Composite material for detecting HCG (human chorionic gonadotropin) protein in sweat, preparation method of composite material and application of composite material in AD (Alzheimer's disease) diagnosis
A high-performance sweat sensor was constructed by combining carbon nanotubes with a 1T phase molybdenum disulfide composite substrate material and peptide molecules. This solved the problems of insufficient non-invasiveness and sensitivity in AD diagnosis, and achieved highly selective detection of HCG protein, supporting early non-invasive diagnosis of AD.
Patent Information
- Application Number
- CN202511629902.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-08
- Publication Date
- 2026-02-27
AI Technical Summary
Existing AD diagnostic methods suffer from problems such as poor non-invasiveness, high cost, and insufficient sensitivity. Traditional sweat sensing materials are inadequate in detecting low-abundance protein biomarkers, and there is a lack of effective biomarkers and sensitive materials.
A composite substrate material consisting of carbon nanotubes and 1T phase molybdenum disulfide was used, and peptide molecules were fixed by a crosslinking agent to form a composite material that can specifically bind HCG protein, which was then integrated into a wearable sweat detection sensor.
It achieves ultrasensitive detection of HCG protein with a detection limit as low as 0.45 pg/mL, exhibits high selectivity and stability, enables early diagnosis of AD, and is non-invasive and portable, improving patient acceptance and screening convenience.
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Figure CN121577908A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biosensing, more particularly, it relates to a composite material for detecting HCG protein in sweat, a preparation method thereof and application in AD diagnosis. BACKGROUND
[0002] Early and accurate diagnosis of Alzheimer's Disease (AD) is crucial for disease intervention and patient management. However, the existing mainstream AD detection methods have significant limitations, making it difficult to achieve convenient, non-invasive, efficient and low-cost screening in the population.
[0003] Current clinical diagnosis methods mainly include: brain imaging examination, cerebrospinal fluid detection, blood detection and gene sequencing. Brain imaging examination (such as MRI, PET) is expensive and costly, and in the early stage of the disease, the specificity of amyloid protein markers is low. Cerebrospinal fluid detection requires sample collection through lumbar puncture, which is an invasive procedure with low patient acceptance and certain risks. Although blood detection has a lighter logistical burden, the concentration of related protein markers such as Aβ and tau in blood is extremely low, requiring extremely sensitive detection techniques and expensive ultra-sensitive detection equipment. Gene sequencing is mainly used for risk assessment, which is costly and complex in data analysis, and the clinical significance of many genetic variations is not clear.
[0004] In view of the shortcomings of the above methods, the development of non-invasive biological samples has become a research hotspot. Among them, sweat shows great potential due to its completely non-invasive collection, low cost and real-time availability. However, there are still two major challenges in applying sweat to AD diagnosis: First, the limitations of existing sweat biomarker research. Current research on sweat has focused on the analysis of electrolytes, metabolic small molecules (such as lactic acid, urea) and other components, while the exploration of protein groups, especially specific protein markers related to neurodegenerative diseases, is severely insufficient. The lack of verified sweat protein targets highly related to AD pathological processes is a key bottleneck restricting the development of this field.
[0005] Second, the performance of traditional sweat sensing materials is insufficient. The core sensitive materials used to build sweat biosensors, such as traditional metal nanomaterials or two-dimensional materials, generally have insufficient sensitivity, limited electrochemical activity, poor biocompatibility and poor stability when facing the extremely low abundance of protein markers in sweat. These performance shortcomings of the materials make it difficult for them to achieve high sensitivity, specificity and stable detection of AD-related trace protein markers.
[0006] Therefore, there is an urgent need in the art for an innovative technical solution that can overcome the drawbacks of the existing AD diagnosis methods and provide a new type of sensitive material for the special scenario of sweat detection. SUMMARY
[0007] To solve the above problems, the present application provides a composite material for detecting HCG protein in sweat, a preparation method thereof and application in AD diagnosis.
[0008] The present application adopts the following technical solutions: In a first aspect, the present application provides a composite material for detecting HCG protein in sweat, comprising: a composite substrate material composed of carbon nanotubes and 1T-phase molybdenum disulfide; and a polypeptide molecule fixed on the composite substrate material by a cross-linking agent; The polypeptide molecule can specifically bind to HCG protein, which is a biomarker in Alzheimer's disease sweat.
[0009] Further, the amino acid sequence of the above-mentioned polypeptide molecule (PR) is (N-)PPLRINRHILTR(-C).
