A hetero-phase immunoassay device based on orthogonal orientation type nanofiber

Abstract

This invention relates to a heterogeneous immunoassay device based on orthogonally oriented nanofibers, comprising an orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a micro-analytical membrane filter, and a micro-injection pump. The component is assembled onto the micro-injection pump. The sterile screw-in syringe is connected to the micro-analytical membrane filter via a threaded valve. The micro-analytical membrane filter has a clamp composed of two O-ring silicone seals that holds the orthogonally oriented nanofiber membrane within it. The outer diameter of the O-ring silicone seals is equal to the diameter of the orthogonally oriented nanofiber membrane. The orthogonally oriented nanofiber membrane is composed of multiple layers of fibers, with all fibers in each layer oriented in a specific direction. The fiber orientations in any two adjacent layers are perpendicular to each other, resulting in a uniform pore structure, high porosity, and strong protein adsorption. Using the device of this invention for heterogeneous immunoassay significantly improves the detection limit, detection sensitivity, and detection accuracy.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay materials technology, and relates to a heterogeneous immunoassay device based on orthogonally oriented nanofibers. Background Technology

[0002] Heterogeneous immunoassay involves immobilizing antibodies (or antigens) on the surface of a solid-phase support. The antigen (or antibody) binds to the support through a specific immune reaction, and the antigen-antibody complex can be separated from the free antigen or antibody through washing. Simultaneously, when the target molecule (the substance to be detected, such as nucleic acids, infectious viruses, bacteria, cardiac markers, drugs, proteins, and small molecule compounds) is captured by the antibody or other recognition reagents, a measurable color signal change occurs on the solid-phase support. This is mainly achieved through the colorimetric reaction of the enzyme with the substrate or the aggregation of the recognition element. Detection methods based on this colorimetric mechanism include colloidal gold lateral flow immunochromatography, photoluminescent enzyme-linked immunosorbent assay (ELISA), and fluorescence immunoassay. Heterogeneous immunoassay can enrich antigens from dilute solutions onto the surface of a solid-phase support, exhibiting higher sensitivity compared to homogeneous immunoassay where the antigen and antibody react in the liquid phase.

[0003] In the past, the solid supports used in heterogeneous immunoassays were mostly polystyrene microplates, cellulose fiber filter paper, and nitrocellulose (NC) membranes.

[0004] When polystyrene microplates are used as solid-phase carriers, the amount of biological reagent used is about 30-50 μL / well / test. The detection process is time-consuming (in order to ensure sufficient diffusion of proteins in the solution, the incubation and blocking time of biomolecules is about 1 hour, and the entire detection process usually takes more than 3 hours). At the same time, an expensive microplate reader is also required as a signal analysis device.

[0005] Using cellulose fiber filter paper as a solid-phase carrier requires cumbersome pretreatment steps. First, a hydrophobic isolation zone is created on the filter paper to control the flow of the test solution within a defined area in the desired direction. Then, biological reagents are modified into the hydrophilic region. However, when modifying the surface of the paper-based carrier with antibodies (antigens), it mainly relies on non-specific adsorption, i.e., the negative charge generated by the swelling and weak ionization of amorphous cellulose and hemicellulose in water binds to positively charged particles. In this case, neutral or negatively charged particles are difficult to be stably adsorbed, and other molecules with weaker binding forces are easily lost during washing. This makes the detection limit and sensitivity of paper-based immunoassays inferior to traditional polystyrene microplates. Moreover, since the binding capacity of cellulose filter paper to different proteins such as antigens, antibodies, and blocking reagents (bovine serum albumin) is not significantly different, the blocking effect is not ideal during assay, resulting in a high background signal and a low signal-to-noise ratio.

[0006] The production process of NC membranes is complex and poses a high risk of fire. The prepared NC membranes have weak light transmittance, poor mechanical properties, low specific surface area, and uneven pore size, which affects the uniformity of biomolecule distribution on them and the accessibility of target molecules (the binding sites on the NC membrane cannot be exposed, making it impossible for most biomolecules to bind to the target molecules). This results in the detection results generally having disadvantages such as low sensitivity, large signal fluctuations, and high background signal.

