A meta-spr optical detection sensor for detecting metabolites in human body fluids

Abstract

The application provides a MetaSPR optical detection sensor for detecting metabolites in human body fluids, and belongs to the technical field of MetaSPR sensors. The MetaSPR optical detection sensor comprises a MetaSPR chip; the application is characterized in that a metal film with a silver mirror effect is deposited on the back of the MetaSPR chip, and a Ti3C2T x The mixed solution of MXene and graphene oxide is dried to obtain the MGMSPR sensor; when the MGMSPR sensor is used for detecting metabolites in human body fluids, corresponding enzymes and protective layers are added first, then the sample to be detected is added, and the corresponding products are detected by an optical or electrochemical method to realize quantitative analysis. The MGMSPR sensor prepared by the application has a unique nanoscale 3D structure on the surface, has higher sensitivity and specificity, and has a lower detection limit compared with traditional MetaSPR sensors, is suitable for detecting lower concentration samples, and has a wide application potential in the field of small molecule metabolites in human body fluids.

Description

Technical Field

[0001] This invention relates to the field of MetaSPR sensor technology, and more specifically to a MetaSPR optical detection sensor for detecting metabolites in human body fluids. Background Technology

[0002] Wearable sensing devices continuously and in real-time acquire physiological information by non-invasively measuring biochemical indicators in biological fluids. They can non-invasively sample and analyze bodily fluids such as sweat, tears, and saliva at specific sites, enabling cost-effective and real-time biomedical monitoring of health and disease. Among these, sweat, containing various biochemical substances including electrolytes, metabolites, and hormones, has become one of the most challenging research areas in biomedical sensing. Monitoring biomarkers in sweat to assess health and disease in real-time is also rapidly developing in the field of wearable sensors. Furthermore, sweat's ease of acquisition makes it the preferred sample for real-time monitoring by wearable sensors.

[0003] Existing studies have shown the dynamic changes in various biomarkers in sweat, including glucose, cortisol, and neuroimmunological markers. Among various metabolites, glucose is an important biomarker for the diagnosis and treatment of diabetes, but its concentration fluctuates greatly and depends on a patient's diet and lifestyle. Therefore, real-time blood glucose monitoring is crucial for the prevention of diabetes and its complications. Although some wearable sensors for real-time glucose monitoring (CGM) have been developed, they still suffer from drawbacks such as low sensitivity and large sensor size.

[0004] Metasurface plasmon resonance (MetaSPR) sensors have proven to be more portable and more sensitive than traditional biosensors. Surface plasmon resonance (SPR) technology is a novel analytical technique based on optical principles. It refers to the resonance phenomenon that may occur when light undergoes total internal reflection on the surface of a prism or metal film, forming an evanescent wave that enters the optically less dense medium. Meanwhile, a certain amount of plasma wave exists in the medium. Based on the principle of energy conservation, these two wavebands may meet. Currently, silver mirrors are widely used in optical systems, but their use in SPR technology to improve detection sensitivity is less common. Silver's low optical loss and clearer, stronger SPR bands in the visible and near-infrared praseodymium range make it an ideal choice for improving the sensitivity and performance of SPR sensors. Due to the inherent characteristics of optical biosensors, despite their unique nanostructures and the silver mirror effect (SME), direct label-free detection of small molecules such as glucose using MetaSPR biosensors remains challenging. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a MetaSPR optical sensor for detecting metabolites in human body fluids, which can detect metabolite concentrations with high sensitivity and in real time.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a MetaSPR optical detection sensor for detecting metabolites in human body fluids, comprising a MetaSPR chip; wherein a metal film with a silver mirror effect is deposited on the back side of the MetaSPR chip, and Ti3C2T is coated on the surface of the MetaSPR chip. x A mixed solution of MXene and graphene oxide (GO) was dried to obtain a MetaSPR optical detection sensor (denoted as MGMSPR sensor) for detecting metabolites in human body fluids.

[0008] This invention enhances the sensitivity of the MetaSPR chip by depositing a metal film of a certain thickness with a silver mirror effect on the back side of the chip and employing a reflection mode for detection. Silver possesses excellent optical properties, including high reflectivity and good conductivity; the silver mirror acts as a reflective layer. When light waves excite surface plasmons on the metal surface of the MetaSPR chip, the reflection effect of the silver mirror causes the electromagnetic field to be reflected and amplified multiple times between multiple metals and the silver mirror, thereby enhancing the signal strength of the MGMSPR sensor.