[0010] Further, the cross-linking agent is NHS / EDC.
[0011] In a second aspect, the present application provides a preparation method of the above-mentioned composite material for detecting HCG protein in sweat, comprising: S1. Using one-pot solvothermal reaction, mixing MoO3, thioacetamide, urea and carbon nanotubes in a solvent to react to generate a CNTs / 1T-MoS2 composite substrate material; S2. Carboxyl-modifying the composite substrate material to obtain a carboxylated composite substrate material CNTs / 1T-MoS2-COOH; S3. Using a cross-linking agent to activate the carboxyl groups on the surface of the carboxylated composite substrate material, and reacting with a polypeptide molecule that specifically recognizes HCG protein, so that the polypeptide molecule is fixed on the surface of the composite substrate material to obtain the composite material.
[0012] Further, in the above step S2, mercaptoacetic acid or mercaptopropionic acid is used for the carboxyl modification.
[0013] Further, in the above step S1, the temperature of the solvothermal reaction is 180-220℃, and the reaction time is 12-24 hours.
[0014] Further, in the above step S1, the mass ratio of MoO3 to thioacetamide and urea is 1:1.5-2.5. The mass ratio of the MoO3 to the carbon nanotube is 10:1.0-1.5.
[0015] In a third aspect, the application provides a sensor for non-invasive detection of Alzheimer's disease, comprising: a working electrode; and the composite material as claimed in any one of claims 1-3 fixed on the surface of the working electrode.
[0016] Further, the above-mentioned sensor further comprises a reference electrode and a counter electrode, and is integrated with a microfluidic chip to form a wearable sweat detection patch.
[0017] In a fourth aspect, the application provides a use of the above-mentioned composite material or sensor in the preparation of a product for diagnosing or assisting in the diagnosis of Alzheimer's disease.
[0018] In summary, the application has the following beneficial effects: In the application, liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology is used for proteomic analysis of sweat samples of AD patients and healthy control groups, and a total of 709 proteins and 2916 peptide segments are identified; through subcellular localization, Gene Ontology (GO) function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis methods, the biological functions of the differentially expressed proteins are analyzed, and it is found that HCG protein is not detected in the sweat of AD patients but is significantly expressed in the healthy control group, thereby providing a specific marker basis for subsequent sensor design.
[0019] 2. The composite material provided by the application, the core of which is composed of carbon nanotubes (CNTs) and 1T-phase molybdenum disulfide (1T-MoS2). CNTs have excellent electrical conductivity and one-dimensional structure, while 1T-MoS2 is a metallic phase with much higher electrical conductivity than the common semiconductor 2H phase. The synergistic effect of the two forms a composite substrate with high electrical conductivity and large specific surface area, greatly enhancing the electron transport efficiency and providing a strong electrochemical signal basis for identifying low-abundance proteins. This enables the sensor constructed from this material to achieve ultra-sensitive detection of HCG protein with a detection limit as low as 0.45 pg / mL, and can accurately capture low-abundance disease markers in sweat, providing a reliable basis for early diagnosis of AD.
[0020] 3. The composite material provided by the application can specifically bind to HCG protein and has a targeted recognition effect, ensuring high selective binding of the composite material to the target protein HCG and effectively avoiding interference from other substances in sweat, thereby ensuring the accuracy of the detection results.
[0021] 4. The high-performance composite material is integrated into the working electrode of the sensor, successfully changing the detection scene from large instruments to portable devices. This makes completely non-invasive sweat detection a reality, completely avoiding the invasive pain of cerebrospinal fluid detection and the high cost of brain imaging, greatly improving patient acceptance and screening convenience.
[0022] 5. Cell viability experiments and acute toxicity experiments in SD rats prove that the composite material provided by the application has no obvious toxicity, good biocompatibility, no adverse effects on cell survival rate and animal liver and kidney function, and no abnormal inflammatory reactions are found in histological examination, ensuring the biological safety of the sensor during long-term wearing and laying a foundation for its clinical application. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 The morphology and structure characterization results of CNTs, 1T-MoS2 and CNTs / 1T-MoS2 composite material are shown: (a) is the SEM image of CNTs; (b) is the SEM image of 1T-MoS2; (c) is the SEM image of CNTs / 1T-MoS2; (d) and (e) are TEM images of CNTs / 1T-MoS2 at different magnifications; (f) is the HRTEM image of CNTs / 1T-MoS2; (g) is the EDS elemental spectrum.
