MetaSPR optical detection sensor for detecting metabolites in human body fluid

Abstract

The invention provides a MetaSPR optical detection sensor for detecting metabolites in human body fluid, and belongs to the technical field of MetaSPR sensors. The MetaSPR optical detection sensor comprises a MetaSPR chip, a light emitting diode chip and a light emitting diode chip, the preparation method comprises the following steps: depositing a metal film with a silver mirror effect on the back surface of a MetaSPR chip, coating the surface of the MetaSPR chip with a mixed solution of Ti3C2Tx MXene and graphene oxide, and drying to obtain an MGMSPR sensor; when the kit is used for detecting human body fluid metabolites, corresponding enzymes and protective layers are added firstly, then a sample to be detected is added, and corresponding products are detected through an optical or electrochemical method so as to realize quantitative analysis. The surface of the prepared MGMSPR sensor has a unique nanoscale 3D structure, and compared with a traditional MetaSPR sensor, the prepared MGMSPR sensor has higher sensitivity and specificity and lower detection limit, is suitable for detecting samples with lower concentration, and has wide application potential in the field of human body fluid small molecule metabolites.

Description

Technical Field

[0001] The present invention relates to the technical field of MetaSPR sensors, and in particular to a MetaSPR optical detection sensor for detecting metabolites in human body fluids. Background Art

[0002] Wearable sensing devices obtain physiological information continuously and in real time by non-invasively measuring biochemical indicators in biological fluids. They can perform non-invasive sampling and analysis of body fluids such as sweat, tears, and saliva at specific sites, thereby performing cost-effective and real-time biomedical monitoring of health and disease. Among them, since sweat contains a variety of biochemical substances, including electrolytes, metabolites, hormones, etc., human sweat has become one of the most challenging research directions in the field of biomedical sensing. Real-time assessment of health and disease by monitoring biomarkers in sweat is also developing rapidly in the field of wearable sensors. In addition, since sweat is easy to obtain, it has become the preferred sample for real-time monitoring of wearable sensors.

[0003] Studies have shown the dynamic changes in various biomarkers in sweat, including glucose, cortisol, and neuroimmunity. Among various metabolites, glucose is an important biomarker for the diagnosis and treatment of diabetes, and the concentration of glucose fluctuates greatly and depends on the patient's diet and lifestyle. Therefore, real-time monitoring of blood sugar is very important for preventing diabetes and preventing diabetic complications. Although some wearable sensors for real-time glucose monitoring (CGM) have been developed, there are still disadvantages such as low sensitivity and large size of sensing devices.

[0004] MetaSPR sensors have been shown to be more portable and more sensitive than traditional biosensors. Surface plasmon resonance (SPR) technology is a new analytical technology developed based on optical principles. It refers to the resonance phenomenon that may occur when light is totally reflected on the surface of a prism and a metal film, forming an evanescent wave that enters a light-scarce medium, and there is a certain plasma wave in the medium. At present, silver mirrors are widely used in optical systems, but there are few cases of using silver mirrors in SPR technology to promote the improvement of detection sensitivity. Since silver has low optical loss and shows clearer and more intense SPR bands in the visible and near-infrared range, this property makes silver an ideal choice for improving the sensitivity and performance of SPR sensors. Due to the natural characteristics of optical biosensors, despite their unique nanostructure and silver mirror effect (SME), direct label-free detection of small molecules such as glucose based on MetaSPR biosensors is still challenging. Summary of the invention

[0005] In view of the above deficiencies in the prior art, the present invention provides a MetaSPR optical detection sensor for detecting metabolites in human body fluids, which can detect the concentration of metabolites in real time with high sensitivity.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are 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; a metal film having a silver mirror effect is deposited on the back of the MetaSPR chip, and Ti3C2T3O4 is coated on the surface of the MetaSPR chip. x The mixed solution of MXene and graphene oxide (GO) was dried to obtain the MetaSPR optical detection sensor (denoted as MGMSPR sensor) for detecting metabolites in human body fluids.