[0009] This invention uses Ti3C2T x MXene and GO were simply mixed, and the resulting solution was coated onto the surface of a MetaSPR chip to fabricate an MGMSPR sensor with a unique 3D surface structure, further enhancing the resonance effect of surface plasmons; among which, Ti3C2T x The two-dimensional nanostructure of MXene endows the MG composite film with an open porous structure and a highly hydrophilic microenvironment; while the GO film exhibits wrinkled microstructure characteristics, Ti3C2T x The MG film obtained by combining MXene and GO has a unique stacked 3D nanosheet structure, and the porosity of its structure can be changed by altering the Ti3C2T x The ratio between MXene and GO is adjusted to suit the detection of different small metabolic molecules.

[0010] Preferably, the metal film consists of titanium, silver, and gold in sequence; wherein the thickness of the titanium layer is 10-30 nm, the thickness of the silver layer is 20-200 nm, and the thickness of the gold layer is 10-30 nm.

[0011] Preferably, the Ti3C2T x The weight ratio of MXene to GO is (0~5):(0~5), and Ti3C2T x MXene and GO are not both 0.

[0012] Preferably, the Ti3C2T x A mixed solution of MXene and GO was obtained by passing Ti3C2T x The MXene dispersion and GO dispersion were mixed uniformly by ultrasonication.

[0013] Secondly, the present invention provides a method for preparing a MetaSPR optical detection sensor for detecting metabolites in human body fluids, comprising the following steps:

[0014] S1. A tapered nanopillar array is molded on a silicon oxide wafer using laser interference lithography and ion etching technology. After the mold is placed in a vacuum dryer filled with hexylsilane for hydrophobic treatment, optical adhesive is uniformly coated on the mold, and a PET film is placed on it. After curing under ultraviolet irradiation, the nanocup array located on the PET film is obtained by peeling. A metal film 1 is deposited on the nanocup array using electron beam evaporation to obtain the MetaSPR chip.

[0015] S2. A metal film 2 with a silver mirror effect is deposited on the back side of the MetaSPR chip using electron beam evaporation to obtain a silver mirror MetaSPR chip.

[0016] S3, Ti3C2T x The MXene dispersion and GO dispersion were mixed evenly, and the mixture was added to the surface of the silver mirror MetaSPR chip and dried to obtain the MGMSPR sensor.

[0017] Preferably, the optical adhesive is Norland optical adhesive NOA-61.

[0018] Preferably, the metal film 1 is composed of titanium and gold in sequence, or the metal film 1 is composed of titanium, silver and gold in sequence.

[0019] Preferably, the metal film 2 consists of titanium, silver, and gold in sequence; wherein the thickness of the titanium layer is 10-30 nm, the thickness of the silver layer is 20-200 nm, and the thickness of the gold layer is 10-30 nm.

[0020] Thirdly, the present invention provides the application of the sensor or the sensor prepared by the method in detecting metabolites in human body fluids.

[0021] Preferably, the human body fluids include, but are not limited to, sweat, tears, and saliva; the metabolites include, but are not limited to, glucose, lactic acid, urea, and cholesterol.

[0022] Preferably, the method for detecting the metabolite includes the following steps:

[0023] (1) Immobilize the enzyme corresponding to the metabolite to be tested on the chip surface of the sensor;

[0024] (2) Add Nafion or chitosan into the sensor chip hole;

[0025] (3) Add the sample to be tested into the sensor chip hole.

[0026] Preferably, when the metabolite to be tested is glucose, the corresponding enzyme is glucose oxidase (GOD); the GOD can convert glucose into gluconic acid and hydrogen peroxide, and quantitative analysis of glucose can be achieved by detecting the generated hydrogen peroxide optically.

[0027] Preferably, when the metabolite to be tested is lactic acid, the corresponding enzyme is lactate oxidase (LOD) or lactate dehydrogenase (LDH); LOD can convert lactic acid into hydrogen peroxide, and quantitative analysis of lactic acid can be achieved by detecting the generated hydrogen peroxide optically; LDH can indirectly measure the lactic acid concentration by detecting the generated NADH (coenzyme).

[0028] Preferably, when the metabolite to be tested is urea, the corresponding enzyme is urease; the urease hydrolyzes urea into ammonia and carbon dioxide, and the concentration of chemical urea can be obtained by detecting the amount of ammonia generated or the change in pH value.

[0029] Preferably, when the metabolite to be tested is cholesterol, the corresponding enzyme is cholesterol oxidase or cholesterol esterase; the cholesterol oxidase or cholesterol esterase can oxidize cholesterol to hydrogen peroxide or react with esters to release cholesterol, and quantitative analysis can be achieved by detecting the generated hydrogen peroxide by optical or electrochemical methods.

