Janus membrane for penicillin allergy detection and preparation method thereof

Abstract

The invention relates to the technical field of biological detection, and provides a Janus membrane for penicillin allergy detection. The Janus membrane for penicillin allergy detection is constructed by combining gradient continuous spinning, freezing burying and functionalization treatment, and a blood filtering area and a signal detection area are sequentially arranged from top to bottom. During use, a blood sample firstly enters from the central position of the blood filtering area of the Janus membrane, the blood sample flows into the signal detection area after being filtered, and after antigen penicillin thiazole acid-bovine serum albumin is captured and is combined with an anti-PTAIgE antibody in the blood sample, the colloidal gold labeled anti-human IgE detection probe is used for detection, so that the signal detection area is red. Results show that the kit has the effects of guiding filtered blood samples into a signal detection area, preventing sample leakage, and being high in specificity, convenient to operate, high in detection efficiency, low in false positive rate and visual in result interpretation, and the kit is expected to be widely applied to pre-detection of penicillin allergy conditions in a home first-aid scene due to the fact that the kit is easy and convenient to operate.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a Janus membrane for penicillin allergy detection and its preparation method. Background Technology

[0002] Penicillin allergy is the most common type of drug allergy worldwide, causing tens of thousands of serious adverse reactions each year, posing a significant challenge to its clinical detection. While traditional skin tests are widely used, they have two major drawbacks: limited sensitivity (only 60-70%), easily missing delayed-type allergies; and, more importantly, the potential to induce life-threatening risks such as anaphylactic shock, especially threatening children and critically ill patients. While laboratory serological testing avoids these safety risks, it relies on large equipment and specialized operation, with testing cycles exceeding four hours, making it unsuitable for rapid screening in emergency situations and before surgery. Crucially, penicillin thiazolic acid (PTA), produced from penicillin metabolism in the body, accounts for over 85% of allergen epitopes. Directly targeting the PTA antigen can significantly improve detection specificity, but current rapid detection technologies have not effectively utilized this key target.

[0003] Current mainstream rapid detection carriers (such as nitrocellulose membranes and homogeneous polymer membranes) have fundamental defects due to the bidirectional free diffusion of functional reagents within the fiber network: firstly, reagent cross-contamination leads to a high false positive rate; secondly, insufficient protein immobilization efficiency reduces sensitivity; and thirdly, the sample area and signal area cannot be spatially separated. Therefore, these defects severely limit the reliability of high-precision POCT diagnosis, especially in critical tests where there is zero tolerance for false negatives, such as those involving penicillin allergy, where these defects could lead to missed diagnoses.

[0004] Janus membranes, as advanced fibrous membrane materials with bilateral asymmetric structures (such as chemical composition, wettability, and pore size / porosity gradients) and differential functional properties (such as hydrophilicity / hydrophobicity and charge characteristics), have demonstrated unique advantages in areas such as directed liquid transport and smart dressings. Their core strength lies in their ability to guide liquids or specific substances to achieve unidirectional transport or interfacial selective permeation. However, their application in rapid diagnostic testing, particularly as a core reaction carrier, is still in its early stages, and their enormous potential remains largely untapped. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing a Janus membrane for penicillin allergy detection.

[0006] The present invention utilizes a Janus membrane, which combines gradient continuous spinning, cryogenic embedding and functionalization to construct a Janus membrane for penicillin allergy detection. The Janus membrane of the present invention is configured from top to bottom as a blood filtration zone and a signal detection zone.

[0007] Specifically, the blood filtration zone is a hydrophilic zone, which has three layers of PAN / PVP / TiO2 gradient fiber membranes with different pore sizes, from top to bottom, with pore sizes of 1500nm, 700nm and 100nm respectively.

[0008] Specifically, the signal detection region is a hydrophobic region with a functionalized coating containing hydrophobic siloxane, penicillin thiazolic acid-bovine serum albumin capture antigen, and colloidal gold-labeled anti-human IgE detection probe.

