malachite green is crucial. Summary of the Invention

[0004] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a surface plasmon resonance sensor chip and its application in detecting malachite green.

[0005] The first object of the present invention is to provide a surface plasmon resonance sensor chip.

[0006] The second object of the present invention is to provide the use of the above-mentioned surface plasmon resonance sensor chip in the preparation of products for detecting malachite green.

[0007] The third object of the present invention is to provide a surface plasmon resonance sensing detection system.

[0008] The fourth object of the present invention is to provide a method for detecting malachite green.

[0009] The present invention claims the following: A surface plasmon resonance sensor chip, comprising a substrate, a metal material layer and an imidazole-based MOF material layer; The metal material layer is deposited on the surface of the substrate, and the imidazole-based MOF material layer is deposited on the surface of the metal material layer.

[0010] Preferably, the imidazole-based MOF material is a zeolite imidazole ester framework material.

[0011] Preferably, the deposition thickness of the imidazole-based MOF material layer is 50 to 250 μm.

[0012] More preferably, the zeolitic imidazolate framework material is ZIF-67.

[0013] More preferably, the ZIF-67 layer is deposited to a thickness of 100 μm. By regulating the deposition thickness, the refractive index sensitivity exhibits a nonlinear variation trend. When the ZIF-67 layer is deposited to a thickness of 100 μm, the surface plasmon resonance sensor chip of the present invention can achieve a maximum refractive index sensitivity of 2570.94 nm / RIU, a 42.30% increase compared to a pure metal material layer. This significantly improves the surface plasmon resonance sensor chip's ability to detect subtle changes in the dielectric environment.

[0014] Preferably, the metal material layer includes a chromium layer and a gold layer, the chromium layer is deposited on the surface of the substrate, and the gold layer is deposited on the surface of the chromium layer.

[0015] Preferably, the deposition thickness of the chromium layer is 4-6 nm, and the deposition thickness of the gold layer is 44-46 nm.

[0016] More preferably, the deposition thickness of the chromium layer is 5 nm, and the deposition thickness of the gold layer is 45 nm.

[0017] Preferably, the substrate is optical glass.

[0018] Use of any of the above-mentioned surface plasmon resonance sensor chips in the preparation of products for detecting malachite green.

[0019] A surface plasmon resonance sensing detection system comprises any of the above-mentioned surface plasmon resonance sensing chips.

[0020] Preferably, the surface plasmon resonance sensing detection system further comprises a prism, a light source, a polarizer, a plano-convex lens and / or a spectrometer.

[0021] A method for detecting malachite green, using the above-mentioned surface plasmon resonance sensing detection system for detection; The method is aimed at non-disease treatment and diagnosis.

[0022] Surface plasmon resonance (SPR) sensing is based on the physical optical phenomenon induced by light at the interface between a metal (such as a gold film) and a dielectric. When polarized light is incident on the interface at a specific angle, total internal reflection occurs, resulting in an evanescent wave that penetrates the metal surface. If the evanescent wave couples with free electrons on the metal surface, it excites surface plasmon resonance, causing a significant decrease in the intensity of the reflected light. The corresponding angle of incidence is called the resonance angle, which is extremely sensitive to the refractive index of the medium adjacent to the metal surface. Therefore, when a ligand (such as protein or DNA) immobilized on the metal surface binds or dissociates with an analyte (such as an antibody or target sequence), the surface changes cause a shift in the refractive index, which in turn leads to a shift in the resonance angle or a change in the intensity of the reflected light. Real-time monitoring of this signal allows for quantitative analysis of molecular interactions. Therefore, when a malachite green solution interacts with ZIF-67 immobilized on a noble metal film, the refractive index of the medium adjacent to the metal surface changes. By monitoring this SPR change in real time, the malachite green solution can be quantitatively detected.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a surface plasmon resonance (SPR) sensor chip and its application in detecting malachite green. The present invention discovered that regulating the thickness of the imidazole-based MOF material layer deposited on a precious metal film can effectively improve the refractive index sensitivity of the SPR sensor chip. Furthermore, the SPR sensor chip of the present invention can specifically detect malachite green. The interaction between the imidazole rings on the imidazole-based MOF material and malachite green significantly alters the surface refractive index of the SPR sensor chip.