[0008] The present invention enhances the sensitivity of the MetaSPR chip by evaporating a metal film with a silver mirror effect of a certain thickness on the back of the MetaSPR chip and adopting a reflection mode for detection. Among them, silver has excellent optical properties, including high reflectivity and good conductor properties; the silver mirror can act as a reflection layer. When the light wave excites the surface plasma on the metal surface of the MetaSPR chip, the reflection effect of the silver mirror can make the electromagnetic field reflect and enhance multiple times between multiple metals and the silver mirror, thereby enhancing the signal strength of the MGMSPR sensor.

[0009] The present invention adopts Ti3C2T x MXene and GO are simply mixed, and the mixed solution is coated on the surface of the MetaSPR chip to prepare an MGMSPR sensor with a MG composite film having a unique 3D structure on the surface, which further enhances the resonance effect of the surface plasmon. x The two-dimensional nanostructure of MXene gives the MG composite film an open porous structure and a highly hydrophilic microenvironment; while the GO film has wrinkled microscopic features, and the Ti3C2T x The MG film obtained by combining MXene with GO has a unique stacked 3D nanosheet structure, and the void size of the structure can be changed by changing the Ti3C2T x The ratio between MXene and GO can be adjusted to be suitable for the detection of different metabolic small molecules.

[0010] Preferably, the metals of the metal film are 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 0 at the same time.

[0012] Preferably, the Ti3C2T x The mixed solution of MXene and GO was prepared by mixing Ti3C2T x The MXene dispersion and the GO dispersion were uniformly mixed by ultrasonic mixing.

[0013] In a second aspect, 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. Use laser interference lithography and ion etching technology to mold a conical nanocolumn array on a silicon oxide wafer, place the mold in a vacuum dryer filled with hexylsilane for hydrophobic treatment, evenly apply optical glue on the mold, and place a PET film on it; after curing under ultraviolet irradiation, peel off to obtain a nanocup array on the PET film; use electron beam evaporation to deposit a metal film 1 on the nanocup array to obtain a MetaSPR chip;

[0015] S2, using electron beam evaporation to deposit a metal film 2 having a silver mirror effect on the back of the MetaSPR chip to obtain a silver mirror MetaSPR chip;

[0016] S3, Ti3C2T x The MXene dispersion and the 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 metals of the metal film 1 are titanium and gold in sequence, or the metals of the metal film 1 are titanium, silver and gold in sequence.

[0019] Preferably, the metals of the metal film 2 are 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] In a third aspect, the present invention provides the use 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 comprises the following steps:

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

[0024] (2) Adding Nafion or chitosan to the sensor chip holes;

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

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

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

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

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

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

[0031] Compared with the prior art, the present invention is beneficial in that:

[0032] 1. The present invention deposits a metal film with a silver mirror effect on the back of the MetaSPR chip, and uses a MG composite film to modify the surface of the MetaSPR chip, so that the prepared MGMSPR sensor has higher sensitivity and specificity and lower detection limit than the traditional MetaSPR sensor. Among them, the metal film with a silver mirror effect can reduce scattering loss, improve signal strength and accuracy, and at the same time, the reflection effect of the silver mirror can make the electromagnetic field reflect and enhance multiple times between multiple metals and silver mirrors, thereby enhancing the signal strength of the MGMSPR sensor; the MG composite film has a unique nanoscale 3D structure, which is conducive to combining with small molecules, further enhancing the detection performance of the sensor.