[0007] Compared to traditional solid-phase carrier materials, electrospun nanofiber membranes have advantages such as simple preparation process, wide range of raw materials, and easy functionalization modification. In particular, they have higher porosity (up to 80% or more), adjustable pore size and distribution, and larger specific surface area, which is three orders of magnitude larger than that of polystyrene microplates. Theoretically, this can immobilize more trapped molecules, increase the reaction probability between target molecules and the carrier, and thus improve sensitivity. Therefore, some studies have attempted to use nanofibers as solid-phase carriers for lateral flow immunochromatographic test strips. For example, Reference 1 (Anal Bioanal Chem 2014, 406, 3297-3304) developed a lateral flow immunochromatographic test strip based on polylactic acid / polyethylene glycol nanofibers, which achieved E. coli detection performance comparable to NC membranes. Reference 2 (Microchimica Acta, 2020, 187, 644.) constructed a lateral flow immunochromatographic test strip for human chorionic gonadotropin (hCG) using nitrocellulose nanofiber membranes as a solid-phase support. The detection limit was 10 mIU / mL, which is 2.5 times that of commercially available test strips. However, previous studies have also shown that when nanofiber membranes are used as solid-phase supports, the sensitivity of the detection system does not exhibit the enhancement effect that should be brought about by the high specific surface area, and there is no significant advantage compared with traditional supports.

[0008] Therefore, it is of great significance to study a heterogeneous immunoassay device that significantly improves detection limit, detection sensitivity, and detection accuracy. Summary of the Invention

[0009] To address the problems existing in the prior art, the present invention provides a heterogeneous immunoassay device based on orthogonally oriented nanofibers;

[0010] To achieve the above objectives, the present invention adopts the following solution:

[0011] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes an orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0012] A sterile screw-in syringe is connected to a microanalytical membrane filter via a threaded valve. The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that hold the orthogonally oriented nanofiber membrane within it, thus sealing the periphery of the orthogonally oriented nanofiber membrane and allowing liquid to flow through the membrane without leakage. The orthogonally oriented nanofiber membrane has a uniform pore structure, requiring O-ring silicone seals on both the upper and lower surfaces of the membrane to maintain a sealed liquid channel. The flow rate through the nanofiber membrane pores is precisely controlled by the sterile screw-in syringe to ensure a more complete and stable immune response, thereby achieving high-sensitivity detection. The assembly consisting of the orthogonally oriented nanofiber membrane, the sterile screw-in syringe, and the microanalytical membrane filter is assembled onto a micro-injection pump to control the flow rate and propel the liquid through the membrane. The orthogonally oriented nanofiber membrane is circular in shape, and the outer diameter of the O-ring silicone seal is equal to the diameter of the orthogonally oriented nanofiber membrane.

[0013] The orthogonal orientation nanofiber membrane is a functionalized orthogonal orientation nanofiber membrane, and the functional groups on the orthogonal orientation nanofiber membrane can covalently couple with protein molecules;

[0014] Orthogonally oriented nanofiber membranes are composed of multiple layers of fibers, with all fibers in each layer arranged in an orthogonal orientation, and the fiber orientations of any two adjacent layers being perpendicular to each other.

[0015] As a preferred technical solution:

[0016] The heterogeneous immunoassay device based on orthogonally oriented nanofibers, as described above, has 100–300 layers of oriented nanofibers in the orthogonally oriented nanofiber membrane, with each layer having a unidirectional orientation parameter S > 0.97 (S = 2cos). 2 θ-1, where θ is the angle between unidirectionally aligned nanofibers. If they are parallel, then θ is 0 and the unidirectional orientation parameter is 1 (Separation and Purification Technology, 2021, 260, 118246).

[0017] The heterogeneous immunoassay device based on orthogonally oriented nanofibers described above has an orthogonally oriented nanofiber membrane that is a circular membrane with a diameter of 13–25 mm; the thickness of the orthogonally oriented nanofiber membrane is 0.1–0.3 μm, the average pore size is 0.5–4 μm, the porosity is >90%, and the permeability is >80%.

[0018] As described above, in a heterogeneous immunoassay device based on orthogonally oriented nanofibers, the functional groups on the orthogonally oriented nanofiber membrane are carboxyl, aldehyde, amino, sulfonic acid, secondary amine, tertiary amine, NHS ester, maleimide, carbodiimide, and imine ester.