[0030] The MGMSPR sensor of this invention is suitable for detecting various small molecule metabolites in human body fluids, including glucose, lactic acid, urea, cholesterol, etc. When using the MGMSPR sensor to detect other small molecule metabolites, the first step is to fix the corresponding enzymes on the surface of the sensor chip. After fixing the corresponding enzymes, this invention also needs to add Nafion or chitosan as a protective layer to prevent enzyme loss, maintain enzyme activity, and prevent the influence of external environmental factors such as pH value and ionic strength changes on the enzymes, thereby further improving the stability of the sensor. Among them, Nafion is a negatively charged ion exchange membrane that can repel charged interferences (such as anionic interferences) from entering the enzyme layer, thereby improving the selectivity of the sensor.

[0031] Compared with the prior art, the advantages of the present invention are:

[0032] 1. This invention utilizes a metal film with a silver mirror effect deposited on the back of a MetaSPR chip and an MG composite film to modify the surface of the MetaSPR chip. The resulting MGMSPR sensor exhibits higher sensitivity and specificity, as well as a lower detection limit compared to traditional MetaSPR sensors. Specifically, the metal film with the silver mirror effect reduces scattering loss, improving signal strength and accuracy. Furthermore, the reflection effect of the silver mirror allows the electromagnetic field to be reflected and amplified multiple times between various metals and the silver mirror, thereby enhancing the signal strength of the MGMSPR sensor. The MG composite film possesses a unique nanoscale 3D structure, which facilitates its binding with small molecules, further enhancing the sensor's detection performance.

[0033] 2. Based on the MGMSPR sensor, this invention selects and immobilizes corresponding enzymes according to different analytes, and also uses Nafion or chitosan as a protective layer to prevent enzyme loss or inactivation, further improving the selectivity and stability of the sensor; finally, the corresponding products are detected by optical or electrochemical methods to achieve quantitative analysis of small molecule metabolites. Attached Figure Description

[0034] Figure 1 This is a flowchart illustrating the fabrication process of the MGMSPR sensor.

[0035] Figure 2 A is a physical image of the MetaSPR chip; Figure 2 B is a top-view SEM image of the MetaSPR chip; Figure 2 C is a SEM image of the cross-section of the MetaSPR chip;

[0036] Figure 3 The relationship between the thickness of the silver mirror layer and the sensor sensitivity;

[0037] Figure 4 A represents the reflection intensity of the MetaSPR chip with a 9 nm Ti + 90 nm Au metal film. Figure 4 B represents the reflection intensity of the MetaSPR chip with a 9nm Ti + 90nm Au metal film. Figure 4 C is the regression fitting curve of the difference in reflection intensity between 605 nm and 660 nm for the MetaSPR chip with a 9 nm Ti + 90 nm Au metal film and the silver mirror MetaSPR chip. Figure 4 D represents the reflection intensity of the MetaSPR chip with a 9 nm Ti + 70 nm Ag + 20 nm Au metal film. Figure 4E represents the reflection intensity of the MetaSPR chip with a 9nm Ti + 70 nm Ag + 20 nm Au metal film. Figure 4 F is the regression fitting curve of the difference in reflection intensity between the MetaSPR chip with a 9 nm Ti + 70 nm Ag + 20 nm Au metal film and the silver mirror MetaSPR chip at 628 nm-650 nm.

[0038] Figure 5 A flowchart illustrating the fabrication of an MGMSPR sensor on a silver mirror MetaSPR chip;

[0039] Figure 6 A is Ti3C2T x TEM image of MXene thin film; Figure 6 B is a TEM image of the GO thin film; Figure 6 C is a TEM image of the MG composite film; Figure 6 D represents different Ti3C2T x SEM images of the MGMSPR sensor surface with MXene to GO weight ratio;

[0040] Figure 7 A represents the relationship between the MG weight ratio and the sensitivity of the MGMSPR sensor; Figure 7 B is the curve showing the relationship between sucrose concentration and the corresponding signal in an M:G=2:1 MGMSPR sensor;

[0041] Figure 8 A represents the real-time binding curve between the MGMSPR sensor and IgG; Figure 8 B is the curve showing the relationship between IgG concentration and response signal; Figure 8 C represents the use of the MGMSPR sensor to monitor the intensity of the binding and dissociation interactions between rapamycin and FKBP12 molecules in real time.