[0033] 2. Based on the MGMSPR sensor, the present invention selects and fixes the corresponding enzyme according to different analytes, and also uses Nafion or chitosan as a protective layer to prevent the loss or inactivation of the enzyme, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The preparation flow chart of MGMSPR sensor;

[0035] Figure 2 A is a physical picture of the silver mirror MetaSPR chip; Figure 2 B is a SEM image of the silver mirror MetaSPR chip viewed from above; Figure 2 C is the SEM image of the cross section of the silver mirror MetaSPR chip;

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

[0037] Figure 4 A is the reflection intensity of the MetaSPR chip with 9 nm Ti + 90 nm Au metal film; Figure 4 B is the reflection intensity of the silver mirror MetaSPR chip with 9 nm Ti + 90 nm Au metal film; Figure 4 C is the regression fitting curve of the difference in reflection intensity between the MetaSPR chip with 9 nm Ti+90 nm Au metal film and the silver mirror MetaSPR chip at 605 nm-660 nm; Figure 4 D is the reflection intensity of the MetaSPR chip with 9 nm Ti+70 nm Ag+20 nm Au metal film; Figure 4E is the reflection intensity of the silver mirror MetaSPR chip with 9 nm 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 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 Flow chart for preparing MG thin film on silver mirror MetaSPR chip to obtain MGMSPR sensor;

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

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

[0041] Figure 8 A is the real-time binding curve of MGMSPR sensor and IgG; Figure 8 B is the relationship curve between IgG concentration and response signal; Figure 8 C is the real-time monitoring of the binding and dissociation interaction response intensity between rapamycin and FKBP12 molecules using the MGMSPR sensor;

[0042] Fig. 9 A is the schematic diagram of the MGMSPR sensor for detecting glucose; Fig. 9 B is the differential spectrum of different concentrations of glucose detected using MGMSPR sensor; Fig. 9 C is the functional relationship between the relative response intensity of the MGMSPR sensor and the glucose concentration;

[0043] Fig.10 A is a schematic diagram of a microfluidic chip based on MGMSPR sensor; Fig.10 B is a physical picture of the microfluidic chip based on the dual-channel MGMSPR sensor; Fig.10 C is the response curve of the MGMSPR sensor monitoring different concentrations of glucose in artificial sweat; Fig.10D is the 4PL regression curve of the response signal of the MGMSPR sensor and the glucose concentration; Fig.10 E is a comparison chart of glucose concentration in artificial sweat detected by commercial blood glucose detector and MGMSPR sensor; Fig.10 F is the selectivity verification result of MGMSPR sensor; Fig.10 G is the nonspecific validation result of the MGMSPR sensor. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The present invention provides a MetaSPR optical detection sensor for detecting metabolites in human body fluids, comprising a MetaSPR chip; a metal film having a silver mirror effect is deposited on the back of the MetaSPR chip, and Ti3C2T3O4 is coated on the surface of the MetaSPR chip. x The mixed solution of MXene and GO was dried to obtain the MetaSPR optical detection sensor for detecting metabolites in human body fluids (denoted as MGMSPR sensor, the preparation process is as follows Figure 1 as shown).

[0046] In some examples, the metals of the metal film are titanium, silver, and gold, respectively; 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.

[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 0 at the same time.

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

[0049] S1. Use laser interference lithography and ion etching technology to mold a conical nanocolumn array on a silicon oxide wafer, place the mold in a vacuum dryer filled with hexylsilane for hydrophobic treatment, evenly apply Norland optical adhesive NOA-61 on the mold, and place a PET film on it; after curing under ultraviolet irradiation, peel off to obtain a nanocup array on the PET film; use electron beam evaporation to deposit a metal film 1 on the nanocup array to obtain a MetaSPR chip;

[0050] S2, using electron beam evaporation to deposit a metal film 2 having a silver mirror effect on the back of the MetaSPR chip to obtain a silver mirror MetaSPR chip;

[0051] S3, Ti3C2T x The MXene dispersion and the 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 of the metal film 1 is titanium and gold in sequence, or the metal of the metal film 1 is titanium, silver and gold in sequence.