[0019] The heterogeneous immunoassay device based on orthogonally oriented nanofibers described above is obtained by preparing a nanofiber membrane through electrospinning of a spinning solution and then performing functionalization treatment. The receiving method is high-speed roller receiving, auxiliary electrode receiving, or additional magnetic field receiving. The first layer of fibers is arranged at 0° as a reference, the second layer of fibers is arranged at 90°, and the 0° and 90° layers are deposited sequentially.

[0020] As described above, in a heterogeneous immunoassay device based on orthogonally oriented nanofibers, the spinning solution is a homogeneous solution obtained by dissolving a polymer in a solvent. The polymer is one or more of the following: polyvinylidene fluoride, poly(vinyl alcohol-co-ethylene), polycarbonate, polyvinyl acetate, polyethylene, polystyrene, polylactic acid, nylon 6, polyethylene oxide, polyacrylonitrile, polyvinyl alcohol, polyethylene glycol, polyaniline, polyurethane, polyhydroxybutyrate, polycaprolactone, polyethersulfone, chitosan, collagen, and cellulose. The solvent is methanol, ethanol, isopropanol, hexafluoroisopropanol, tetraethylene glycol, triethylene glycol, formic acid, etc. One or more of the following: acid, phenol, methyl formate, ethyl formate, propyl formate, methyl n-acetone, diisopropanone, diisobutyl ketone, acetone, hexafluoroacetone, chloromethane, chloroethane, dichloromethane, chloroform, p-chlorotoluene, 1,1-dichloroethane, 1,2-dichloroethane, dichloropropane, trichloromethane, trichloroethane, dibromoethane, dibromopropane, bromomethane, bromoethane, bromopropane, acetic acid, benzene, toluene, hexane, cyclohexane, cyclohexanone, cyclopentane, o-xylene, p-xylene, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, pyridine, and water.

[0021] As described above, in a heterogeneous immunoassay device based on orthogonally oriented nanofibers, the concentration of the spinning solution is 5–30 wt%.

[0022] The heterogeneous immunoassay device based on orthogonally oriented nanofibers described above has the following electrospinning process parameters: spinning voltage 8–30 kV, spinning solution flow rate 0.5–5 mL / h, humidity 30–70%, temperature 25–40 °C, distance between spinneret and receiving device 5–30 cm, and receiving speed 1000–3000 rpm.

[0023] As described above, the heterogeneous immunoassay device based on orthogonally oriented nanofibers has a deposition time of 2–5 minutes for each layer of fibers.

[0024] As described above, a heterogeneous immunoassay device based on orthogonally oriented nanofibers includes an orthogonally oriented nanofiber membrane immobilized with a first recognition substance that reacts with target substances in the test solution. The immobilized region consists of one or more dots or stripes, the specific number and arrangement of which can be flexibly selected according to the number of target substances. The method for immobilizing the first recognition substance on the nanofiber membrane can be appropriately selected based on the polymer material used for the nanofibers. It can be achieved through chemical bonding between functional groups, coupling of biotin and avidin, or hydrophobic interaction, but is not limited to these forms. The specific immobilization method can be appropriately selected by those skilled in the art.

[0025] The heterogeneous immunoassay device based on orthogonally oriented nanofibers described above, compared to existing heterogeneous immunoassay techniques including lateral flow chromatography and speckled gold immunoassay, commonly used indicators for evaluating immunoassay techniques include limit of detection, sensitivity, specificity, false positive rate, and false negative rate. Using the aforementioned heterogeneous immunoassay device based on orthogonally oriented nanofibers, the detection limit is 0.1–100 ng / mL, representing a 10–1000-fold improvement over existing heterogeneous immunoassay techniques, increasing the detection limit from nM to pM level. The sensitivity is above 90%, and both the false positive and false negative rates are below 5%.

[0026] The specific steps for assembling the heterogeneous immunoassay device based on orthogonal orientation nanofibers and performing heterogeneous immunoassay using the orthogonal orientation nanofibers-based heterogeneous immunoassay device are as follows:

[0027] Step 1: Prepare orthogonally oriented nanofiber membranes and perform functionalization treatment;

[0028] Step 2: Immobilize a first identification substance that can react with the target substance in the test solution in a certain area of ​​the above orthogonal oriented nanofiber membrane. The immobilization area is one or more dots or strips. The specific number of areas can be flexibly selected and arranged according to the number of targets to be tested.

