Visual allergy-infection duplex detection device based on partitioned nanofiber membrane and preparation method of visual allergy-infection duplex detection device
The partitioned nanofiber membrane is constructed by electrospinning technology, which solves the problem of the existing technology that the detection device cannot quickly distinguish between allergies and infections in COPD patients, and realizes efficient and accurate allergy and infection detection. It solves the problem of the existing technology and the existing technology device, and realizes efficient and accurate detection of allergy and infection markers. It has good long-term stability and easy operation, and is suitable for personalized prognosis management of COPD patients.
Patent Information
- Application Number
- CN202510828884.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-09-19
AI Technical Summary
Existing technologies make it difficult to quickly distinguish between the causes of allergies and infections in COPD patients, leading to the abuse of hormones and antibiotics. In addition, existing detection devices lack sensitivity, have high equipment costs, and easily inactivated color development systems, making it difficult to achieve efficient and accurate non-invasive dual testing.
Electrospinning technology is used to construct partitioned nanofiber membranes, using PVA nanofibers as the substrate, combined with hydrophilic and hydrophobic materials to encapsulate antibodies and nanozymes respectively, realizing visual dual detection of allergy and infection markers.
It achieves efficient, accurate, and cross-reaction-free detection of allergy and infection markers, has good long-term stability and ease of operation, and is suitable for personalized prognostic management of COPD patients.
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Figure CN120668936A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological detection technology, and in particular to a visualized allergy-infection dual detection device based on a partitioned nanofiber membrane and a preparation method thereof. Background Art
[0002] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by persistent airflow limitation. Due to chronic airway inflammation and damage to alveolar structure, COPD patients experience chronic symptoms such as dyspnea, cough, and sputum production, and in severe cases, even become unable to function effectively. Recurrent acute exacerbations (AECOPD) during the course of the disease are a major cause of accelerated lung function deterioration, increased hospitalization rates, and increased mortality. Approximately 40% of these exacerbations are triggered by a combination of allergen exposure and respiratory infections (bacterial or viral). However, current technologies struggle to quickly distinguish these triggers, leading to the overuse of steroids and antibiotics. For patients, COPD not only deteriorates physiologically but also carries significant psychosocial and economic burdens. 60% of severe COPD patients are forced to stay home due to restricted mobility, and social isolation leads to depression and anxiety rates as high as 50%. A single AECOPD hospitalization can cost over $15,000 USD, and approximately 30% of patients are repeatedly readmitted within 30 days of discharge. Therefore, prognostic management of COPD patients is necessary. The allergy marker eosinophil cationic protein (ECP), a direct effector protein of eosinophil activation, has a salivary concentration that specifically reflects the Th2 inflammatory load of COPD patients and is significantly associated with the risk of acute exacerbations and responsiveness to hormone therapy. Infection detection studies using H2O2 as a marker have important clinical value for COPD management. As a core molecule of oxidative stress, H2O2 can promote the release of proinflammatory cytokines such as IL-6 and TNF-α by activating the NF-κB pathway, exacerbating airway inflammation and enhancing the adhesion and colonization of pathogens (such as Streptococcus pneumoniae), making it a key driver of infection-induced AECOPD.
[0003] Existing dual-detection technology devices in the medical field primarily include dual immunochromatographic test strips (such as HIV / syphilis combined test strips), microfluidic chips (for dual nucleic acid / protein detection), and multi-parameter biosensors (such as portable blood glucose / lactate meters). These devices integrate immunochromatographic, electrochemical, and optical detection principles to enable rapid analysis of multiple parameters. However, they still have significant limitations: First, traditional nitrocellulose (NC) membrane-based immunochromatographic test strips are susceptible to environmental humidity, and their reliance on physical adsorption results in inefficient antibody immobilization, leading to insufficient sensitivity and nonspecific cross-reactions. Second, microfluidic chips rely on precision instrumentation to drive fluids and detect signals, resulting in high equipment costs and complex maintenance. Third, existing colorimetric systems (such as colloidal gold / enzyme catalysis) are prone to false negatives due to premature substrate oxidation or enzyme inactivation. Therefore, there is a need for a test device with high detection efficiency, good long-term stability, high accuracy, no cross-reactivity, non-invasiveness, ease of use, and specificity for COPD patients, to provide guidance for prognostic management of COPD patients. Summary of the Invention
[0004] The purpose of the present invention is to address the above technical problems and provide a visual allergy-infection dual detection device based on partitioned nanofiber membranes.