[0024] The SPR sensor chip preparation method of the present invention is simple and easy to operate. At the same time, the prepared SPR sensor chip has high sensitivity and good linear correlation, and has broad application prospects in the field of biological environment detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation process of the SPR sensor chip.

[0026] Figure 2Schematic diagram of the overall structure of the SPR biosensor detection system; 1-SPR sensor chip, 2-BK7 prism, 3-light source, 4-polarizer, 5-plano-convex lens, 6-spectrometer, 7-incident light, 8-reflected light.

[0027] Figure 3 This is the reflection spectrum curve of the SPR sensor chip at different refractive indices when the number of ZIF-67 deposition times on the SPR sensor chip is 2.

[0028] Figure 4 is the refractive index sensitivity of ZIF-67 with different deposition times on the SPR sensor chip.

[0029] Figure 5 This is the process of the resonance wavelength responding over time after 100 μg / mL malachite green solution was added to the SPR sensor chip.

[0030] Figure 6 These are the linear fitting results of the resonance wavelength shift and the malachite green solution concentration after 0, 20, 40, 60, 80, and 100 μg / mL of malachite green solution were added to the SPR sensor chip.

[0031] Figure 7 It is the resonance wavelength shift after 100 μg / mL malachite green solution, methylene blue solution, crystal violet solution, leucomalachite green solution, and diethylstilbestrol solution are added to the SPR sensor chip respectively.

[0032] Figure 8 The detection results of lead by SPR sensor chips with different deposition times; A: ZIF-67 was deposited once on the SPR sensor chip, B: ZIF-67 was deposited twice on the SPR sensor chip, C: ZIF-67 was deposited three times on the SPR sensor chip, D: ZIF-67 was deposited four times on the SPR sensor chip. DETAILED DESCRIPTION

[0033] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0035] Example 1 Preparation and performance testing of SPR biosensor detection system based on ZIF-67 1. Experimental Methods The preparation process of SPR sensor chip is as follows Figure 1 As shown, the specific preparation method is: A 5 nm thick chromium film was vacuum-evaporated onto a glass substrate. A 45 nm thick gold film was then vacuum-evaporated onto the chromium film to form a precious metal film. A 100 μg / mL ZIF-67 solution was drop-coated onto the precious metal film. After drop-coating, the film was exposed to air to allow the solvent to evaporate. The film was then rinsed with deionized water, dried, and then drop-coated with the ZIF-67 solution once more. After evaporation of the solvent, washing, and drying, an SPR sensor chip was prepared. The ZIF-67 layer was deposited twice (i.e., dropped onto the film), resulting in a thickness of approximately 100 μm (each ZIF-67 deposition cycle resulted in a thickness of approximately 50 μm).

[0036] The schematic diagram of the SPR biosensor detection system is shown in Figure 2 As shown, the SPR sensor chip 1 includes a BK7 prism 2, a light source 3, a polarizer 4, a plano-convex lens 5, and a spectrometer 6. Incident light, emitted by light source 3, is first converted into polarized light by the polarizer 4. It is then focused by the plano-convex lens 5 and incident on the BK7 prism 2. The BK7 prism 2 couples to the glass substrate of the SPR sensor chip 1 via matching oil (to isolate it from air). The incident light generates the SPR phenomenon at the interface of the noble metal thin film. Reflected light exits the BK7 prism 2, is then dispersed by the plano-convex lens 5, and is finally received and analyzed by the spectrometer 6.