[0029] Step 3: Cut the nanofiber membrane into discs and assemble them into the heterogeneous immunoassay analyzer;

[0030] Step 4: After the colorimetric label binds to the second recognition substance, it is added to the test solution and incubated. Then, the test solution is transferred to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0031] Step 5: Select the pore size of the solid support according to the size of the second identified substance being labeled. The selection principle is that the complex of the target substance and the second identified substance being labeled should not pass through, while the second identified substance that is not bound to the target substance can pass through.

[0032] Step 6: Control the flow rate of the sterile screw-type syringe to 0.5-5 mL / h so that the test solution can complete the immune reaction through the solid-phase carrier. The reaction time varies depending on the affinity of the recognition substance, the specific surface area of ​​the fiber membrane, and the pore structure, and is usually less than 10 minutes, preferably less than 3 minutes. After washing, high-sensitivity detection is achieved through signal acquisition.

[0033] The target substance for detection in heterogeneous immunoassay devices based on orthogonally oriented nanofibers is not specifically limited. It can be used for the immunoassay of nucleic acids, infectious viruses, bacteria, cardiac markers, drugs, proteins, and small molecule compounds, but is not limited to these. It can be widely used in general immunoassay methods in this field for human, animal and plant medical clinical testing, as well as environmental monitoring and food safety testing. The first and second recognition substances are generally antigens, antibodies, or aptamers (aptamers are single strands of nucleic acids). Both the first and second recognition substances can specifically bind to the target substance. As long as they can simultaneously bind to the target substance, they can be the same or different. The colorimetric labels include colloidal gold, colloidal selenium, carbon nanotubes, carbon nanoparticles, latex microspheres, cellulose microspheres, magnetic nanoparticles, or quantum dots.

[0034] The principle of this invention is:

[0035] After analyzing the principle of lateral flow immunochromatographic test strips and the structural characteristics of nanofiber membranes, it is known that when nanofibers are applied to lateral flow immunoassay, the analyte flows laterally based on chromatography, essentially utilizing the capillary action of the horizontal pores in the nanofiber membrane. Electrospun nanofiber membranes are formed by the random, layer-by-layer stacking of horizontal nanofiber networks; therefore, the pore structure characteristics in the horizontal and vertical directions are completely different. The conventionally described pore structure tunability mainly refers to the vertical direction of the membrane. In the horizontal direction, the pore size is small and the structure is difficult to control, making it difficult to alter the movement path of the analyte solution through pore structure regulation. The diffusion rate of the target substance migrating to the active center within the horizontal pores is inevitably hindered. However, in the vertical direction of the nanofiber membrane, it plays a role in immobilizing recognition molecules. Because the pore structure of the top layer of fiber mesh is blocked by the subsequent stacking of fiber mesh, the effective pore size for mass transfer in the fiber membrane is significantly smaller than the measured pore size. This hinders the diffusion of biorecognition molecules, causing most to bind only to the outer layer of the nanofiber membrane. Increased ligand density on the membrane surface leads to a significant spatial congestion effect and low binding efficiency of the target substance. Furthermore, among the three pore structures formed by the disordered stacking of fibers in the vertical direction, only through-pores facilitate protein molecule diffusion; blind and closed pores do not contribute to the diffusion or partitioning of biomolecules into the membrane. Therefore, as shown in reference 3 (ACS Applied Polymer Materials 2021 3, 1618-1627), the structural advantage of high porosity does not play a positive role in the mass transfer and diffusion of recognition molecules. In conclusion, previous solid-phase supports cannot be considered ideal materials for heterogeneous immunoassay.

[0036] This invention relates to both the optimization of the bulk structure of nanofiber solid-phase carriers and the design of a heterogeneous immunoassay device with a specially matched nanofiber structure. This device is a tool for the quantitative or qualitative detection of target substances in the test solution, thereby fully utilizing the structural properties of nanofibers, such as large specific surface area and high porosity, to significantly improve sensitivity. This is of great significance for practical applications in the field of in vitro point-of-care diagnostics.