[0009] The mechanism of action of the Janus membrane for penicillin allergy detection in this invention is as follows: When in use, the blood sample first enters from the center of the blood filtration zone of the Janus membrane, and after filtration, it flows to the signal detection zone. After capturing the antigen penicillin thiazolic acid-bovine serum albumin and binding with the anti-PTAIgE antibody in the blood sample, the colloidal gold-labeled anti-human IgE detection probe detects it, causing the signal detection zone to turn red.

[0010] This invention also provides a method for preparing a Janus membrane for penicillin allergy detection, specifically including the following steps:

[0011] (1) Preparation of gradient composite fiber membrane: 0.1 wt% titanium dioxide (TiO2) was dispersed in N,N-dimethylformamide (DMF) and ultrasonically treated for 20 min. Then, 8 wt% polyvinylpyrrolidone (PVP) was added and magnetically stirred for 2 h. Finally, 12 wt% polyacrylonitrile (PAN) was added and magnetically stirred for another 12 h to obtain spinning solution 1. Using this spinning solution, uniaxial electrospinning was performed at 25℃ and 60% relative humidity using a 20G needle. Gradient continuous spinning was carried out by adjusting parameters in three stages. After the solvent in the resulting three-layer gradient composite fiber membrane with different pore sizes was completely evaporated, it was placed in a 50% glutaraldehyde environment for steam crosslinking for 3 h. After drying, a PAN / PVP / TiO2 composite fiber membrane was obtained.

[0012] (2) Preparation of a single-sided trehalose porous gel protective layer: Prepare a 40% (w / v) trehalose aqueous solution, filter it through 0.22 μm and set it aside. Then, spray it on one side of the gradient composite fiber membrane prepared in step (1) to form a thick liquid layer. Immediately freeze dry it to form a porous gel layer that completely covers the single-sided fiber and achieves physical isolation.

[0013] (3) Stepwise vacuum adsorption functionalization of the unprotected side: The single-sided protected sample (protected side facing up) was placed in a vacuum chamber. First, a 1% heptadecafluorodecyltrimethoxysilane / heptane solution was introduced and permeated under a vacuum of -85 kPa for 20 min. After reacting at 25°C for 2 h, the sample was washed with anhydrous ethanol and cured with UV. Then, a pH 7.4 PBS solution containing 10 μg / mL penicillin thiazolic acid-bovine serum albumin (PTA-BSA), 1% trehalose, and 0.5% casein was injected. The sample was adsorbed under a vacuum of -80 kPa for 15 min, fixed at 4°C for 12 h, and then blocked with 2% casein / PBS for 1 h. Finally, OD was introduced. 520 The colloidal gold-anti-human IgE probe solution (containing 0.5% trehalose / 0.2% casein) was adsorbed at -80 kPa for 10 min, fixed at 25℃ in the dark for 1.5 h, gently washed three times with PBS buffer containing 0.05% Tween 20 / 0.3% casein / 5% trehalose, dried under nitrogen to complete the functionalization process, immersed the functionalized sample in 37℃ deionized water and shaken to dissolve the protective layer (5 min, 3 times), rinsed, freeze-dried, and sealed for storage.

[0014] Furthermore, in step (1), the first stage of gradient spinning has a spinning speed of 0.005 mm / s, a voltage of 20 kV, and a receiving distance of 9 cm; the second stage has a spinning speed of 0.003 mm / s, a voltage of 15 kV, and a receiving distance of 11 cm; and the third stage has a spinning speed of 0.001 mm / s, a voltage of 10 kV, and a receiving distance of 13 cm.

[0015] Furthermore, the parameters for pneumatic spraying in step (2) are 0.3 MPa and 12 cm.

[0016] Furthermore, the thickness of the thick liquid layer in step (2) is 80 μm.

[0017] Furthermore, the UV curing parameters in step (3) are 254 nm and 30 min.

[0018] The advantages of this invention are:

[0019] 1. This invention is the first to adopt a gradient continuous spinning technology to composite fiber substrates. A trehalose porous gel protective layer is constructed on one side by cryogenic burial to achieve physical isolation. Subsequently, the non-protected side is functionalized by stepwise vacuum adsorption to construct a Janus membrane based on the detection of penicillin metabolites and capable of penicillin allergy detection.