[0042] Figure 9 A is a schematic diagram of the principle of glucose detection by the MGMSPR sensor; Figure 9 B shows the differential spectra of glucose at different concentrations detected using an MGMSPR sensor; Figure 9 C represents the functional relationship between the relative response intensity of the MGMSPR sensor and the glucose concentration;

[0043] Figure 10 A is a schematic diagram of a microfluidic chip based on the MGMSPR sensor; Figure 10 B is a physical image of a microfluidic chip based on a dual-channel MGMSPR sensor; Figure 10 C represents the response curve of the MGMSPR sensor monitoring different concentrations of glucose in artificial sweat; Figure 10D is the 4PL regression curve of the response signal of the MGMSPR sensor versus glucose concentration; Figure 10 E is a comparison graph showing the glucose concentration in artificial sweat detected using a commercial blood glucose meter and an MGMSPR sensor, respectively. Figure 10 F represents the selective verification result of the MGMSPR sensor; Figure 10 G represents the non-specific verification result of the MGMSPR sensor. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] This invention provides a MetaSPR optical sensor for detecting metabolites in human body fluids, comprising a MetaSPR chip; a metal film with a silver mirror effect is deposited on the back side of the MetaSPR chip, and Ti3C2T is coated on the surface of the MetaSPR chip. x A mixed solution of MXene and GO was dried to obtain a MetaSPR optical sensor (denoted as MGMSPR sensor) for detecting metabolites in human body fluids; the preparation process is as follows: Figure 1 (As shown).

[0046] In some examples, the metal film consists of titanium, silver, and gold in sequence; wherein the titanium layer has a thickness of 10-30 nm, the silver layer has a thickness of 20-200 nm, and the gold layer has a thickness of 10-30 nm.

[0047] In some examples, the Ti3C2T x The weight ratio of MXene to GO is (0~5):(0~5), and Ti3C2T x MXene and GO are not both 0.

[0048] The method for preparing the MetaSPR optical detection sensor for detecting metabolites in human body fluids includes the following steps:

[0049] S1. A tapered nanopillar array is molded on a silicon oxide wafer using laser interference lithography and ion etching technology. After the mold is placed in a vacuum dryer filled with hexylsilane for hydrophobic treatment, Norland optical adhesive NOA-61 is uniformly coated on the mold, and a PET film is placed on it. After curing under ultraviolet irradiation, the nanocup array located on the PET film is obtained by peeling. A metal film 1 is deposited on the nanocup array using electron beam evaporation to obtain the MetaSPR chip.

[0050] S2. A metal film 2 with a silver mirror effect is deposited on the back side of the MetaSPR chip using electron beam evaporation to obtain a silver mirror MetaSPR chip.

[0051] S3, Ti3C2T x The MXene dispersion and GO dispersion were mixed evenly, and the mixture was added to the surface of the silver mirror MetaSPR chip and dried to obtain the MGMSPR sensor.

[0052] In some examples, the metal film 1 is composed of titanium and gold in sequence, or the metal film 1 is composed of titanium, silver and gold in sequence.

[0053] In some examples, the metal film 2 is composed of titanium, silver, and gold in sequence; wherein the thickness of the titanium layer is 10-30 nm, the thickness of the silver layer is 20-200 nm, and the thickness of the gold layer is 10-30 nm.

[0054] Example 1

[0055] MetaSPR optical detection sensor for detecting metabolites in human body fluids

[0056] I. Investigating the effect of silver mirror layer thickness on the sensitivity of the silver mirror MetaSPR chip.

[0057] A tapered nanopillar array was molded on a 12-inch silicon oxide wafer using laser interference lithography and ion etching. After the mold was placed in a vacuum dryer filled with hexylsilane for 12 hours for hydrophobic treatment, Norland optical adhesive (NOA-61) was uniformly coated on the mold, and a PET film was placed on it. After curing by ultraviolet irradiation for 3 minutes, the nanocup array on the PET film was obtained by peeling. A Ti layer with a thickness of 9 nm and an Au layer with a thickness of 90 nm were sequentially deposited on the nanocup array using electron beam evaporation to obtain the MetaSPR chip.

[0058] A 10 nm Ti film, a 20-100 nm Ag film, and a 10 nm gold film were deposited on the back side of the MetaSPR chip using electron beam evaporation to obtain a silver mirror MetaSPR chip. The morphology of the silver mirror MetaSPR chip was characterized by SEM.