[0037] This invention discloses a heterogeneous immunoassay device based on orthogonally oriented nanofibers. The orthogonally oriented nanofiber membrane serves as the solid-phase support. The membrane is prepared by alternately depositing nanofibers with a unidirectional ordered structure along two orthogonal directions. Using the first fiber layer as a reference (0°), the second fiber layer forms a 90° angle with the first, and deposition is performed sequentially at 0° and 90° angles. Unlike porous membranes composed of conventional disordered nanofibers, the spacing between fibers in the vertical direction of the orthogonally oriented nanofibers is regular, reducing the probability of pore blockage, resulting in high porosity, improved and adjustable pore size uniformity. When a substance capable of binding to a target substance (antibody is used as an example below) is immobilized on this support, it facilitates antibody diffusion or distribution into the membrane, improving the binding efficiency of the target substance.

[0038] The specific reasons for improving the binding efficiency of the target substance are as follows: The nanofiber membrane is a three-dimensional structure with layers stacked. When the first recognition substance is coated on the surface, the high porosity of the fiber membrane allows the recognition molecules to be distributed vertically on the fiber membrane rather than just on the membrane surface. This avoids the spatial crowding effect caused by the recognition substance being distributed only on the surface. The high porosity of the fiber membrane can naturally fix more recognition substances, and the uniformity of the pore size structure can maintain the stability of the sample flow. This allows the target molecules in the sample to react stably with the recognition substances on the fiber membrane, thereby improving the binding efficiency.

[0039] Furthermore, the heterogeneous immunoassay device of the present invention with a special matching nanofiber solid-phase carrier requires the nanofibers to be clamped in the device. The target substance sample is vertically injected into the solid-phase carrier at a certain flow rate. The filterability of the high-porosity nanofiber membrane is utilized to play the role of affinity chromatography concentration, so that the target substance in the test solution is in close contact with the antibody on the membrane, the color signal is more concentrated, and the signal can be measured after washing, thus achieving the purpose of high-sensitivity detection.

[0040] Beneficial effects

[0041] (1) Using orthogonally oriented nanofiber membranes as solid-phase carriers for heterogeneous immunoassay can effectively solve the problem of significant spatial crowding effect of recognition molecules immobilized on the membrane and low binding rate of target substances caused by the blockage of interlayer mesh structure and low porosity of traditional nanofiber membranes when used as carriers. By forming periodic controlled deposition of unidirectional ordered nanofibers along two orthogonal directions, the pore size uniformity and porosity can be effectively improved without losing the high specific surface area of ​​the nanofiber membrane. The pore structure characteristics can be flexibly controlled by the single-layer deposition time and the number of deposition layers, which can improve the binding kinetics of heterogeneous immunoassay and thus improve the reaction efficiency of target substances with solid-phase carriers.

[0042] (2) By designing a heterogeneous immunoassay device with a specially matched orthogonal-oriented nanofiber membrane, the test liquid is vertically injected into the solid support at a certain flow rate. This solves the problem of uncontrolled mass transfer and reaction process caused by the transverse flow of the target substance when the nanofiber membrane is used as a solid support. The orthogonal-oriented nanofiber membrane in this device plays a role in the concentration of affinity chromatography, allowing the target substance in the test liquid to come into close contact with the antibody on the membrane, resulting in a more concentrated color signal. At the same time, due to the high mechanical strength and adjustable pore size and porosity of the orthogonal-oriented nanofiber membrane, it can also be filtered in this device to eliminate interference signals and further improve the accuracy of detection. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of a heterogeneous immunoassay device using orthogonally oriented nanofibers.

[0044] Figure 2 This is a partial schematic diagram of a heterogeneous immunoassay device using orthogonally oriented nanofibers;

[0045] Among them, 1-micro-injection pump, 2-sterile screw-in syringe, 3-micro-analytical membrane filter, 4-O-ring silicone seal, 5-orthogonal oriented nanofiber membrane. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0047] The test methods / calculation methods used in the embodiments are as follows:

[0048] (1) Unidirectional orientation parameter S: According to the literature (Separation and Purification Technology[J], 2021, 260, 118246), the calculation formula is: S=2cos 2 θ-1; The specific measurement process of this invention is as follows: First, the morphology of the uppermost fiber membrane is observed using a scanning electron microscope. Then, the orientation angle of the fiber is measured from the scanning electron microscope image using Image Pro-Plus software. The angle between a single fiber and the vertical direction is the fiber orientation angle θ. Each time, 50 fibers are measured. Based on the frequency distribution of the fiber orientation angle, the average orientation angle of a layer of fibers can be obtained. Substituting this into the calculation formula gives the unidirectional orientation parameter of the fiber membrane.