[0037] After assembling the SPR biosensor system, the refractive index sensing characteristics were tested. At room temperature, refractive index matching solutions of 1.334, 1.337, 1.340, and 1.343 RIU were added to the SPR sensor chip. Spectra were measured and recorded after each addition. After each test, the chip was rinsed with deionized water to prevent residual molecules. The next refractive index matching solution was then added and the spectra were measured and recorded.

[0038] 2. Experimental Results The reflection spectrum curves under different refractive indices are as follows Figure 3 As shown, as the refractive index of the added refractive index matching liquid increases, the resonance valley wavelength of the reflection spectrum increases, and there is a good linear correlation between the resonance valley wavelength and the refractive index.

[0039] Example 2 Effect of deposition times on the refractive index sensitivity of the SPR sensor chip 1. Experimental Methods The SPR sensor chip was prepared according to Example 1, except that the deposition times of ZIF-67 were set to 0, 1, 2, 3 and 4 times, respectively, that is, the deposition thickness of ZIF-67 was 0, 50 μm, 100 μm, 150 μm and 200 μm, respectively.

[0040] An SPR biosensor detection system was assembled according to Example 1, and refractive index sensing characteristics were tested. At room temperature, refractive index matching solutions of 1.334, 1.337, 1.340, and 1.343 RIU were added to each SPR sensor chip. After addition, spectral measurements were performed and the resonant wavelengths of the SPR sensor chip under different refractive index matching solutions were recorded. The resonant wavelength was linearly fitted with the refractive index to obtain the refractive index sensitivity.

[0041] 2. Experimental Results The refractive index sensitivity results for different deposition times are shown in Figure 2. Figure 4 As shown in the figure, with the increase of the number of deposition times, the refractive index sensitivity shows a trend of first increasing and then decreasing. When the number of deposition times is 2, the refractive index sensitivity reaches the highest 2570.94 nm / RIU, which is 42.30% higher than that of pure noble metal film. Therefore, 2 times is selected as the deposition number of ZIF-67 on the SPR sensor chip.

[0042] Example 3 Detection of Malachite Green by SPR Sensor Chip 1. Experimental Methods At room temperature, 100 μg / mL of malachite green solution was added dropwise to the SPR sensor chip prepared in Example 1, and a spectral test was performed using the SPR biosensor detection system assembled in Example 1. The spectra were received and recorded by the spectrometer 6 every 2 minutes for a total of 30 minutes.

[0043] At room temperature, 0, 20, 40, 60, 80 and 100 μg / mL of malachite green solutions were dripped onto the SPR sensor chip prepared in Example 1, and a spectral test was performed using the SPR biosensor detection system assembled in Example 1. The resonance wavelength shift under different concentrations of malachite green solutions was received and analyzed by spectrometer 6 (i.e., the difference in resonance wavelength at the moment of dripping and after stabilization of the malachite green solution, where the moment of dripping is equivalent to no reaction and the moment of stabilization is equivalent to complete reaction), and a linear fit was performed between the resonance wavelength shift and the concentration of the malachite green solution.

[0044] 2. Experimental Results The resonance wavelength responds to time as follows: Figure 5 As shown, spectral recording begins immediately after the malachite green solution is added. Within the first 16 minutes, the spectral line shifts significantly, with the rate of line shift decreasing over time. After 16 minutes, the absorption of malachite green slowly reaches saturation, the reaction approaches completion, and the spectral line essentially stops shifting.

[0045] The linear fitting results of the resonance wavelength shift and the concentration of malachite green solution are as follows: Figure 6As shown in Figure 2, with the increase of malachite green solution concentration, the corresponding increase in the sensor resonance wavelength shift shows a good linear correlation (R 2 =0.99121).