[0005] The device uses electrospinning technology to integrate functional nanofiber membranes, with hydrophilic PVA (polyvinyl alcohol) nanofibers as the base membrane. It achieves dual-functional integration through precise zoning (allergy detection area, sample collection area, and infection detection area) design combined with immunochromatography and color development mechanisms.
[0006] Specifically, an allergy detection area fiber membrane, an infection detection area fiber membrane and a sample collection area are provided on the nanofiber membrane basement membrane; the sample collection area is located at the central axis of the basement membrane, and the allergy detection area fiber membrane and the infection detection area fiber membrane are respectively distributed on both sides of the sample collection area in an axially symmetrical manner.
[0007] Specifically, the fiber membrane in the allergy detection area uses PVA nanofibers to encapsulate anti-human ECP capture antibodies and colloidal gold-anti-ECP detection antibodies to form an "antibody-antigen" complex to capture ECP in saliva and display a red signal.
[0008] Specifically, the fiber membrane in the infection detection area uses gelatin and PLGA (polylactic acid-co-glycolic acid) nanofibers to encapsulate Fe3O4 nanozymes and TMB (3,3',5,5'-tetramethylbenzidine).
[0009] The infection detection area nanofiber membrane provided by the present invention utilizes the hydrophobic sustained-release properties of PLGA to prevent premature oxidation of the TMB substrate. When H2O2 is present, the Fe3O4 nanoenzyme efficiently catalyzes TMB to generate a blue product. The three-dimensional network structure of the PVA nanofiber not only gives the device an excellent capillary effect, but its covalent coupling properties also increase the antibody loading capacity.
[0010] At present, there are no research reports on the simultaneous detection of allergy and infection markers based on electrospinning partitioned nanofiber membranes. This technology can dynamically monitor the allergic status and infection risk of COPD patients through non-invasive saliva samples, providing an efficient and portable solution for personalized prognosis management (such as determining the timing of hormone use and anti-infection warning), and has significant clinical translation value.
[0011] The present invention also provides a method for preparing a visual allergy-infection dual detection device based on a partitioned nanofiber membrane, which specifically comprises the following steps:
[0012] (1) Preparation of PVA substrate membrane: PVA was dissolved in 10% (w / v) deionized water, heated in an 80°C water bath with stirring for 6 h to obtain spinning solution 1, and uniaxial electrospinning was performed using spinning solution 1 as a raw material to obtain a PVA substrate membrane;
[0013] (2) Preparation of fiber membrane in the allergy detection area: anti-human ECP capture antibody, colloidal gold-anti-ECP detection antibody and glycerol were dissolved in PBS buffer and stirred for 2 h to obtain spinning solution 2. A circular mold was used to cover the PVA basement membrane, and spinning solution 1 and spinning solution 2 were used as raw materials to perform coaxial electrospinning on the surface to obtain a fiber membrane in the allergy detection area;
[0014] (3) Preparation of fiber membrane for allergy-infection dual detection: Gelatin and PLGA were dissolved in hexafluoroisopropanol and stirred for 4 h to obtain spinning solution 3. Fe3O4 nanozyme and TMB were added thereto and stirred for 2 h to obtain spinning solution 4. The fiber membrane of the allergy detection area obtained in step (2) was covered with a circular mold, and uniaxial electrospinning was performed on the surface of the spinning solution 4 as a raw material. After spinning, the spinning was freeze-dried to obtain the fiber membrane for allergy-infection dual detection.
[0015] Furthermore, in step (2), the concentration of the anti-human ECP capture antibody is 0.2 mg / mL, and the concentration of the colloidal gold-anti-ECP detection antibody is 0.1 mg / mL.
[0016] Furthermore, the volume ratio of glycerol in the spinning solution 2 in step (2) is 10%.
[0017] Furthermore, the flow rate ratio of the coaxial electrospinning to the core-shell in step (2) is 1:7.
[0018] Furthermore, in step (3), the gelatin and PLGA are in a mass ratio of 3:7.
[0019] Furthermore, in the spinning solution 4 of step (3), the concentration of Fe3O4 nanozyme is 1 mg / mL, and the concentration of TMB is 15 mg / mL.
[0020] The present invention also provides an application of a visualized allergy-infection dual detection device based on a partitioned nanofiber membrane in the preparation of a kit for detecting allergies and / or infections in COPD patients; the test sample of the kit is saliva.