[0059] Figure 2 A is a physical image of the MetaSPR chip with a silver mirror. It can be observed that the MetaSPR chip with a silver mirror exhibits a visual color change phenomenon. This is the result of the collective oscillation of free electrons in the metal nanostructure excited when exposed to light. This phenomenon has a strong response to the wavelength of the incident light, causing the visual color to change with the wavelength of the light. Figure 2 B is a top-view SEM image of the MetaSPR chip. Figure 2 B shows that the MetaSPR chip exhibits a uniform nanoarray; Figure 2 C is a SEM image of the cross-section of the MetaSPR chip. Figure 2 C also shows that the MetaSPR chip has a nanocup array structure.

[0060] Considering that the silver mirror effect varies depending on the thickness of the metal on the back of the MetaSPR chip, the inventors compared the thickness of the Ag layer on the back of the MetaSPR chip in Example 1. In the back metal film, a 10 nm Ti layer serves as a binder between the substrate and the silver, and a 10 nm Au layer acts as a surface layer to prevent the silver from being oxidized by air. Compared to the Ag layer, the Ti and Au layers have relatively smaller impacts on the sensitivity of the MetaSPR chip; therefore, the silver layer thickness was tested in the range of 20-100 nm. Sensitivity tests were conducted on silver layers of different thicknesses using sucrose solutions of different concentrations (0-5% w / v).

[0061] Sucrose solutions with an RI range of 1.3328–1.34023 (RI of water = 1.3328, RI of 5% sucrose = 1.3403) were prepared using water. The performance of silver layers of different thicknesses was evaluated using a high-resolution spectrometer within a narrow wavelength range (500–700 nm) based on the dual-wavelength difference reflectance intensity. The results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the performance of the 80 nm Ag layer is close to that of the 100 nm Ag layer, indicating that the silver mirror effect reaches the saturation point at a thickness of 80 nm. Therefore, the silver mirror metal film composed of 10 nm Ti, 80 nm Ag and 10 nm Au has the highest reflection intensity and can clearly distinguish the low sugar solution concentration.

[0062] II. Investigating the Influence of Different Sensing Metal Films in the MetaSPR Chip on the Sensitivity of the MetaSPR Chip for Silver Mirrors

[0063] A tapered nanopillar array was molded on a 12-inch silicon oxide wafer using laser interference lithography and ion etching. After the mold was placed in a vacuum dryer filled with hexylsilane for 12 hours for hydrophobic treatment, Norland optical adhesive (NOA-61) was uniformly coated on the mold, and a PET film was placed on it. After curing by ultraviolet irradiation for 3 minutes, the nanocup array on the PET film was obtained by peeling. 9 nm Ti and 90 nm Au and 9 nm Ti, 70 nm Ag and 20 nm Au were deposited on the nanocup array by electron beam evaporation, respectively, to obtain two types of MetaSPR chips.

[0064] A silver mirror MetaSPR chip was obtained by depositing a 10 nm Ti film, an 80 nm Ag film, and a 10 nm Au film on the back side of the MetaSPR chip using electron beam evaporation.

[0065] Sensitivity tests were performed on silver mirror MetaSPR chips with different MetaSPR chips using sucrose solutions of varying concentrations (0-5% w / v). Sucrose solutions with an RI range of 1.3328–1.34023 were prepared using water. The sensitivity of both the MetaSPR chip and the silver mirror MetaSPR chip was tested using a high-resolution spectrometer within a narrow wavelength range (500–700 nm). The results are as follows: Figure 4 As shown; where, Figure 4 A and 4B represent the reflection intensities of a MetaSPR chip with a 9 nm Ti + 90 nm Au metal film and a silver mirror MetaSPR chip, respectively. Figure 4 C is the regression fitting curve of the difference in reflection intensity between 605 nm and 660 nm for the MetaSPR chip with a 9 nm Ti + 90 nm Au metal film and the silver mirror MetaSPR chip. Figure 4 D and 4E represent the reflection intensities of a MetaSPR chip with a 9 nm Ti + 70 nm Ag + 20 nm Au metal film and a silver mirror MetaSPR chip, respectively. Figure 4 F is the regression fitting curve of the difference in reflection intensity between a MetaSPR chip with a 9 nm Ti + 70 nm Ag + 20 nm Au metal film and a silver mirror MetaSPR chip at 628 nm-650 nm. Figure 4 A, 4B, 4D, and 4E show that, due to the absence of the silver mirror effect, the MetaSPR chip can detect the reflectance spectrum at a sucrose concentration of 0.2%, while the silver mirror MetaSPR chip can detect a significant reflectance spectrum at a sucrose concentration of 0.04%. Figure 4As shown in C, the detection limits of the MetaSPR chip with a 9 nm Ti + 90 nm Au metal film and the MetaSPR chip with a silver mirror are RI = 1.334053 (0.78%) and RI = 1.333143 (0.16%), respectively, with a sensitivity difference of approximately 4.8 times. Figure 4 As shown in F, the detection limits of the MetaSPR chip with a 9 nm Ti + 70 nm Ag + 20 nm Au metal film and the silver mirror MetaSPR chip are RI = 1.333776 (0.59%) and RI = 1.332969 (0.047%), respectively, with a sensitivity difference of approximately 12.5 times. Figure 4 C and Figure 4 As shown in F, the MetaSPR chip with a silver mirror containing a 9 nm Ti + 70 nm Ag + 20 nm Au metal film exhibits higher sensitivity compared to the MetaSPR chip with a 9 nm Ti + 90 nm Au metal film. These results demonstrate that the MetaSPR chip with enhanced silver mirror effect possesses better liquid sensing performance and higher sensitivity. Furthermore, the addition of the double silver layer not only improves the high reflectivity and good conductivity of the MetaSPR chip but also further enhances the reflection intensity through multiple reflections and amplifications between the various metals and the silver mirror.