[0049] (2) Porosity: According to the literature (Journal of the Korean Wood Science and Technology[J],2019,47(1):8-20), the formula for calculating porosity ε is: Among them, V 实 V represents the actual measured volume (the ratio of sample mass to the standard density of the polymer). 测 The volume obtained using a gas specific gravity bottle;

[0050] (3) Porosity: The nanofiber membrane sample was immersed in a Galwick solution (a perfluoroalkane liquid compound) in a beaker and placed in a vacuum chamber. At this point, liquid could flow into both the through-pores and blind pores in the sample. Then, pressure was applied to the membrane, which was completely permeated by the wetting solution. As the pressure increased, the wetted liquid was slowly squeezed out; however, because one end of each blind pore was closed, the liquid inside the blind pore was not squeezed out. The weight of the liquid impregnating the blind pores was m. 盲 It is calculated by subtracting the initial sample weight m0 from the weight m1 of the pressed sample, using the known liquid density ρ. 液 This allows us to obtain the volume and porosity of blind pores. Through porosity is the difference between porosity and blind porosity. Among them, V 实 V is the actual measured volume. 测 This refers to the volume measured using a gas specific gravity bottle.

[0051] (4) Limit of Detection (LOD): The limit of detection of the test strip was tested according to the method in patent CN201520796221.4 (A Fluorescent Immunochromatographic Test Strip). The LOD was determined using the standard deviation method, LOD = 3σ / S, where σ is the standard deviation of the y-intercept of the standard curve, and S represents the slope of the standard curve. Error bars were obtained by performing three parallel experiments to confirm the results.

[0052] Establishing the standard curve: First, the color intensity is quantified using a smartphone, digital camera, or desktop scanner to obtain the photoelectric signal value (which can be the R, G, or B value in RGB, or a grayscale value). The target substance concentration is used as the x-axis, and the photoelectric signal value is used as the y-axis. Based on the linear relationship between the target substance concentration and the photoelectric signal value within a certain range, a fitting standard curve y = Sx + b is established, where x is between x0 and x1 (x0 is the minimum target substance concentration in the standard curve, and x1 is the maximum target substance concentration in the standard curve), S is the slope of the standard curve, and b is the intercept of the standard curve. The standard deviation σ of the y-intercept is obtained through three parallel experiments, and the detection limit LOD = 3σ / S.

[0053] (5) Sensitivity: According to GB / T 40369-2021, under the test conditions, the percentage of positive samples that detected positive results out of the total number of positive samples, and the number of test samples is 120.

[0054] (6) False positive rate: The percentage of positive results detected in negative samples under the detection limit level according to GB / T 40369-2021, with 120 samples tested.

[0055] (7) False negative rate: The percentage of positive samples that are detected as negative when the detection limit level is set under the test conditions according to GB / T 40369-2021, with 120 samples tested.

[0056] (8) Average pore size: The average pore size of the fiber membrane is measured using a capillary flow pore size analyzer. The testing principle is as follows: Under a certain pressure, by calculating the gas flow changes through the dry and wet samples, the computer automatically integrates the pore size and distribution curve of the sample, thereby obtaining the average pore size. During the testing process, the sample is completely wetted with Galwick wetting solution and then placed in the test chamber. Before the test, the pore size of the sample should be estimated in advance to select the test range. The specific process is as follows: When selecting the pore size distribution of nanofibers using a capillary flow pore size analyzer, you can first select "1μm and above" in the computer for testing. If most of the pore size distributions exported by the computer are below 1μm, then you need to select the pore size range "0.1~1μm" for measurement. Click start in the computer, and the pore size distribution of the sample can be measured after a certain period of time.

[0057] Polymer manufacturers, molecular weight / purity

[0058]

[0059]

[0060] Example 1

[0061] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0062] (1) Preparation of raw materials:

[0063] Polymer: Polyvinylidene fluoride;

[0064] Solvent: Acetone;

[0065] (2) A homogeneous solution obtained by dissolving polyvinylidene fluoride in acetone, i.e. a spinning solution with a concentration of 5 wt%;

[0066] (3) After preparing a nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonally oriented nanofiber membrane grafted with aldehyde groups.