[0021] The advantages of the present invention are:
[0022] 1. The present invention is the first to simultaneously distribute anti-human ECP capture antibodies, colloidal gold-anti-ECP detection antibodies, Fe3O4 nanozymes and TMB on a nanofiber membrane to construct an allergy-infection dual detection fiber membrane. The fiber membrane of the present invention has the performance of allergy-infection visual dual detection, and does not require physical separation of detection areas, reducing the cross-reaction rate to 0, which facilitates the prognosis management of COPD patients.
[0023] 2. The allergy-infection dual detection fiber membrane of the present invention uses PVA basement membrane instead of the NC membrane of traditional test paper, and has excellent detection efficiency, mechanical properties, and long-term storage ability.
[0024] 3. The allergy-infection dual detection fiber membrane of the present invention uses PLGA nanofibers to encapsulate Fe3O4 nanozyme and TMB, effectively reducing the risk of premature exposure and oxidation of TMB, improving the load stability, and further enhancing the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 Schematic diagram of the fiber membrane device of the present invention.
[0026] Figure 2 is the contact angle data of the fiber membrane of the present invention.
[0027] Figure 3 It is the mechanical property of the fiber membrane of the present invention.
[0028] Figure 4 The detection performance of the fiber membrane of the present invention is: Figure 4 (a) is the change of R value in the allergy detection area under different concentrations of ECP; Figure 4 (b) is the change of R value in the infection detection area under different H2O2 concentrations.
[0029] Figure 5 The stability of the fiber membrane of the present invention is shown in FIG5(a). FIG5(a) shows the change of the detection performance of the allergy detection area with the storage time. Figure 5 (b) is the change of detection performance of the infection detection area with storage time; Figure 5 (c) is the change of fiber membrane loading stability over time.
[0030] Figure 6 This is the detection effect of the fiber membrane of the present invention. DETAILED DESCRIPTION
[0031] The technical solutions described in the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described in this specification are only part of the feasible technical solutions of the present invention. Other implementation methods obtained by ordinary technicians in this field based on the embodiments of the present invention without any creative work should be deemed to fall within the scope of protection of the present invention.
[0032] Example 1: Preparation of a visualized allergy-infection dual detection device based on partitioned nanofiber membranes.
[0033] (1) Preparation of PVA substrate membrane: PVA was dissolved in 10% (w / v) deionized water, heated in a water bath at 80°C with stirring for 6 h to obtain spinning solution 1. Spinning solution 1 was used as a raw material for uniaxial electrospinning to obtain a PVA substrate membrane. The electrospinning parameters were set as follows: propulsion speed of 0.003 mm / s, spinning needle of 20 G, voltage of 15 kV, receiving distance of 13 cm, temperature of 25°C, and relative humidity of 60%;
[0034] (2) Preparation of fiber membrane in the allergy detection area: anti-human ECP capture antibody, colloidal gold-anti-ECP detection antibody and glycerol were dissolved in PBS buffer and stirred for 2 h to obtain spinning solution 2. The PVA basement membrane obtained in step (1) was covered with a circular mold, and coaxial electrospinning was performed on the surface of the PVA basement membrane using spinning solution 1 and spinning solution 2 as raw materials to obtain a fiber membrane in the allergy detection area. The electrospinning parameters were set as follows: the core layer propulsion speed was 0.001 mm / s, the shell layer propulsion speed was 0.007 mm / s, the spinning needles were 20G and 17G, the voltage was 15 kV, the receiving distance was 13 cm, the temperature was 25°C, and the relative humidity was 60%;
[0035] (3) Preparation of allergy-infection dual detection fiber membrane: Gelatin and PLGA were dissolved in hexafluoroisopropanol at a mass ratio of 3:7, stirred and mixed for 4 hours to obtain spinning solution 3, Fe3O4 nanozyme (1 mg / mL) and TMB (15 mg / mL) were added thereto, stirred for 2 hours to obtain spinning solution 4, and the allergy detection area fiber membrane obtained in step (2) was covered with a circular mold, and uniaxial electrospinning was performed on its surface using spinning solution 4 as a raw material. After spinning, it was freeze-dried to obtain an allergy-infection dual detection fiber membrane. The electrospinning parameters were set as follows: propulsion speed of 0.003 mm / s, spinning needle of 20 G, voltage of 15 kV, receiving distance of 13 cm, temperature of 25 ° C, and relative humidity of 60%.