[0024] Figure 2 The XRD detection results of CNTs, 1T-MoS2 and CNTs / 1T-MoS2 composite material are shown.
[0025] Figure 3 The CV (a) and EIS curve (b) of the HCG sensor provided in Example 8 are measured in 5mM K3[Fe(CN)6] and 0.1M KCl after each surface modification step.
[0026] Figure 4 The CV curve of the HCG sensor provided in Example 8 at different scan rates in 0.1M PBS solution is shown.
[0027] Figure 5 The cyclic stability CV curves of the HCG sensor provided in Example 8 loaded with PP (a) and unloaded with PP (b) electrodes are shown.
[0028] Figure 6 The DPV curve (a) and linear fitting graph (b) of different HCG concentrations are shown.
[0029] Figure 7 The performance test results of the HCG sensor provided in Example 8 are shown: (a) selectivity; (b) repeatability; (c) mechanical stability; (d) long-term stability.
[0030] Figure 8 Differential protein results of proteomic analysis in Example 1 are shown.
[0031] Figure 9 A double staining diagram of cell viability experiment of CNTs / 1T-MoS2 electrode material is shown.
[0032] Figure 10 A flow cytometry analysis diagram of cell apoptosis experiment of CNTs / 1T-MoS2 electrode material is shown.
[0033] Figure 11 A pathological section diagram of main organs of SD rats of CNTs / 1T-MoS2 electrode material is shown.
[0034] Figure 12 A pathological section diagram of reproductive organs of SD rats of CNTs / 1T-MoS2 electrode material is shown. DETAILED DESCRIPTION
[0035] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.
[0036] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.
[0037] Example 1
[0038] Proteomic analysis of sweat of Alzheimer's disease patients: S1, subject screening: 6 cases of AD patients diagnosed by neurologists and 6 cases of healthy controls were included, and strictly matched according to age, race and gender, and individuals with mental illness and drug abuse history were excluded, and the average ages of the two groups were 85 years and 84.5 years, respectively, with no significant difference; Number Age Gender Race Group DGF1 88 Female Han Patient DGF2 88 Female Han Patient DGF3 82 Female Han Patient CGF1 84 Female Han Control CGF2 95 Female Han Control CGF3 88 Female Han Control DGM1 85 Male Han Patient DGM2 87 Male Han Patient DGM3 80 Male Han Patient CGM1 82 Male Han Control CGM2 79 Male Han Control CGM3 79 Male Han Control S2, stimulate the palmar sweat glands of the forearm with pilocarpine iontophoresis, collect 50-60 μL undiluted pure exocrine sweat samples, extract proteins after lysis, shaking and centrifugation, quantify by BCA method, then perform SDS-PAGE electrophoresis analysis, and then obtain protein digestion products by trypsin digestion.
[0039] S3, DIA mass spectrometry detection was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology, combined with Spectronaut™ software and Uniprot database for data analysis, and the biological functions of the differential proteins were analyzed by bioinformatics methods such as subcellular localization, Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis S4, 709 unique proteins and 2916 peptides were identified, and more than 99% of the proteins contained 10 or fewer peptides, 91.9% of the proteins had a molecular weight of 100 kDa or less. After filling in the missing values by seqknn method and removing the proteins with more than 90% missing values, subsequent analysis was performed.
[0040] S5, hierarchical clustering analysis showed that the protein expression patterns of samples within the group were highly consistent (the correlation coefficient was generally more than 0.9), while the correlation coefficient between the AD patient group and the healthy control group was about 0.4, which was significantly different; volcano plot analysis showed that 90 differentially expressed proteins were identified by comparing all patient groups with all control groups, of which 65 were up-regulated and 25 were down-regulated.
[0041] S6, GO enrichment analysis: the differential proteins were mainly enriched in biological processes such as cell processes, biological regulation, and metabolic processes, molecular functions such as binding and catalytic activity, and cellular components such as protein complexes and cell anatomical entities.
[0042] S7, KEGG enrichment analysis: the differential proteins were involved in signaling, neurodegenerative diseases, immune system, etc. Among them, 6 differential proteins related to neurodegenerative disease pathway were found in male and female AD patients, such as RTN4, PIK3C3, PSMB7, PPIB, PSMA1 and HCG, as shown in Figure 8 .