[0066] III. Research on different Ti3C2T x The effect of MXene to GO weight ratio on MGMSPR sensor performance

[0067] A tapered nanopillar array was molded on a 12-inch silicon oxide wafer using laser interference lithography and ion etching. After the mold was placed in a vacuum dryer filled with hexylsilane for 12 hours for hydrophobic treatment, Norland optical adhesive (NOA-61) was uniformly coated on the mold, and a PET film was placed on it. After curing by ultraviolet irradiation for 3 minutes, the nanocup array on the PET film was obtained by peeling. 9 nm Ti, 70 nm Ag, and 20 nm Au were deposited on the nanocup array by electron beam evaporation to obtain the MetaSPR chip.

[0068] A silver mirror MetaSPR chip was obtained by depositing a 10 nm Ti film, an 80 nm Ag film, and a 10 nm Au film on the back side of the MetaSPR chip using electron beam evaporation.

[0069] Ti3C2T x The MXene dispersion (1.5 mg / mL) and GO dispersion (1.5 mg / mL) were mixed thoroughly, wherein Ti3C2T xThe weight ratios of MXene to GO were 3:1, 2:1, 1:1, 1:2, and 1:3, respectively; the dispersion mixture was then sonicated (40 Hz, 70 W); the silver mirror MetaSPR chip was washed twice with anhydrous ethanol and then sonicated for 5 minutes.

[0070] 10 μL Ti3C2T x MXene dispersion (1.5 mg / mL), GO dispersion (1.5 mg / mL), and a mixture of five dispersions were respectively added dropwise to the surface of a silver mirror MetaSPR chip and vacuum dried at 60 °C for 20 minutes to obtain seven MGMSPR sensors. The process for preparing MG films on the surface of the silver mirror MetaSPR chip is as follows: Figure 5 As shown.

[0071] Figure 6 A is Ti3C2T x TEM image of MXene thin film, showing Ti3C2T x MXene films have a smooth surface; Figure 6 B is a TEM image of the GO thin film, which shows that the GO thin film has wrinkled features. Figure 6 C is a TEM image of the MG composite film, which shows that the MG composite film has a stacked nanosheet-like 3D structure. Figure 6 D represents different Ti3C2T x SEM image of the MGMSPR sensor surface with MXene to GO weight ratio.

[0072] This invention employs simple mixing and drying steps to prepare a MG composite film with a stacked nanosheet 3D structure on the surface of a silver mirror MetaSPR chip. Transmission photoelectron microscopy was used to analyze the Ti3C2T... x Detection of MXene nanosheets and GO nanosheets showed that the original Ti3C2T x MXene films exhibit a smooth surface, while GO films show wrinkled characteristics. In contrast, the MG composite film formed by mixing the two has a tightly packed, stacked nanosheet-like 3D structure; among them, the hydrophilic Ti3C2T x MXene nanofilms endow MG composite films with an open porous structure and a highly hydrophilic microenvironment.

[0073] The sensitivity of the MGMSPR sensor was also tested by preparing sucrose solutions of different concentrations (0-5% w / v), and the results were as follows: Figure 7 As shown. Figure 7 A represents the relationship between the MG weight ratio and the sensitivity of the MGMSPR sensor. Figure 7A shows that when the sucrose concentration is 0.04%, five MGMSPR sensors with different MG weight ratios (M:G = 3:1, 2:1, 1:1, 1:2, 1:3) and Ti3C2T modified alone can achieve the desired effect. x Both MXene (M) and GO (G) sensors exhibited significant response signals; among them, the MGMSPR sensor with MG = 2:1 showed the strongest signal strength. Linear regression analysis was performed on the response signal of the MGMSPR sensor with MG = 2:1, and the results are as follows... Figure 7 As shown in B, the correlation coefficient R... 2 = 0.977, indicating a strong linear relationship. Furthermore, the regression curve shows that the limit of detection (LOD) of this MGMSPR sensor is 1.33285, representing a 36% increase in signal strength compared to the unmodified MG sensor. The following tests were conducted on the molecular binding capacity and applications of an MGMSPR sensor with an M:G = 2:1 ratio.