[0036] Comparative Example 1: Preparation of a visual allergy-infection dual detection device based on NC membrane
[0037] The difference between this comparative example and Example 1 is that the basement membrane is an NC membrane.
[0038] Preparation of NC substrate membrane: NC was dissolved in 10% (w / v) acetone and stirred for 6 hours to obtain spinning solution 1. The spinning solution 1 was used as a raw material for uniaxial electrospinning to obtain the NC substrate membrane.
[0039] Comparative Example 2: Preparation of a visual allergy-infection dual detection device based on partitioned nanofiber membranes
[0040] Compared with Example 1, this comparative example is different in that the Fe3O4 nanozyme is loaded by electrostatic spraying in the preparation of the allergy-infection dual detection fiber membrane.
[0041] Preparation of allergy-infection dual detection fiber membrane: TMB (15 mg / mL) was added to the spinning solution 3 and stirred for 2 h to obtain spinning solution 4. The allergy detection area fiber membrane obtained in step (2) was covered with a circular mold, and uniaxial electrospinning was performed on its surface using spinning solution 4 as a raw material. Subsequently, Fe3O4 nanozyme (1 mg / mL) was dissolved in hexafluoroisopropanol, stirred and mixed for 4 h, and electrostatic sprayed on the surface of the fiber membrane. After freeze-drying, the allergy-infection dual detection fiber membrane was obtained.
[0042] Test Example 1: Characterization of the hydrophilicity and hydrophobicity of the allergy-infection dual detection fiber membranes prepared in Example 1, Comparative Example 1 and Comparative Example 2
[0043] The fiber membrane was tested using a water contact angle tester, and the results were as follows: Figure 2 As shown, Example 1 and Comparative Example 2, whose basement membranes are PVA, are hydrophilic, with contact angles of 40°. This is primarily due to the high hydrophilic hydroxyl groups in the PVA basement membrane. In contrast, Comparative Example 1, whose basement membrane is NC, is hydrophobic, with a contact angle of 110°. This is because the NC basement membrane is nitrated, with a large number of hydrophobic nitrate groups substituted on the molecular chain. Furthermore, the hydrophilicity of the basement membrane increases the speed at which the sample travels from the collection zone to the detection zone. These results demonstrate that the detection fiber membrane prepared in Example 1 possesses excellent hydrophilicity.
[0044] Test Example 2: Mechanical properties characterization of the allergy-infection dual detection fiber membranes prepared in Example 1, Comparative Example 1 and Comparative Example 2
[0045] The fiber membrane was tested using a universal mechanical tensile tester. The results are as follows: Figure 3As shown, Example 1 and Comparative Example 2, with PVA as the base film, exhibited excellent elongation at break, reaching 210% and 214%, respectively. Comparative Example 1, with NC as the base film, exhibited an elongation at break of only 16%, indicating that the NC base film exhibited poor mechanical properties. On the other hand, the PVA base films in Example 1 and Comparative Example 2 exhibited fracture strengths of 77 and 81 MPa, respectively, while the fracture strength of Comparative Example 1, with NC as the base film, was 32 MPa. These results demonstrate that the PVA base film prepared in Example 1 possesses excellent mechanical properties.
[0046] Experimental Example 3: Characterization of the allergy and infection detection performance of the allergy-infection dual detection fiber membranes prepared in Example 1, Comparative Example 1, and Comparative Example 2
[0047] 100 μL of different concentrations of ECP (1, 5, 10, 50, 150 ng / mL) and different concentrations of H2O2 (2, 4, 6, 8, 10 mM) were added to the cross center of the middle sample collection area of the fiber membrane. After a certain period of time, the red and blue channel values of the color development area were extracted using an RGB (red, green, and blue) color picker. The R value was negatively correlated with the color development intensity of red, while the B value was negatively correlated with the color development intensity of blue. Figure 4 As shown in (a), the R value of the allergy detection area decreases with the increase of substrate concentration, and the red color deepens. However, the R value of Comparative Example 1 is larger than that of Example 1, indicating that the color intensity of Comparative Example 1 is lighter under the same detection time. This further indicates that the NC membrane as a basement membrane affects its color development effect. This is because it is more difficult for the sample to be transported to the detection area on the NC basement membrane in the same time, resulting in a lower color intensity. Figure 4 As shown in (b), the B value of the infection detection area decreases with increasing substrate concentration, and the blue color deepens. However, the B value of Comparative Example 2 is larger than that of Example 1, indicating that the color intensity of Comparative Example 2 is lighter under the same detection time. This further indicates that the loading of Fe3O4 nanozyme on the fiber surface affects the color development reaction rate of the infection detection area, thereby affecting its color intensity. The above results show that the detection fiber membrane prepared in Example 1 has good detection sensitivity and detection efficiency.