[0043] From a biological perspective, HCG (human chorionic gonadotropin) is not only related to reproduction, but recent studies have also shown that it may play a role in maintaining the health of the central nervous system in adults, including potential neuroprotective effects. The absence of HCG in the sweat of AD patients may reflect a systemic or local (skin / sweat gland) protein expression disorder or depletion mechanism in the pathological state of AD.
[0044] Therefore, HCG protein can be used as a specific biological target, and its "presence / absence" or "high / low" pattern in sweat provides an unprecedented, intuitive and specific indicator for AD diagnosis.
[0045] Example 2
[0046] The embodiment provides a composite material CNTs / 1T-MoS2 / PR for detecting HCG protein in sweat, and a preparation method thereof, which comprises the following steps: (1) Preparation of CNTs / 1T-MoS2: 100 mg urea, 50 mg MoO3 and 100 mg thioacetamide (TAA) are dissolved in a mixed solution of 60 mL deionized water and ethanol, and mechanically stirred for 30 minutes.
[0047] After 6 mg of CNTs powder is added and ultrasonic treatment is performed for 30 minutes, the mixed solution is transferred into a polytetrafluoroethylene-lined autoclave, and reaction is performed at 200°C for 18 hours. After cooling, the CNTs / 1T-MoS2 black powder is obtained through multiple centrifugal cleaning with ethanol and deionized water and drying.
[0048] (2) Preparation of CNTs / 1T-MoS2 / PR: 2 mL of mercaptoacetic acid (TA) is added to 20 mg of CNTs / 1T-MoS2 suspension, and stirring is performed for 24 hours. The accumulated CNTs / 1T-MoS2 is removed through centrifugation, the supernatant is collected, and CNTs / 1T-MoS2-COOH powder is obtained through freeze-drying.
[0049] CNTs / 1T-MoS2-COOH is mixed with N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and the carboxyl group is activated into an NHS ester. A polypeptide sequence PR (sequence: PPLRINRHILTR) which specifically recognizes HCG protein is added, and reaction is performed at room temperature for 1 hour. Unbound PR is blocked with BSA, and the CNTs / 1T-MoS2 / PR biological composite material is obtained after centrifugal washing and resuspension in a PBS buffer for preservation.
[0050] Example 3
[0051] The embodiment provides a composite material CNTs / 1T-MoS2 / PR for detecting HCG protein in sweat, which is different from the embodiment 2 in that the feeding amount is different. The feeding amount of the embodiment is: 75 mg urea, 50 mg MoO3 and 75 mg thioacetamide, and 5 mg of CNTs powder.
[0052] Example 4
[0053] The embodiment provides a composite material CNTs / 1T-MoS2 / PR for detecting HCG protein in sweat, which is different from the embodiment 2 in that the feeding amount is different. The dosing amount of this embodiment is: 125 mg urea, 50 mg MoO3, and 125 mg thioacetamide, 7.5 mg CNTs powder.
[0054] Example 5
[0055] This embodiment provides a composite material CNTs / 1T-MoS2 / PR for detecting HCG protein in sweat, which is different from example 2 in that the condition parameters of the solvothermal reaction are different, specifically: the reaction temperature is 180°C, and the reaction time is 24 hours.
[0056] Example 6
[0057] This embodiment provides a composite material CNTs / 1T-MoS2 / PR for detecting HCG protein in sweat, which is different from example 2 in that the condition parameters of the solvothermal reaction are different, specifically: the reaction temperature is 200°C, and the reaction time is 12 hours.
[0058] Example 7
[0059] This embodiment provides a composite material CNTs / 1T-MoS2 / PR for detecting HCG protein in sweat, which is different from example 2 in that the carboxyl activating agent is 2 ml of mercaptopropionic acid.
[0060] Example 8
[0061] This embodiment provides an HCG sensor for non-invasive detection of Alzheimer's disease, and the preparation method thereof comprises: (1) Using soft lithography technology to prepare a microfluidic chip: after the silicon wafer is cleaned and plasma treated, SU-8 2100 photoresist is spin-coated, exposed and developed to form a microfluidic channel. PDMS is molded with the photoresist, and after curing, it is peeled off, cut, punched, and bonded with a glass substrate to form a 200 μm thick microfluidic chip; (2) Preparing a sensing electrode: cleaning the screen-printed electrode, and using cyclic voltammetry (CV) to electrochemically deposit Prussian blue (PB) on the electrode, with the optimized number of cycles being 20 times. After deposition, the electrode is subjected to 10 cyclic voltammetry scans in 0.1 M KCl and 0.1 M HCl solutions, and after drying, a PB electrode is obtained. The CNTs / 1T-MoS2 / PR composite material provided in the foregoing example 2 is loaded onto the PB electrode to form a complete sensing electrode. The CNTs / 1T-MoS2 / PR is dispersed in a diluted PEDOT:PSS (PP) solution, ultrasonically mixed, and then dropped and coated onto the surface of the PB modified electrode, and after drying at room temperature, the loading is completed.