[0074] IV. Testing the bonding capability of MGMSPR sensors

[0075] Protein A was immobilized on the surface of the MGMSPR sensor chip, and different concentrations of IgG (0.125 ~ 32 nM) were added to the chip wells. The response signal was monitored in real time, and the detection results are shown below. Figure 8 ,in, Figure 8 A represents the real-time binding curve over a 10-minute period; Figure 8 B is the curve showing the relationship between IgG concentration and response signal, indicating a strong relationship between the response signal and IgG concentration (R). 2 The sensor, with a limit of detection (LOD) of 0.055 nM (= 0.999), exhibits a 6.7-fold increase in binding sensitivity compared to the unmodified MG sensor (LOD 0.37 nM). Therefore, MG modification significantly enhances the sensor's sensitivity in detecting IgG molecules.

[0076] Furthermore, small molecule dynamics has always been a focus of label-free MetaSPR research. Among these, the interaction between rapamycin and FKBP12 molecules exhibits rapid kinetic characteristics; however, due to their relatively low molecular weight, this interaction is difficult to detect. To verify the applicability of the MGMSPR sensor of this invention in detecting small molecule dynamic interactions, this invention uses a model of the rapamycin-FKBP12 interaction for testing, and the results are as follows... Figure 8As shown in Figure C. The results show that the dynamic binding and dissociation phases of the rapamycin-FKBP12 interaction change rapidly, indicating its fast kinetic characteristics. Based on the equilibrium affinity calculated from the binding phase endpoints, the calculated value of the dissociation constant (KD) is 5.98E-8 M, consistent with the data obtained from the commercial instrument Biacore (KD = 3.9E-8 M). These results demonstrate that the MGMSPR sensor of this invention has broad application potential in the field of small molecule interactions.

[0077] V. Application of MGMSPR sensor in blood glucose monitoring

[0078] S1. First, thoroughly clean the MGMSPR sensor chip with ethanol and deionized water, and then dry it for later use.

[0079] S2. Apply sodium acetate buffer containing GOD to the chip surface and dry it to promote GOD fixation on the chip surface.

[0080] S3. 10 μL of 1.25% Nafion solution was coated on the chip surface as a protective layer, and then the response signal of interaction with different concentrations of glucose was detected by a spectrometer. Figure 9 A is a schematic diagram of the principle of glucose detection by the MGMSPR sensor; Figure 9 B shows the differential spectra of glucose at different concentrations detected using an MGMSPR sensor; Figure 9 C represents the relative response intensity of the MGMSPR sensor as a function of glucose concentration. The results indicate that the MGMSPR sensor possesses good sensitivity.

[0081] To verify the MGMSPR sensor's ability to detect glucose in sweat, artificial sweat was monitored in real time. Figure 10 As shown in Figure A, the microfluidic chip based on the MGMSPR sensor, from bottom to top, consists of: a 3D-printed chip card, an adhesive layer, a biosensor layer (two MGMSPR chips: one for testing and one for reference), a flow channel adhesive layer, and a PET protective layer. The microfluidic chip has two inlets and one outlet. A physical image of the microfluidic biochip based on the dual-channel MGMSPR sensor is shown in Figure S. Figure 10 B. When sweat flows through two MGMSPR chips, the GOD on the surface of the test chip reacts with glucose and PAT in the sweat, causing a change in the RI value. At the same time, the sweat in the reference chip and other metabolites or ions in the sweat cause changes in the RI value. A series of glucose concentrations can be detected with ultrasensitive sensitivity within 10 minutes, and clear and distinguishable results can be obtained within 100 seconds. Figure 10 C). The standard curve was fitted using 4PL regression, and the results are as follows: Figure 10As shown in Figure D, the results indicate a strong correlation between the response signal and different glucose concentrations, R 2 = 0.993 (low concentration of R) 2 = 0.902), with a detection limit of 106.8 μM, which significantly improves sensitivity compared to traditional glucose detection instruments.