[0020] 2. The Janus membrane of the present invention is a Janus-structured fiber membrane carrier with precise unilateral functionalization capability. It ensures efficient fixation of PTA antigen and spatially controllable reaction by blocking lateral permeation. For the first time, the Janus membrane is used for pre-detection of penicillin allergy. The top layer is a gradient fiber network that provides mechanical support and guides filtered blood samples into the signal region. The bottom layer is a hydrophobically modified biofunctional complex region that can prevent sample leakage and capture antibodies for signal expression.

[0021] 3. The Janus membrane of the present invention is based on the detection of penicillin metabolites and has the ability to pre-detect penicillin allergy. Experiments have shown that it has the effects of high specificity, convenient operation, high detection efficiency, low false positive rate and visual interpretation of results. Moreover, its simple operation makes it likely to be widely used in the pre-detection of penicillin allergy in home emergency scenarios. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a Janus membrane for penicillin allergy detection according to the present invention.

[0023] Figure 2 This is a characterization of the pore size of the Janus membrane of the present invention.

[0024] Figure 3 This is a characterization of the hydrophilicity and hydrophobicity of the Janus membrane of this invention.

[0025] Figure 4 This is a protein adhesion characterization of the Janus membrane of the present invention.

[0026] Figure 5 The mechanical properties of the Janus membrane of this invention are described.

[0027] Figure 6 The liquid conductivity of the Janus membrane of this invention is as follows: Figure 6 (a) is the wetting ring of the present invention; Figure 6 (b) is the wetting ring of Comparative Example 2; Figure 6 (c) is the statistics of wetted area.

[0028] Figure 7 The specific properties of the Janus membrane of this invention are as follows: Figure 7 (a) is the false positive rate; Figure 7 (b) is the cross-reactivity rate of different antibiotics. Detailed Implementation

[0029] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0030] Example 1: Preparation of Janus membrane for penicillin allergy detection

[0031] (1) Preparation of gradient composite fiber membrane: 0.1 wt% titanium dioxide (TiO2) was dispersed in N,N-dimethylformamide (DMF) and ultrasonically treated for 20 min. Then, 8 wt% polyvinylpyrrolidone (PVP) was added and magnetically stirred for 2 h. Finally, 12 wt% polyacrylonitrile (PAN) was added and magnetically stirred for another 12 h to obtain spinning solution 1. Using this spinning solution, uniaxial electrospinning was performed using a 20G needle at a temperature of 25℃ and a relative humidity of 60%. Gradient continuous spinning was performed by adjusting parameters in three stages: the first stage had a spinning speed of 0.005 mm / s, a voltage of 20 kV, and a receiving distance of 9 cm; the second stage had a spinning speed of 0.003 mm / s, a voltage of 15 kV, and a receiving distance of 11 cm; and the third stage had a spinning speed of 0.001 mm / s, a voltage of 10 kV, and a receiving distance of 13 cm. After the solvent in the obtained three-layer gradient composite fiber membrane with different pore sizes has completely evaporated, it is placed in a 50% glutaraldehyde environment for steam crosslinking for 3 hours, and then dried to obtain a PAN / PVP / TiO2 composite fiber membrane.

[0032] (2) Preparation of a single-sided trehalose porous gel protective layer: Prepare a 40% (w / v) trehalose aqueous solution, filter it through 0.22 μm and set it aside. Pneumatically spray one side of the gradient composite fiber membrane prepared in step (1) (0.3 MPa, 12 cm) to form an 80 μm thick liquid layer. Immediately freeze dry to form a porous gel layer that completely covers the single-sided fiber and achieves physical isolation.