[0062] (3) Assembly of the sensor: Assembling the above-mentioned sensing electrode with the microfluidic chip to obtain a microfluidic wearable sensor for detecting HCG protein, referred to as: CNTs / 1T-MoS2 / PR / PB / PP.
[0063] Comparative Example 1 The difference between this comparative example and Example 2 is that CNTs are not added, and only 1T-MoS2 nanosheets are synthesized using MoO3, TAA and urea as a control.
[0064] Performance detection test
[0065] I. Material characterization The morphology and structure of CNTs, 1T-MoS2 (provided by Comparative Example 1) and CNTs / 1T-MoS2 (provided by Example 2) composite materials were characterized respectively, and the results are as follows: SEM characterization: As shown in Figure 1 (a) is the SEM image of CNTs, showing the typical tubular structure of CNTs, which is randomly distributed; (b) is the SEM image of 1T-MoS2, showing that the flaky 1T-MoS2 is stacked together; (c) is the SEM image of CNTs / 1T-MoS2, which clearly shows that the 1T-MoS2 nanosheets are uniformly distributed on the surface of CNTs, and there is no obvious agglomeration phenomenon. This uniform distribution of core-shell structure helps to improve the electrochemical performance of the material.
[0066] TEM characterization: As shown in Figure 1 (d) and (e) reveal the internal structure of CNTs / 1T-MoS2 at different magnifications, and the multilayer 1T-MoS2 nanosheets are well assembled on the CNTs; the HRTEM image (f) further reveals the crystal structure of the material, in which the interplanar spacing of CNTs is 0.34 nm, corresponding to its (002) crystal plane, and the lattice spacing of 1T-MoS2 is 0.92 nm, corresponding to the (002) crystal plane. This larger lattice spacing is related to the lattice expansion caused by the intercalation of NH4⁺ during the hydrothermal process.
[0067] EDS test: As shown in Figure 1 the energy spectrum (g) shows that C, S and Mo are uniformly distributed in CNTs / 1T-MoS2, further confirming the successful preparation of the composite material.
[0068] The above characterization results show that the CNTs / 1T-MoS2 composite material has good structure and composition, providing a solid foundation for subsequent electrochemical performance testing and biological safety evaluation.
[0069] XRD test: As shown in Figure 2As shown, it shows the XRD patterns of CNTs, 1T-MoS2 and CNTs / 1T-MoS2 composites. As can be seen from the figure, two strong peaks of CNTs appear at 25.8° and 42.9°, respectively, which correspond to the characteristic diffraction peaks of CNTs, indicating that CNTs have good crystallinity. For 1T-MoS2, a new peak appears at 9.6°, which corresponds to the (002) crystal plane of 1T-MoS2, and the interplanar spacing is 0.92 nm. This larger lattice spacing may be due to the intercalation of NH4⁺ during the hydrothermal process, causing lattice expansion and forming 1T phase. In addition, the XRD patterns of 1T-MoS2 and CNTs / 1T-MoS2 also show obvious sharp and broad diffraction peaks, indicating that there are nanoscale structures of 1T-MoS2 crystals in all samples. These results further confirm the successful preparation of CNTs / 1T-MoS2 composites, and show that 1T-MoS2 retains good crystal structure in the composite, providing a strong structural basis for subsequent electrochemical performance tests.
[0070] II. Performance test of HCG sensor The performance of the HCG sensor provided in Example 8 was tested, and the test method was as follows: The performance of the HCG sensor was tested by cyclic voltammetry, electrochemical impedance spectroscopy, differential pulse voltammetry, simulated sweat applicability, verification of anti-interference ability, and stability test.
[0071] Figure 3 After each surface modification step, CV (a) and EIS curve (b) were measured in 5mM K3[Fe(CN)6] and 0.1M KCl.