[0046] Example 4 Effects of different dyes and interfering substances on the SPR sensor chip 1. Experimental Methods At room temperature, 100 μg / mL of malachite green solution, methylene blue solution, crystal violet solution, leucomalachite green solution, and diethylstilbestrol solution were respectively added dropwise to the SPR sensor chip prepared in Example 1. Spectral testing was performed using the SPR biosensor detection system assembled in Example 1, and the resonance wavelength shift under different solutions was received and analyzed by the spectrometer 6.

[0047] 2. Experimental Results The results are as follows Figure 7 As shown, the SPR biosensor detection system prepared in Example 1 exhibited minimal response to other dyes and interfering substances at the same concentration. The SPR shifts of methylene blue solution, crystal violet solution, leucomalachite green solution, and diethylstilbestrol solution at a concentration of 100 μg / mL were 3.93, 3.57, 1.32, and 1.31 nm, respectively. At the same concentration, the wavelength shift of malachite green solution was much greater than that of nonspecific substances, demonstrating that the SPR sensor prepared in Example 1 has good selectivity.

[0048] Comparative Example 1 1. Experimental Methods An SPR sensor chip was prepared according to Example 1, except that the deposition times of ZIF-67 were set to 1, 2, 3, and 4 times, respectively, that is, the deposition thickness of ZIF-67 was 50 μm, 100 μm, 150 μm, and 200 μm, respectively. The SPR sensor chip was prepared and assembled into an SPR biosensor detection system according to Example 1.

[0049] At room temperature, 200 μM Pb was added to the SPR sensor chip. 2+ The solution was tested spectrally by the SPR biosensor detection system, and the spectra were received and recorded by the spectrometer 6 every 1 min for a total of 6 min.

[0050] 2. Experimental Results According to the study "Study on the Adsorption Performance of Zeolite Imidazole Framework Material ZIF-67 on Heavy Metal Ions Cadmium, Copper and Lead" (published on February 28, 2020), ZIF-67 material has good adsorption capacity for lead. However, Figure 8Results A–D in Figure 3 show that the SPR response of Pb does not change with increasing ZIF-67 deposition times, and therefore cannot be used to detect Pb. These results indicate that although there is an interaction between two substances, it does not necessarily cause an SPR change.

[0051] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A surface plasmon resonance sensor chip, characterized in that: The surface plasmon resonance sensor chip includes a substrate, a metal material layer and an imidazole-based MOF material layer; The metal material layer is deposited on the surface of the substrate, and the imidazole-based MOF material layer is deposited on the surface of the metal material layer.

2. The surface plasmon resonance sensor chip according to claim 1, characterized in that: The imidazole-based MOF material is a zeolite imidazole ester framework material.

3. The surface plasmon resonance sensor chip according to claim 2, characterized in that: The zeolite imidazolate framework material is ZIF-67.

4. The surface plasmon resonance sensor chip according to claim 1, wherein The deposition thickness of the imidazole-based MOF material layer is 50 to 250 μm.

5. The surface plasmon resonance sensor chip according to claim 1, characterized in that: The metal material layer includes a chromium layer and a gold layer. The chromium layer is deposited on the surface of the substrate, and the gold layer is deposited on the surface of the chromium layer.

6. The surface plasmon resonance sensor chip according to claim 5, wherein: The deposition thickness of the chromium layer is 4-6 nm, and the deposition thickness of the gold layer is 44-46 nm.

7. Use of the surface plasmon resonance sensor chip according to any one of claims 1 to 6 in the preparation of a product for detecting malachite green.

8. A surface plasmon resonance sensing detection system, characterized in that: The surface plasmon resonance sensing detection system comprises the surface plasmon resonance sensing chip according to any one of claims 1 to 6.

9. The surface plasmon resonance sensing system according to claim 8, characterized in that: The surface plasmon resonance sensing detection system further includes a prism, a light source, a polarizer, a plano-convex lens and / or a spectrometer.

10. A method for detecting malachite green, characterized in that: Detection is performed using the surface plasmon resonance sensing detection system according to claim 8 or 9; The method is aimed at non-disease treatment and diagnosis.

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