[0082] To verify the accuracy of the MGMSPR sensor, blood glucose levels of six standard samples were measured using both a commercial blood glucose meter and the MGMSPR sensor. The results are as follows: Figure 10 As shown in Figure E, the results indicate that the MGMSPR sensor's measurement values ​​are very close to those of commercial blood glucose meters, with a correlation coefficient r = 0.943, indicating a strong positive correlation. This confirms the reliability and practicality of the MGMSPR sensor of this invention for glucose detection.

[0083] To verify the selectivity of the MGMSPR sensor, the inventors added other metabolites (100 μM lactic acid, 10 μM ascorbic acid, and 59 μM uric acid) to the sweat buffer solution. The detection results are as follows: Figure 10 As shown in Figure F (a: 110 μM glucose, b: 100 μM lactic acid, c: 10 μM ascorbic acid, d: 59 μM uric acid), the results show that only the addition of 110 μM glucose resulted in a significant change in RU, while the addition of other metabolites did not cause a significant change in RU. Furthermore, the sensor RU value changed again after the addition of 110 μM glucose. These results indicate that the MGMSPR sensor has good selectivity for glucose detection. In addition, to test the specificity of the MGMSPR sensor, the inventors compared the sensor's response to glucose with five other different solutions, including sweat metabolites (lactic acid, uric acid, dopamine, NaCl, and KCl), with the results shown below. Figure 10 As shown in Figure G, the results indicate that the MGMSPR sensor's maximum response to lactic acid, uric acid, dopamine, NaCl, and KCl is only 52 RU, demonstrating the sensor's good specificity. These experimental results confirm the feasibility of the MGMSPR biosensor of this invention in identifying glucose in sweat, indicating its great potential for further clinical application.

[0084] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A MetaSPR optical detection sensor for detecting metabolites in human body fluids, characterized in that, Includes a MetaSPR chip; Ti3C2T is coated on the surface of the MetaSPR chip by depositing a metal film with a silver mirror effect on the back side of the MetaSPR chip. x A mixed solution of MXene and graphene oxide was dried to obtain a MetaSPR optical sensor for detecting metabolites in human body fluids; the Ti3C2T x The weight ratio of MXene to graphene oxide is 2:1; The metal film consists of titanium, silver, and gold in sequence; wherein the thickness of the titanium layer is 10-30 nm, the thickness of the silver layer is 20-200 nm, and the thickness of the gold layer is 10-30 nm.

2. The MetaSPR optical detection sensor for detecting metabolites in human body fluids according to claim 1, characterized in that, The Ti3C2T x A mixed solution of MXene and graphene oxide is obtained by passing Ti3C2T x The MXene dispersion and the graphene oxide dispersion were mixed uniformly by ultrasonication.

3. A method for preparing a MetaSPR optical detection sensor for detecting metabolites in human body fluids as described in claim 1 or 2, characterized in that, Includes the following steps: S1. A tapered nanopillar array is molded on a silicon oxide wafer using laser interference lithography and ion etching technology. After the mold is placed in a vacuum dryer filled with hexylsilane for hydrophobic treatment, optical adhesive is uniformly coated on the mold, and a PET film is placed on it. After curing under ultraviolet irradiation, the nanocup array located on the PET film is obtained by peeling. A metal film 1 is deposited on the nanocup array using electron beam evaporation to obtain the MetaSPR chip. S2. A metal film 2 with a silver mirror effect is deposited on the back side of the MetaSPR chip using electron beam evaporation to obtain a silver mirror MetaSPR chip. S3, Ti3C2T x The MXene dispersion and the graphene oxide dispersion were mixed evenly, and the mixture was added to the surface of the silver mirror MetaSPR chip and dried to obtain the MetaSPR optical detection sensor for detecting metabolites in human body fluids.

4. The method for preparing a MetaSPR optical detection sensor for detecting metabolites in human body fluids according to claim 3, characterized in that, The metal film 1 is composed of titanium and gold in sequence, or the metal film 1 is composed of titanium, silver and gold in sequence.

5. The application of the sensor as described in claim 1 or 2, or the sensor prepared by the method described in claim 4, in the detection of metabolites in human body fluids.

6. The application according to claim 5, characterized in that, The human body fluids include sweat, tears, or saliva; the metabolites include glucose, lactic acid, urea, or cholesterol.

7. The application according to claim 6, characterized in that, The method for detecting the metabolite includes the following steps: (1) Immobilize the enzyme corresponding to the metabolite to be tested on the chip surface of the sensor; (2) Add Nafion or chitosan into the sensor chip hole; (3) Add the sample to be tested into the sensor chip hole.

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