[0072] a The redox peak current of the bare electrode and the CNTs modified electrode is low, indicating poor conductivity; while the peak current of the CNTs / 1T-MoS2 electrode is significantly improved, proving that the metallic phase characteristics of 1T-MoS2 can enhance the electron transport efficiency; the peak current further increases after loading PB, because PB as a redox probe can amplify the electrical signal; the peak current slightly decreases after loading PR, because PR is a biological molecule that slightly hinders electron transport, but has no significant effect; the peak current is significantly enhanced after finally loading PP, confirming that PP can further reduce the charge transfer resistance and optimize the electrode conductivity.
[0073] b is the maximum Rct of the bare electrode, Rct decreases after CNTs modification; Rct further decreases for CNTs / 1T-MoS2 electrode, verifying the promotion of 1T-MoS2 to electron transfer; Rct continues to decrease after loading PB due to the redox characteristics of PB accelerating interface charge transfer; Rct slightly increases after loading PR (consistent with the CV results), but significantly decreases after loading PP, again proving that PP can optimize the electrode interface and improve the efficiency of electron transfer.
[0074] Figure 4 The CV curves at different scan rates in 0.1M PBS solution. With the increase of scan rate, the potential difference between oxidation peak and reduction peak gradually expands, but the peak current ratio (Ipa / Ipc) is close to 1, indicating that the quasi-reversible redox reaction occurs on the electrode surface without obvious side reaction; the oxidation peak current and the reduction peak current show a linear relationship with the square root of the scan rate; the CV curve shape is stable at different scan rates without abnormal peaks, providing an electrochemical basis for the signal stability of subsequent HCG detection.
[0075] Figure 5 The CV curves of HCG sensor with and without loading PP electrode for cycle stability.
[0076] a is the oxidation peak current of CNTs / 1T-MoS2 / PB / PR electrode significantly attenuates after 50 cycles, because PB is directly in contact with the solution, which is easy to dissolve or oxidize and reduce.
[0077] b is the oxidation peak current of the electrode with PP loading only slightly decreases after 50 cycles, proving that PP can form a protective film to wrap PB and composite sensing materials, avoiding direct contact between PB and the solution.
[0078] Figure 6 The DPV curves and linear fitting graph of different HCG concentrations.
[0079] a is that with the increase of HCG concentration, the reduction peak current of DPV curve decreases regularly, after HCG specifically binds to PR, it will hinder the redox reaction of PB on the electrode surface, and the binding amount increases with the increase of HCG concentration, the current decrease amplitude is positively correlated with the HCG concentration.
[0080] b is that the peak current and the logarithm of HCG concentration show a good linear relationship in the range of 0~10μg / mL, and the calculated LOD is 0.45pg / mL, which is much lower than the expression level of HCG in sweat of healthy control group.
[0081] Figure 7 The results of HCG sensor selectivity and stability test.
[0082] a is the selective test, the results show that the sensor has high selectivity to HCG even in the presence of a large amount of interferents (such as β-lactoglobulin, BSA, uric acid, glucose and lactic acid, the concentration is 10 times that of HCG), indicating that it has good anti-interference ability.
[0083] b shows the repeatability test results of the electrodes prepared by the same method and tested for detection stability, the relative standard deviation (RSD) is 6.14% when detecting 10 ng / mL HCG, indicating that the sensor has good repeatability.
[0084] c depicts the detection stability of the sensor for 0.1 mg / mL HCG after different numbers of bending (bending radius is 1.5 cm), the results show that even after bending 300 times, the detection performance of the sensor remains stable, the oxidation peak current remains 96.97% of that before bending, which indicates that the sensor has good mechanical stability and anti-deformation ability.
[0085] d presents the stability test of the sensor for 1 μg / mL HCG at different storage times, the results show that even after 30 days of storage, the detection performance of the sensor only decreases slightly, the peak current remains 96.41% of the initial value, further confirming its long-term stability.
[0086] These test results show that the HCG sensor provided in Example 8 not only has high selectivity and good repeatability, but also has excellent mechanical stability and long-term storage stability, providing reliable guarantee for practical application.
[0087] Biological safety test To further investigate the biological safety of the CNTs / 1T-MoS2 electrode material provided in the present application, and to provide support for the subsequent development of wearable sensors for specific applications, the inventors tested the in vitro cytotoxicity and in vivo acute toxicity of the electrode material.