[0053] In some examples, the metals of the metal film 2 are titanium, silver, and gold, respectively; 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] A MetaSPR optical detection sensor for detecting metabolites in human body fluids

[0056] 1. Study the effect of silver mirror layer thickness on the sensitivity of silver mirror MetaSPR chip

[0057] A conical nanocolumn array was molded on a 12-inch silicon oxide wafer using laser interference lithography and ion etching technology. The mold was placed in a vacuum desiccator filled with hexylsilane for 12 hours for hydrophobic treatment, and then Norland optical adhesive (NOA-61) was evenly applied on the mold, and a PET film was placed on it. After curing with ultraviolet light for 3 minutes, the nanocup array on the PET film was peeled off. Electron beam evaporation was used to deposit Ti with a thickness of 9 nm and Au with a thickness of 90 nm on the nanocup array in sequence to obtain a 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 of the MetaSPR chip by 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 picture of the silver mirror MetaSPR chip. It can be observed that the silver mirror MetaSPR chip has a visual color change phenomenon. This is the result of the collective oscillation of free electrons in the metal nanostructure excited by light. This phenomenon has a strong response to the wavelength of the incident light, causing the visual color to change with the change of the light wavelength. Figure 2 B is a SEM image of the silver mirror MetaSPR chip viewed from above. Figure 2 B shows that the MetaSPR chip presents a uniform nanoarray; Figure 2 C is the SEM image of the cross section of the silver mirror MetaSPR chip. Figure 2 C also shows that the MetaSPR chip has a nanocup array structure.

[0060] Considering that the silver mirror effect of metals of different thicknesses on the back of the MetaSPR chip is different, the inventors compared the thickness of the Ag layer on the back of the MetaSPR chip in Example 1. In the metal film on the back, the 10 nm Ti layer is used as an adhesive between the substrate and the silver, and the 10 nm Au layer is used as a surface layer to prevent silver from being oxidized by air; compared with the Ag layer, the Ti layer and the Au layer have relatively little effect on the sensitivity of the MetaSPR chip, so the thickness test range of the silver layer is 20-100nm. Sucrose solutions of different concentrations (0-5% w / v) were used to test the sensitivity of silver layers with different thicknesses.

[0061] Sucrose solutions with RI range of 1.3328~1.34023 (RI of water = 1.3328, RI of 5% sucrose = 1.3403) were prepared in water. The performance of silver layers of different thicknesses was evaluated by dual wavelength difference reflection intensity using a high-resolution spectrometer in a narrow wavelength range (500 to 700 nm). The results are shown in Figure 2. Figure 3 As shown. 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 a saturation point at a thickness of 80 nm. Therefore, the silver mirror metal film composed of 10 nm Ti, 80 nm Ag, and 10 nmAu has the highest reflection intensity and can clearly distinguish low sugar solution concentrations.

[0062] 2. Study the influence of different sensing metal films in MetaSPR chip on the sensitivity of silver mirror MetaSPR chip

[0063] A conical nanocolumn array was molded on a 12-inch silicon oxide wafer using laser interference lithography and ion etching technology. The mold was placed in a vacuum desiccator filled with hexylsilane for 12 hours for hydrophobic treatment, and then Norland optical adhesive (NOA-61) was evenly applied on the mold, and a PET film was placed on it. After curing with ultraviolet light for 3 minutes, the nanocup array on the PET film was peeled off. Electron beam evaporation was used to deposit 9 nm Ti, 90 nm Au and 9 nm Ti, 70 nm Ag, and 20 nm Au on the nanocup array, respectively, to obtain two MetaSPR chips.

[0064] Electron beam evaporation was used to deposit a 10 nm Ti film, an 80 nm Ag film, and a 10 nm Au film on the back of the MetaSPR chip to obtain a silver mirror MetaSPR chip.