[0048] Test Example 4: Stability Characterization of the Allergy-Infection Dual Detection Fiber Membranes Prepared in Example 1, Comparative Example 1, and Comparative Example 2
[0049] (1) Characterization of long-term stability: After the fiber membrane was stored at 4°C for 100, 200, and 300 days, the color intensity of the fiber membrane was detected with 150 ng / mL ECP and 10 mM H2O2, respectively. Figure 5As shown in (a, b), the R value of the allergy detection area of Comparative Example 1 increased significantly with the extension of storage time, indicating that the NC membrane significantly affected its long-term stability. The B value of the infection detection area of Comparative Example 2 decreased significantly with the extension of storage time, indicating that the loading of Fe3O4 nanozyme on the fiber surface caused TMB to oxidize and develop color prematurely. This shows that the loading method of Fe3O4 nanozyme in the fiber membrane destroyed the long-term stability of the detection fiber membrane. Fe3O4 nanozyme exposed on the fiber surface easily caused TMB to oxidize prematurely, while coating the nanozyme in the fiber can prolong the long-term stability of the detection fiber membrane.
[0050] (2) Characterization of load stability: The fiber membrane was placed in deionized water for 7 days. 3 mL of sample was taken every other day and supplemented with an equal amount of fresh deionized water. The UV absorbance of the sample at 240 nm was measured. Figure 5 As shown in (c), Fe3O4 nanozyme was found in the sample of Comparative Example 2 after 3 days. This is because loading Fe3O4 nanozyme on the fiber surface weakens the stability of the fiber-loaded nanozyme. Fe3O4 nanozyme was found in the experimental group samples of Example 1 and Comparative Example 1 on the 6th day, proving that encapsulation inside the fiber can improve the loading stability of the nanozyme.
[0051] The above experiments demonstrate that the detection fiber membrane prepared in Example 1 has good stability.
[0052] Experimental Example 5: Characterization of the detection efficiency of the allergy-infection dual detection fiber membranes prepared in Example 1, Comparative Example 1 and Comparative Example 2
[0053] Saliva samples from 20 groups of COPD patients after allergic infection were collected, and the allergy-infection dual detection fiber membranes prepared in Example 1, Comparative Example 1 and Comparative Example 2 were used to detect the saliva samples, and the detection speed was recorded. Figure 6 As shown, the detection time of Comparative Example 1 reaches 16 minutes, which is significantly higher than that of Example 1 and Comparative Example 2, indicating that the PVA film significantly improves the detection efficiency.
[0054] Experimental Example 6: Detection Accuracy and COPD Patient Specificity Characterization of the Allergy-Infection Dual Detection Fiber Membrane Prepared in Example 1
[0055] In order to compare the accuracy of the allergy-infection dual detection fiber membrane prepared in Example 1 and the allergy and infection detection results of the prior art (blood sample detection), 20 groups of saliva and blood samples were collected from COPD patients (stable phase), COPD patients (acute exacerbation phase-allergy), COPD patients (acute exacerbation phase-infection), COPD patients (acute exacerbation phase-mixed), asthma patients and healthy people, and the saliva samples were detected by the allergy-infection dual detection fiber membrane prepared in Example 1. The blood samples were tested for allergy and infection by serum ECP-ELISA and PCT (procalcitonin), respectively. The test results and positive rates were recorded, and the correlation (R 2 ) and consistency analysis (Kappa) to judge the consistency of the test results of the two detection methods, where R 2 A Kappa value greater than 0.7 was considered to indicate a good linear relationship, and a Kappa value greater than 0.6 was considered to indicate an acceptable consistency.