[0088] Figure 9 The double staining schematic diagram for the cell viability experiment of the CNTs / 1T-MoS2 electrode material, the cells in the concentration group are mainly green fluorescence, the red fluorescence signal is weak and has no obvious enhancement, indicating that the CNTs / 1T-MoS2 electrode material has no significant cytotoxicity in the concentration range of 10-1000 μg / mL.
[0089] Figure 10The flow cytometry analysis chart of the apoptosis experiment of the CNTs / 1T-MoS2 electrode material shows that the cell apoptosis / necrosis ratio (Q1+Q2+Q3) of each treatment group and the control group has no significant difference (P>0.05), and the survival cells (Q4) are mainly used, which further verifies that the CNTs / 1T-MoS2 material does not induce cell apoptosis and has good biological safety.
[0090] Figure 11 The SD rat main organ pathological section chart of the CNTs / 1T-MoS2 electrode material shows that the myocardial cells of the two groups are arranged regularly, the liver cells are complete in structure, the spleen corpuscles are clear, the lung alveoli are not damaged, the glomerulus / kidney tubule is normal, the hippocampus nerve cells are closely ordered, and there is no inflammatory cell infiltration or tissue necrosis, which proves that the material has no organic toxicity to the main organs of the rat.
[0091] Figure 12 The SD rat reproductive organ pathological section chart of the CNTs / 1T-MoS2 electrode material shows that the oviduct walls of the two groups are complete, the testis seminiferous tubules are arranged regularly (there are complete spermatogenic cell layers in the lumen), and the sperm in the epididymal lumen is dense, and there is no tissue damage or inflammatory reaction, which shows that the material has no toxic effect on the reproductive system of the SD rat, and further improves the biological safety verification.
[0092] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application, and those skilled in the art can make non-creative contribution modifications to the embodiments according to the needs after reading the present specification, but as long as the present application is within the scope of the claims, it is protected by the patent law.
Claims
1. A composite material for detecting HCG protein in sweat, characterized by, It includes: A composite substrate material composed of carbon nanotubes and 1T phase molybdenum disulfide; And, polypeptide molecules fixed on the composite substrate material by a crosslinking agent; The polypeptide molecule can specifically bind to HCG protein, which is a biomarker in Alzheimer's sweat.
2. The composite material for detecting HCG protein in sweat according to claim 1, characterized by, The amino acid sequence of the polypeptide molecule is: PPLRINRHILTR.
3. The composite material for detecting HCG protein in sweat according to claim 1, characterized by, The crosslinking agent is NHS / EDC.
4. A method for the preparation of a composite material for the detection of HCG protein in sweat as claimed in any one of claims 1 to 3, characterized in that, It includes: S1. A one-pot solvothermal reaction was used to mix MoO3, thioacetamide, urea and carbon nanotubes in a solvent to generate CNTs / 1T-MoS2 composite substrate material. S2. The composite substrate material is modified by carboxylation to obtain the carboxylated composite substrate material CNTs / 1T-MoS2-COOH; S3. Activate the carboxyl groups on the surface of the carboxylated composite substrate material using a crosslinking agent, and react them with polypeptide molecules that specifically recognize HCG protein, so that the polypeptide molecules are fixed on the surface of the composite substrate material to obtain the composite material.
5. The method for preparing a composite material for detecting HCG protein in sweat according to claim 4, characterized by, In step S2, the carboxylation modification is performed using mercaptoacetic acid or mercaptopropionic acid.
6. The method for preparing the composite material for detecting HCG protein in sweat according to claim 4, characterized in that, In step S1, the temperature of the solvothermal reaction is 180-220℃, and the reaction time is 12-24 hours.
7. The method for preparing the composite material for detecting HCG protein in sweat according to claim 4, characterized in that, In step S1, the mass ratio of MoO3 to thioacetamide and urea is 1:1.5-2.5; The mass ratio of MoO3 to carbon nanotubes is 10:1.0-1.
5.
8. A biosensor for non-invasive detection of Alzheimer's disease, characterized in that, include: Working electrode; as well as The composite material as described in any one of claims 1-3 is fixed to the surface of the working electrode.
9. The sensor for non-invasive detection of Alzheimer's disease according to claim 8, characterized in that, It also includes a microfluidic chip, with a working electrode with a surface modified with composite material integrated with the microfluidic chip to form a wearable sweat detection patch.
10. Use of the composite material as described in any one of claims 1-3 or the sensor as described in claim 8 or 9 in the preparation of products for diagnosing or assisting in the diagnosis of Alzheimer's disease.