[0033] (3) Stepwise vacuum adsorption functionalization of the unprotected side: The single-sided protected sample (protected side facing up) was placed in a vacuum chamber. First, a 1% heptadecafluorodecyltrimethoxysilane / heptane solution was introduced and permeated under a vacuum of -85 kPa for 20 min. After reacting at 25 °C for 2 h, the sample was washed with anhydrous ethanol and UV cured (254 nm, 30 min). Then, a PBS solution (pH 7.4) containing 10 μg / mL PTA-BSA, 1% trehalose, and 0.5% casein was injected and adsorbed under a vacuum of -80 kPa for 15 min. After fixing at 4 °C for 12 h, the sample was blocked with 2% casein / PBS for 1 h. Finally, OD was introduced. 520The colloidal gold-anti-human IgE probe solution (containing 0.5% trehalose / 0.2% casein) was adsorbed at -80 kPa for 10 min, fixed at 25℃ in the dark for 1.5 h, gently washed three times with PBS buffer containing 0.05% Tween 20 / 0.3% casein / 5% trehalose, dried under nitrogen to complete the functionalization process, and then immersed in 37℃ deionized water with shaking to dissolve the protective layer (5 min, 3 times), rinsed, freeze-dried, and sealed for storage.

[0034] This embodiment prepared a Janus membrane for penicillin allergy detection, the structure of which is as follows: Figure 1 As shown, the blood filtration zone and the signal detection zone are stacked sequentially from top to bottom, with the blood sample being dropped into the center of the blood filtration zone.

[0035] Comparative Example 1: Preparation of Janus membrane for penicillin allergy detection using non-gradient spinning

[0036] The difference between this comparative example and Example 1 is that the PAN / PVP / TiO2 composite fiber membrane was not obtained by a three-stage gradient continuous spinning technique. The spinning parameters were as follows: temperature 25°C, relative humidity 60%, needle size 20G, spinning speed 0.005mm / s, voltage 20kV, and receiving distance 9cm.

[0037] Comparative Example 2: Preparation of a penicillin allergy detection membrane without Janus structure

[0038] The difference between this comparative example and Example 1 is that the gradient composite fiber membrane was not protected with unilateral trehalose porous gel, but was placed in a vacuum chamber and directly subjected to stepwise vacuum adsorption functionalization.

[0039] Comparative Example 3: Preparation of Janus membrane for penicillin allergy detection without TiO2 loading

[0040] The difference between this comparative example and Example 1 is that TiO2 is loaded in the gradient composite fiber membrane.

[0041] Comparative Example 4: Preparation of Janus membrane for penicillin allergy detection with penicillin-BSA as the capture antigen.

[0042] The difference between this comparative example and Example 1 is that PTA-BSA in the non-protected side stepwise vacuum adsorption functionalization treatment is replaced with penicillin-BSA.

[0043] Experimental Example 1: Pore size characterization of the Janus membrane prepared in Example 1

[0044] The microstructure of the Janus membrane was characterized using field emission scanning electron microscopy (SEM). After the samples were fixed with conductive adhesive, their surface and cross-section were observed, and the pore size was statistically analyzed.

[0045] The results are as follows Figure 2 As shown, the pore sizes of the three-stage gradient nanofiber membrane from top to bottom in this Janus membrane are 1500 nm, 700 nm and 100 nm, respectively.

[0046] Experimental Example 2: Characterization of the hydrophilicity and hydrophobicity of the Janus membrane prepared in Example 1

[0047] A water contact angle tester was used to test the surface of the blood filtration zone and signal detection zone of the detection membrane in Example 1 and Comparative Example 2, respectively.

[0048] The results are as follows Figure 3 As shown, the contact angles of the upper surface of the blood filtration zone of the test membranes in Example 1 and Comparative Example 2 are 46° and 117°, respectively, while the contact angles of the lower surface of the signal detection zone of the test membranes in Example 1 and Comparative Example 2 are 142° and 138°, respectively. This is because Comparative Example 2 did not perform unilateral trehalose porous gel protection treatment on the gradient composite fiber membrane before the stepwise vacuum adsorption functionalization treatment. Therefore, it can be concluded that the upper side of the Janus membrane prepared in Example 1, i.e., the blood filtration zone, is hydrophilic, while the lower side of the Janus membrane, i.e., the signal detection zone, is hydrophobic.