[0056] The evaluation of the accuracy of allergy detection is shown in Table 1. By comparing the test results of the allergy detection area of the fiber membrane with those of serum ECP-ELISA, it was found that in COPD patients in the stable period and infection-induced acute exacerbation period, the positive rates of both methods were less than 60%, indicating that the fiber membrane has the specific recognition ability for allergy markers. The detection of the allergy detection area of the fiber membrane and the serum ECP-ELISA in the allergy-dominated COPD exacerbation and asthma patient groups were highly consistent, which confirmed the reliability of Th2 inflammation detection, and the positive rate of the asthma patient group reached 90-100%, which was higher than that of the allergy-dominated COPD exacerbation group, which is consistent with the more significant Th2 inflammation characteristics of asthma patients, and also proved that the detection fiber membrane can effectively distinguish allergy-induced COPD acute exacerbation from asthma. In addition, the detection rate of both detection methods for healthy samples was 0-5%, which is because healthy samples have almost no allergy markers. Therefore, the above results show that the allergy detection area of the fiber membrane has the ability to specifically detect allergy markers, and can distinguish COPD patients from ordinary asthma patients, and the correlation between the allergy detection results of the two detection methods is R 2 >0.7, the detection results of the detection fiber membrane of Example 1 and the conventional blood detection technology are highly consistent, proving that the allergy detection area of the detection fiber membrane of Example 1 has high accuracy.
[0057] Table 1: Positive rates of allergy-infection dual detection fiber membrane and serum ECP-ELISA for samples prepared in Example 1
[0058]
[0059] The evaluation of the accuracy of infection detection is shown in Table 2. In patients with stable COPD, the positive rates of the detection fiber membrane in the infection detection area and PCT detection were both low. In the infection-dominated COPD exacerbation group, the positive rates of the detection fiber membrane and PCT reached approximately 85-95% and 80-90%, respectively, and the two were highly consistent. Because it can detect metabolites such as H2O2, the fiber membrane can warn of infection earlier, and its ability to identify mixed exacerbations is better than PCT. The detection positive rate of the detection fiber membrane for asthma patients is 15-25%, indicating that it can distinguish between patients with acute exacerbation of COPD infection and asthma patients. In addition, its detection positive rate for healthy human samples is only 0-5%, further confirming its good specificity for COPD detection, and the Kappa values of the infection detection results of the two detection methods are both >0.6. The detection results of the detection fiber membrane of Example 1 and conventional blood detection technology are highly consistent, proving that the accuracy of the infection detection area of the detection fiber membrane of Example 1 is high.
[0060] Table 2: Positive rate of allergy-infection dual detection fiber membrane and PCT for samples prepared in Example 1
[0061]
[0062] In order to further verify the specificity of the allergy-infection dual detection fiber membrane prepared in Example 1 for COPD patients, the RGB color picker was used to extract the R / B channel value of the test results of COPD patients (acute exacerbation-mixed), asthma patients and healthy people and perform color analysis. The quantitative analysis results are shown in Table 3. The test results of the allergy-infection dual detection fiber membrane prepared in Example 1 for COPD patients (acute exacerbation-mixed) showed that the R value and B value were 180 and 175 respectively, and the color intensity was medium. Allergy and infection were both positive. This is because the acute exacerbation of COPD patients caused by mixed reasons of allergy and infection; the color intensity of the allergy area of the test results of asthma patients was high, and the R value reached 80, proving that the allergy of asthma patients was strongly positive, while the color intensity of the test results in the infection area was low, and the B value was 210, which shows that asthma patients are usually very allergic and have a relatively mild infection; the test results of healthy people showed no color development, which shows that the fiber membrane can specifically detect allergies and infections. The quantitative analysis results showed that the fiber membrane prepared in Example 1 can specifically detect allergies and infections in COPD patients through visual color development intensity.
[0063] Table 3 Test results of the allergy-infection dual detection fiber membrane prepared in Example 1 on COPD patients (acute exacerbation-mixed), asthma patients and healthy people
[0064]
[0065] The above results show that the allergy-infection dual detection fiber membrane prepared in Example 1 has consistent results in the detection of saliva samples and the allergy and infection detection results of blood samples by serum ECP-ELISA and PCT, proving that the detection fiber membrane has high detection accuracy. In addition, the detection fiber membrane has different positive rates in COPD patients, asthma patients and healthy people, so it can specifically detect COPD patients and warn COPD patients of acute exacerbations caused by allergies and infections.
[0066] In summary, the allergy-infection dual detection fiber membrane of the present invention has the ability to jointly detect allergies and infections. Experiments have shown that it has good detection efficiency, long-term stability, detection accuracy, no cross-reaction, non-invasiveness and specific detection effect, and is easy to operate and is expected to be widely used in the prognosis management of COPD patients.