[0065] The sensitivity of the silver mirror MetaSPR chip with different MetaSPR chips was also tested using sucrose solutions of different concentrations (0-5% w / v). Sucrose solutions with an RI range of 1.3328~1.34023 were prepared in water, and the sensitivity of the MetaSPR chip and the silver mirror MetaSPR chip was tested using a high-resolution spectrometer in a narrow wavelength range (500 to 700 nm). The results are shown in Figure 2. Figure 4 As shown; among them, Figure 4 A and 4B are the reflection intensities of the MetaSPR chip with 9 nm Ti + 90 nm Au metal film and the silver mirror MetaSPR chip, respectively; Figure 4 C is the regression fitting curve of the difference in reflection intensity between the MetaSPR chip with 9 nm Ti+90 nm Au metal film and the silver mirror MetaSPR chip at 605 nm-660 nm; Figure 4 D and 4E are the reflection intensities of the MetaSPR chip with 9 nm Ti+70 nm Ag+20 nm Au metal film and the silver mirror MetaSPR chip, respectively; Figure 4 F is the regression fitting curve of the difference in reflection intensity between the MetaSPR chip with 9 nm Ti+70 nm Ag+20 nm Au metal film and the 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 a reflection spectrum when the sucrose concentration is 0.2%, while the silver mirror MetaSPR chip can detect an obvious reflection spectrum when the sucrose concentration is 0.04%. Figure 4C shows that the minimum detection limits of the MetaSPR chip with 9 nm Ti + 90 nm Au metal film and the silver mirror MetaSPR chip are RI = 1.334053 (0.78%) and RI = 1.333143 (0.16%), respectively, and the sensitivity difference between the two is about 4.8 times. Figure 4 It can be seen that the minimum detection limits of the MetaSPR chip with 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, and the sensitivity difference between the two is about 12.5 times. Figure 4 C and Figure 4 It can be seen that the silver mirror MetaSPR chip with 9 nm Ti+70 nm Ag+20 nm Au metal film has higher sensitivity than the silver mirror MetaSPR chip with 9 nm Ti+90 nm Au metal film. The above results show that the MetaSPR chip with enhanced silver mirror effect has better liquid sensing performance and higher sensitivity; in addition, the addition of double silver layer not only improves the high reflectivity and good conductivity of MetaSPR chip, but also further enhances the reflection intensity through multiple reflections and amplification between multiple metals and silver mirror.

[0066] 3. Study of different Ti3C2T x Effect of MXene to GO weight ratio on the performance of MGMSPR sensors

[0067] A conical nanocolumn array was molded on a 12-inch silicon oxide wafer using laser interference lithography and ion etching technology. The mold was placed in a vacuum desiccator filled with hexylsilane for 12 hours for hydrophobic treatment, and then Norland optical adhesive (NOA-61) was evenly applied on the mold, and a PET film was placed on it. After curing with ultraviolet light for 3 minutes, the nanocup array on the PET film was peeled off. Electron beam evaporation was used to deposit 9 nm Ti, 70 nm Ag, and 20 nm Au on the nanocup array to obtain a MetaSPR chip.

[0068] Electron beam evaporation was used to deposit a 10 nm Ti film, an 80 nm Ag film, and a 10 nm Au film on the back of the MetaSPR chip to obtain a silver mirror MetaSPR chip.

[0069] Ti3C2T x MXene dispersion (1.5 mg / mL) and GO dispersion (1.5 mg / mL) were mixed evenly, among which 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 treated with ultrasound (40 Hz, 70 W); the silver mirror MetaSPR chip was washed twice with anhydrous ethanol and then treated with ultrasound for 5 minutes.

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

[0071] Figure 6 A is Ti3C2T x TEM image of MXene film, showing that Ti3C2T x MXene films have a smooth surface; Figure 6 B is a TEM image of the GO film, which shows that the GO film has wrinkle-like 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 is different Ti3C2T x SEM images of MGMSPR sensor surface with respect to MXene to GO weight ratio.

[0072] The present invention adopts simple mixing and drying steps to prepare MG composite films with stacked nanosheet 3D structures on the surface of silver mirror MetaSPR chip, and uses transmission light electron microscopy to observe the Ti3C2T x MXene nanosheets and GO nanosheets were tested, and the results showed that the original Ti3C2T x The MXene film exhibits a smooth surface, while the GO film exhibits wrinkled features. In contrast, the MG composite film formed by mixing the two has a compact, stacked nanosheet 3D structure; among them, the hydrophilic Ti3C2T x The MXene nanomembrane endows the MG composite film with an open porous structure and a highly hydrophilic microenvironment.