[0049] Experimental Example 3: Characterization of protein adhesion in the Janus membrane prepared in Example 1

[0050] Protein adhesion was assessed on the blood filtration and signal detection areas of the detection membranes in Example 1 and Comparative Example 2, respectively. The detection membranes were incubated in 1 mL of fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) solution (2 mg / mL) for 2 hours in the dark, and the sample surfaces were washed three times with PBS solution. The samples were observed and fluorescence images were recorded and calculated using a laser confocal microscope.

[0051] The results are as follows Figure 4As shown, the fluorescence intensity of the upper surface of the blood filtration zone of the test membrane in Example 1 reached 89.6 au, while the fluorescence intensity of Comparative Example 2 was 34.1 au. The fluorescence intensities of the lower surfaces of the signal detection zones of the test membranes in Example 1 and Comparative Example 2 reached 11.4 and 16.3 au, respectively. This is because the main component of the upper surface of the blood filtration zone in Example 1 is a PAN / PVP / TiO2 composite fiber membrane, which has a certain degree of hydrophilicity and can adsorb BSA-FITC solution. In contrast, the upper surface of Comparative Example 2 is hydrophobic because the gradient composite fiber membrane was not protected with a unilateral trehalose porous gel, making it difficult to adsorb BSA-FITC solution. Therefore, the Janus membrane prepared in Example 1 has different protein adhesion characteristics on the surfaces of the blood filtration zone and the signal detection zone, and the signal detection zone has anti-protein adhesion ability.

[0052] Experimental Example 4: Characterization of the mechanical properties of the Janus membrane prepared in Example 1

[0053] The mechanical properties of the Janus films prepared in Example 1 and Comparative Example 3 were tested using a universal tensile testing apparatus. The results are as follows: Figure 5 As shown, Example 1 exhibits a good elongation at break, reaching 207%, while Comparative Example 3 has an elongation at break of only 78%, indicating that Comparative Example 3 has poor mechanical properties. On the other hand, the tensile strength of Example 1 reaches 79 MPa, while the tensile strength of Comparative Example 3 is 28 MPa. This is because TiO2 was added to Example 1 to enhance the mechanical properties of the fiber membrane. Therefore, the Janus membrane prepared in Example 1 has good mechanical properties.

[0054] Experimental Example 5: Characterization of the liquid conductivity of the Janus membrane prepared in Example 1

[0055] The fluid-wicking performance of the dressing was evaluated by the wetting ring test. The detection membranes prepared in Example 1 and Comparative Example 2 with a size of 4cm×4cm were placed flat on the table. 50μL of methylene blue solution was dropped onto the surface of the blood filtration area and the signal detection area, respectively. The wetting and penetration process of the droplets on the different dressing surfaces was recorded by video. The wetting area was counted by ImageJ software.

[0056] The results are as follows Figure 6 As shown in (a), a wetting ring (circled) was clearly observed in the blood filtration zone of Example 1 within 1 second, indicating that the liquid could rapidly penetrate from the blood filtration zone to the signal detection zone. However, as the time increased to 7 seconds, no obvious wetting ring was observed in the signal detection zone. Figure 6 As shown in (b), a wetting ring was only observed in the blood filtration area of ​​Comparative Example 2 after 7 seconds, but no obvious wetting ring was observed in the signal detection area as the time was extended to 7 seconds. Figure 6As shown in (c), the difference in wetting area in the blood filtration zone between Example 1 and Comparative Example 2 is 573 mm and 137 mm, respectively. 2 The signal detection area is 0mm. 2 This indicates that the blood filtration zone of Example 1 has good wettability and fluid conductivity. This is because the surface of the blood filtration zone in Example 1 is a PAN / PVP / TiO2 composite fiber membrane with a certain degree of hydrophilicity, while the surface of the blood filtration zone in Comparative Example 2 has a partially hydrophobic functionalized coating. Therefore, the Janus membrane prepared in Example 1 has fluid conductivity, enabling droplets to quickly penetrate from the blood filtration zone to the signal detection zone.