[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principles of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.
Claims
1. A visual allergy-infection dual detection device based on partitioned nanofiber membrane, characterized in that: The detection device comprises a nanofiber membrane basement membrane, on which are provided an allergy detection area fiber membrane, an infection detection area fiber membrane and a sample collection area, wherein the sample collection area is located at the central axis of the nanofiber membrane; the allergy detection area fiber membrane and the infection detection area fiber membrane are respectively distributed on both sides of the sample collection area in an axisymmetric manner; The nanofiber membrane basement membrane is formed by uniaxial electrospinning of hydrophilic PVA nanofibers; The allergy detection zone fiber membrane comprises PVA nanofiber-encapsulated anti-human ECP capture antibody and colloidal gold-anti-ECP detection antibody, and is formed by coaxial electrospinning; The infection detection area fiber membrane comprises gelatin and PLGA nanofiber-encapsulated Fe3O4 nanozyme and TMB, and is formed by uniaxial electrospinning.
2. A visual allergy-infection dual detection device based on partitioned nanofiber membrane according to claim 1, characterized in that: The test sample of the visual allergy-infection dual detection device is saliva; The allergy detection area combines with the saliva sample to form an antibody-antigen complex, captures the ECP in the saliva and displays a red signal; the infection detection area combines with the saliva sample and drives the Fe3O4 nanozyme to catalyze TMB and display a blue signal.
3. A method for preparing a visual allergy-infection dual detection device based on a partitioned nanofiber membrane as claimed in claim 1, characterized in that: The preparation method comprises the following steps: (1) Preparation of PVA substrate membrane: PVA was dissolved in deionized water with a mass volume ratio of 10%, heated in a water bath at 80°C with stirring for 6 h to obtain spinning solution 1, and uniaxial electrospinning was performed using spinning solution 1 as a raw material to obtain a PVA substrate membrane; (2) Preparation of fiber membrane in the allergy detection area: anti-human ECP capture antibody, colloidal gold-anti-ECP detection antibody and glycerol were dissolved in PBS buffer and stirred for 2 h to obtain spinning solution 2. A circular mold was used to cover the PVA basement membrane, and spinning solution 1 and spinning solution 2 were used as raw materials to perform coaxial electrospinning on the surface to obtain a fiber membrane in the allergy detection area; (3) Preparation of fiber membrane for allergy-infection dual detection: Gelatin and PLGA were dissolved in hexafluoroisopropanol and stirred for 4 h to obtain spinning solution 3. Fe3O4 nanozyme and TMB were added thereto and stirred for 2 h to obtain spinning solution 4. The fiber membrane of the allergy detection area obtained in step (2) was covered with a circular mold, and uniaxial electrospinning was performed on the surface of the spinning solution 4 as a raw material. After spinning, the spinning was freeze-dried to obtain the fiber membrane for allergy-infection dual detection.
4. The method for preparing a visual allergy-infection dual detection device based on a partitioned nanofiber membrane according to claim 3, characterized in that: The concentration of the anti-human ECP capture antibody in step (2) is 0.2 mg / mL, and the concentration of the colloidal gold-anti-ECP detection antibody is 0.1 mg / mL.
5. The method for preparing a visual allergy-infection dual detection device based on a partitioned nanofiber membrane according to claim 3, characterized in that: The volume ratio of glycerol in the spinning solution 2 in step (2) is 10%.
6. The method for preparing a visual allergy-infection dual detection device based on a partitioned nanofiber membrane according to claim 3, characterized in that: The flow rate ratio of the coaxial electrospinning to the core-shell in step (2) is 1:
7.
7. The method for preparing a visual allergy-infection dual detection device based on a partitioned nanofiber membrane according to claim 3, characterized in that: In step (3), the gelatin and PLGA are in a mass ratio of 3:
7.
8. The method for preparing a visual allergy-infection dual detection device based on a partitioned nanofiber membrane according to claim 3, characterized in that: In the spinning solution 4 of step (3), the concentration of Fe3O4 nanozyme is 1 mg / mL, and the concentration of TMB is 15 mg / mL.
9. Application of a visual allergy-infection dual detection device based on partitioned nanofiber membrane in the preparation of a kit for detecting allergies and / or infections in COPD patients.
10. The use according to claim 9, characterized in that The test sample of the kit is saliva.