[0073] The sensitivity of the MGMSPR sensor was also tested by preparing sucrose solutions with different concentrations (0-5% w / v). Figure 7 shown. Figure 7 A is 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 have good x The sensors of MXene (M) or GO (G) showed significant response signals; among them, the signal intensity of the MGMSPR sensor with MG = 2: 1 was the strongest. 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, and from the regression curve, we can get the minimum detection limit LOD of the MGMSPR sensor = 1.33285, and compared with the sensor without MG modification, its signal intensity is enhanced by 36%. The MGMSPR sensor with M: G = 2: 1 was selected for molecular binding ability and application test.

[0074] 4. Binding ability detection of MGMSPR sensor

[0075] Protein A was fixed on the surface of the MGMSPR sensor chip, and different concentrations of IgG (0.125 ~ 32 nM) were added to the chip wells to monitor the response signal in real time. The detection results are shown in Figure 8 ,in, Figure 8 A is the real-time binding curve within 10 minutes; Figure 8 B is the relationship curve between IgG concentration and response signal, indicating that there is a strong relationship between the response signal and IgG concentration (R 2 = 0.999), the lowest detection limit LOD of the sensor was 0.055 nM; compared with the sensor without MG modification (LOD of 0.37 nM), the binding sensitivity of the MGMSPR sensor increased by 6.7 times. Therefore, MG modification can significantly improve the sensitivity of the sensor to detect IgG molecules.

[0076] In addition, small molecule dynamics has been the focus of label-free MetaSPR technology research. Among them, the interaction between rapamycin and FKBP12 molecules has fast kinetic characteristics; however, due to their relatively low molecular weight, this interaction is difficult to detect. In order to verify the applicability of the MGMSPR sensor of the present invention in detecting small molecule dynamic interactions, the present invention uses a model of rapamycin-FKBP12 interaction for testing, and the results are as follows Figure 8C. The results show that the dynamic binding and dissociation phases of the rapamycin-FKBP12 interaction change rapidly, indicating its fast kinetic characteristics. According to the equilibrium affinity calculated from the binding phase endpoint, the calculated value of the dissociation constant (KD) is 5.98E-8 M, which is consistent with the data obtained by the commercial instrument Biacore (KD = 3.9E-8 M). The above results show that the MGMSPR sensor of the present invention has the potential for wide application in the field of small molecule interactions.

[0077] 5. Application of MGMSPR sensor in blood glucose monitoring

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

[0079] S2, applying sodium acetate buffer containing GOD on the chip surface and drying to facilitate the fixation of GOD on the chip surface;

[0080] S3. Coat 10 μL of 1.25% Nafion solution on the chip surface as a protective layer, and then use a spectrometer to detect the response signals interacting with different concentrations of glucose. Fig. 9 A is the schematic diagram of the MGMSPR sensor for detecting glucose; Fig. 9 B is the differential spectrum of different concentrations of glucose detected using MGMSPR sensor; Fig. 9 C is the function relationship between the relative response intensity of the MGMSPR sensor and the glucose concentration. The results show that the MGMSPR sensor has good sensitivity.

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

[0082] In order to verify the accuracy of the MGMSPR sensor, the blood glucose levels of 6 standard samples were tested using a commercial blood glucose tester and the MGMSPR sensor. Fig.10 As shown in Figure E, the results show that the measurement values ​​of the MGMSPR sensor and the commercial blood glucose detector are very close, and the correlation coefficient r = 0.943, indicating a strong positive correlation, confirming the reliability and practicality of the MGMSPR sensor of the present invention for detecting glucose.