[0057] Experimental Example 6: Specific characterization of the Janus membrane prepared in Example 1

[0058] (1) Characterization of false positive rate: 100 groups of PTA-free blood samples were tested using the detection membranes prepared in Example 1 and Comparative Examples 1 and 4, and the false positive rate (number of positive results / number of samples tested) was evaluated based on the test results. Results are as follows: Figure 7 As shown in (a), Example 1 has a false positive rate of only 3% for identifying blood samples without PTA, because it can specifically identify PTA for detection; the false positive rates of Comparative Examples 1 and 4 are both greater than 22%, because they are difficult to specifically screen PTA to enter the signal detection layer for detection and are easily interfered with by other impurities.

[0059] (2) Cross-reactivity characterization: To compare the detection performance of the Janus membrane for other antibiotics, 100 groups of blood samples containing penicillin G (10 μg / mL), ceftriaxone (150 μg / mL), and vancomycin (30 μg / mL) were taken from the Janus membrane prepared in Example 1 for testing, and the cross-reactivity rate was evaluated based on the test results. The results are as follows: Figure 7 As shown in (b), the cross-reactivity rates of the three drugs in Example 1 were all less than 5%, because the Janus membrane can specifically recognize PTA.

[0060] The above results all indicate that the false positive rate of the Janus membrane in Example 1 is only 3%, and the probability of identifying a negative sample as a positive sample is extremely low; the cross-reactivity rate for different antibiotics is less than 5%, and the probability of identifying other antibiotics as positive is very low. Therefore, the Janus membrane in Example 1 can accurately and specifically identify PTA.

[0061] Experimental Example 7: Characterization of the accuracy of penicillin allergy predetection using Janus membrane prepared in Example 1

[0062] To compare the accuracy of the Janus membrane prepared in Example 1 with the existing skin test (intradermal penicillin injection) for pre-detection of penicillin allergy, 50 μL of fingertip blood samples were collected from 20 groups of penicillin-allergic patients and healthy individuals. The blood samples were added to the center of the blood filtration zone of the Janus membrane prepared in Example 1. The signal detection zone was then used to detect the blood samples. After the detection signal (red indicates a positive result) stabilized, the average detection time, result, and positive rate were recorded. Subsequently, 0.1 mL of 500 U / mL penicillin G sodium salt was injected intradermally into the aforementioned penicillin-allergic patients and healthy individuals. After the local skin reaction stabilized, a positive / negative result was determined, and the average detection time was recorded.

[0063] The test results are shown in Table 1. Example 1 achieved an 83% positive rate in blood samples from penicillin-allergic patients, while the positive rate in healthy individuals was only 3%, and the testing time was within 10 minutes. This is because the Janus membrane in Example 1 directly and specifically detects the penicillin metabolite PTA, resulting in higher accuracy and efficiency. In contrast, the existing skin test method showed a 70% positive rate in penicillin-allergic patients and a 10% positive rate in healthy individuals, with a testing time of approximately 30 minutes. This is because the skin test method is based on skin condition and targets the penicillin drug itself; individual responses can vary, leading to a higher false positive rate. In summary, compared to the existing skin test method, the Janus membrane prepared in Example 1 has higher accuracy, shorter testing time, and is more convenient to operate.

[0064] Table 1. Positive rates of penicillin allergy pre-detection in Janus membranes and skin tests prepared in Example 1.

[0065]

[0066] In summary, this invention utilizes the Janus membrane, which detects penicillin metabolites, to pre-detect penicillin allergy. Experiments have demonstrated that it exhibits high specificity, ease of operation, high detection efficiency, low false positive rate, and visual interpretation of results. Furthermore, its simple operation makes it promising for widespread application in pre-detection of penicillin allergy in home emergency scenarios.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A Janus membrane for penicillin allergy detection, characterized in that, The Janus membrane has an asymmetric structure, and from top to bottom, it is configured as a blood filtration zone and a signal detection zone. The blood filtration zone is a hydrophilic zone, and it is equipped with three layers of PAN / PVP / TiO2 gradient fiber membranes with different pore sizes, from top to bottom, the pore sizes are 1500nm, 700nm and 100nm respectively; The signal detection region is a hydrophobic region with a functionalized coating containing hydrophobic siloxane, penicillin thiazolic acid-bovine serum albumin capture antigen, and colloidal gold-labeled anti-human IgE detection probe.

2. The Janus membrane for penicillin allergy detection according to claim 1, characterized in that, The contact angle of the blood filtration zone is 46°, and the contact angle of the signal detection zone is 142°.

3. A method for preparing a Janus membrane as described in any one of claims 1-2, characterized in that, The preparation method includes the following steps: (1) Preparation of gradient composite fiber membrane: 0.1 wt% titanium dioxide was dispersed in N,N-dimethylformamide and ultrasonically treated for 20 min. Then, 8 wt% polyvinylpyrrolidone was added and magnetically stirred for 2 h. Finally, 12 wt% polyacrylonitrile was added and magnetically stirred for another 12 h to obtain spinning solution 1. Using this spinning solution, uniaxial electrospinning was performed at 25℃ and 60% relative humidity using a 20G needle. Gradient continuous spinning was carried out by adjusting the parameters in three stages. After the solvent in the resulting three-layer gradient composite fiber membrane with different pore sizes was completely evaporated, it was placed in a 50% glutaraldehyde environment for steam crosslinking for 3 h. After drying, a PAN / PVP / TiO2 composite fiber membrane was obtained. (2) Preparation of a single-sided trehalose porous gel protective layer: Prepare a trehalose aqueous solution with a mass-volume ratio of 40%, filter it through 0.22μm and set it aside. Then, spray one side of the gradient composite fiber membrane prepared in step (1) with air to form a thick liquid layer. Immediately freeze dry to form a porous gel layer that completely covers the single-sided fiber and achieves physical isolation. (3) Stepwise vacuum adsorption functionalization of the unprotected side: The single-sided protected sample with the protective side facing up was placed in a vacuum chamber. First, a 1% heptadecafluorodecyltrimethoxysilane / heptane solution was introduced and permeated under a vacuum of -85 kPa for 20 min. After reacting at 25°C for 2 h, the sample was washed with anhydrous ethanol and cured with UV. Then, a pH 7.4 PBS solution containing 10 μg / mL penicillin thiazolic acid-bovine serum albumin, 1% trehalose, and 0.5% casein was injected and adsorbed under a vacuum of -80 kPa for 15 min. After fixing at 4°C for 12 h, the sample was blocked with 2% casein / PBS for 1 h. Finally, OD was introduced. 520 The colloidal gold-anti-human IgE probe solution was adsorbed at -80 kPa for 10 min, fixed at 25°C in the dark for 1.5 h, gently washed three times with PBS buffer containing 0.05% Tween 20 / 0.3% casein / 5% trehalose, dried under nitrogen to complete the functionalization process, immersed the functionalized sample in 37°C deionized water and shaken to dissolve the protective layer, rinsed three times, then freeze-dried and sealed for storage.

4. The method for preparing the Janus membrane according to claim 3, characterized in that, In step (1), the first stage of gradient spinning has a spinning speed of 0.005 mm / s, a voltage of 20 kV, and a receiving distance of 9 cm; the second stage has a spinning speed of 0.003 mm / s, a voltage of 15 kV, and a receiving distance of 11 cm; and the third stage has a spinning speed of 0.001 mm / s, a voltage of 10 kV, and a receiving distance of 13 cm.

5. The method for preparing the Janus membrane according to claim 3, characterized in that, The parameters for pneumatic spraying in step (2) are 0.3 MPa and 12 cm.

6. The method for preparing the Janus membrane according to claim 3, characterized in that, The thickness of the thick liquid layer in step (2) is 80 μm.

7. The method for preparing the Janus membrane according to claim 3, characterized in that, The UV curing parameters in step (3) are 254 nm and 30 min.

8. The use of the Janus membrane as described in claim 1 or 2 in the preparation of products for penicillin allergy testing, characterized in that, The product is configured to test blood samples.

9. The use according to claim 8, characterized in that, The allergy marker targeted in the product testing is anti-PTAIgE antibody.

10. The use according to claim 8, characterized in that, The products include chips, sensors, wearable devices, test strips, patches, and reagent kits.

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