[0083] To verify the selectivity of the MGMSPR sensor, the inventors also added other metabolites (100 μM lactic acid, 10 μM ascorbic acid, 59 μM uric acid) to the sweat buffer. The test results are as follows: Fig.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 RU changed significantly only when 110 μM glucose was added, while RU did not change significantly when other metabolites were added; and, when 110 μM glucose was added again, the RU value of the sensor changed again. The above results indicate that the MGMSPR sensor has good selectivity for the detection of glucose. In addition, in order to test the specificity of the MGMSPR sensor, the inventors also compared the responses of the sensor to glucose and five other different solutions, including sweat metabolites (lactic acid, uric acid, dopamine, NaCl and KCl). The results are shown in Figure 5. Fig.10 As shown in Figure G, the results show that the maximum response of the MGMSPR sensor to lactic acid, uric acid, dopamine, NaCl, and KCl is only 52 RU, indicating that the sensor has good specificity. The above experimental results confirm the feasibility of the MGMSPR biosensor of the present invention to identify glucose in sweat, indicating that it has great potential for further clinical application.

[0084] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A MetaSPR optical detection sensor for detecting metabolites in human body fluids, characterized in that: The invention comprises a MetaSPR chip; a metal film having a silver mirror effect is deposited on the back of the MetaSPR chip, and Ti3C2T is coated on the surface of the MetaSPR chip. x The mixed solution of MXene and graphene oxide is dried to obtain the MetaSPR optical detection sensor for detecting metabolites in human body fluids.

2. A MetaSPR optical detection sensor for detecting metabolites in human body fluids according to claim 1, characterized in that: The metals of the metal film are 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.

3. The MetaSPR optical detection sensor for detecting metabolites in human body fluids according to claim 1, characterized in that: The Ti3C2T x The weight ratio of MXene to graphene oxide is (0~5): (0~5), and Ti3C2T x MXene and graphene oxide are not the same and are 0.

4. The MetaSPR optical detection sensor for detecting metabolites in human body fluids according to claim 1, characterized in that: The Ti3C2T x The mixed solution of MXene and graphene oxide was prepared by mixing Ti3C2T x The MXene dispersion and the graphene oxide dispersion are uniformly mixed by ultrasonic mixing.

5. A method for preparing a MetaSPR optical detection sensor for detecting metabolites in human body fluids, characterized in that: The following steps are involved: S1. Use laser interference lithography and ion etching technology to mold a conical nanocolumn array on a silicon oxide wafer, place the mold in a vacuum dryer filled with hexylsilane for hydrophobic treatment, evenly apply optical glue on the mold, and place a PET film on it; after curing under ultraviolet irradiation, peel off to obtain a nanocup array on the PET film; use electron beam evaporation to deposit a metal film 1 on the nanocup array to obtain a MetaSPR chip; S2, using electron beam evaporation to deposit a metal film 2 having a silver mirror effect on the back of the MetaSPR chip to obtain a silver mirror MetaSPR chip; S3, Ti3C2T x The MXene dispersion and the graphene oxide dispersion are mixed evenly, and the mixed solution is added to the surface of the silver mirror MetaSPR chip and dried to obtain a MetaSPR optical detection sensor for detecting metabolites in human body fluids.

6. The method for preparing a MetaSPR optical detection sensor for detecting metabolites in human body fluids according to claim 5, characterized in that: The metals of the metal film 1 are titanium and gold in sequence, or the metals of the metal film 1 are titanium, silver and gold in sequence.

7. The method for preparing a MetaSPR optical detection sensor for detecting metabolites in human body fluids according to claim 5, characterized in that: The metals of the metal film 2 are 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.

8. Use of the sensor according to any one of claims 1 to 4 or the sensor prepared by the method according to any one of claims 5 to 7 in detecting metabolites in human body fluids.

9. The use according to claim 8, characterized in that: 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.

10. The use according to claim 9, characterized in that: The method for detecting the metabolite comprises the following steps: (1) Immobilizing the enzyme corresponding to the metabolite to be detected on the surface of the sensor chip; (2) Adding Nafion or chitosan to the sensor chip holes; (3) Add the sample to be tested into the hole of the sensor chip.

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