[0067] The electrospinning process parameters are as follows: spinning voltage 8kV, spinning solution flow rate 0.5mL / h, humidity 30%, temperature 25℃, distance between spinneret and receiving device 5cm, receiving speed 1000rpm, receiving method is high-speed roller receiving, with the first layer of fiber arrangement direction as 0° as a reference, the second layer of fiber arrangement direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time of each fiber layer is 2min.

[0068] The orthogonally oriented nanofiber membrane has 100 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.973. The orthogonally oriented nanofiber membrane is a disc with a diameter of 13 mm, a thickness of 0.1 μm, an average pore size of 0.5 μm, a porosity of 90.2%, and a through-porosity of 80.2%.

[0069] Example 2

[0070] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0071] (1) Preparation of raw materials:

[0072] Polymer: Poly(vinyl alcohol-co-ethylene);

[0073] Solvent: Isopropanol;

[0074] (2) A homogeneous solution obtained by dissolving poly(vinyl alcohol-co-ethylene) in isopropanol, i.e. a spinning solution with a concentration of 7.5 wt%;

[0075] (3) After preparing nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonal oriented nanofiber membrane grafted with NHS ester groups.

[0076] The electrospinning process parameters are as follows: spinning voltage 10kV, spinning solution flow rate 1mL / h, humidity 35%, temperature 27℃, distance between spinneret and receiving device 10cm, receiving speed 1200rpm, receiving method is auxiliary electrode receiving, with the first layer of fiber arrangement direction as 0° as a reference, the second layer of fiber arrangement direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time of each fiber layer is 2.4min.

[0077] The orthogonally oriented nanofiber membrane has 120 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.976. The orthogonally oriented nanofiber membrane is a disc with a diameter of 13 mm, a thickness of 0.12 μm, an average pore size of 0.55 μm, a porosity of 90.3%, and a through-porosity of 82%.

[0078] Example 3

[0079] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0080] (1) Preparation of raw materials:

[0081] Polymer: Polystyrene;

[0082] Solvent: N,N-dimethylformamide;

[0083] (2) Dissolve polystyrene in N,N-dimethylformamide to obtain a homogeneous solution, i.e. a spinning solution with a concentration of 10 wt%;

[0084] (3) After preparing a nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonal oriented nanofiber membrane grafted with sulfonic acid groups.

[0085] The electrospinning process parameters are as follows: spinning voltage 12kV, spinning solution flow rate 1.5mL / h, humidity 40%, temperature 30℃, distance between spinneret and receiving device 15cm, receiving speed 1400rpm, receiving method is additional magnetic field reception, with the first layer of fiber alignment direction as 0° as a reference, the second layer of fiber alignment direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time for each layer of fiber is 2.8min.

[0086] The orthogonally oriented nanofiber membrane has 160 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.978. The orthogonally oriented nanofiber membrane is a disc with a diameter of 14 mm, a thickness of 0.16 μm, an average pore size of 0.6 μm, a porosity of 90.6%, and a through-porosity of 82.4%.

[0087] Example 4

[0088] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0089] (1) Preparation of raw materials:

[0090] Polymer: Polycaprolactone;

[0091] Solvent: chloroform;

[0092] (2) Dissolve polycaprolactone in chloroform to obtain a homogeneous solution, i.e. a spinning solution with a concentration of 12.5 wt%.

[0093] (3) After preparing nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonal oriented nanofiber membrane grafted with NHS ester groups.

[0094] The electrospinning process parameters are as follows: spinning voltage 14kV, spinning solution flow rate 2mL / h, humidity 45%, temperature 32℃, distance between spinneret and receiving device 20cm, receiving speed 1600rpm, receiving method is high-speed roller receiving, with the first layer of fiber arrangement direction as 0° as a reference, the second layer of fiber arrangement direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time of each fiber layer is 3min.

[0095] The orthogonally oriented nanofiber membrane has 180 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.981. The orthogonally oriented nanofiber membrane is a disc with a diameter of 15 mm, a thickness of 0.18 μm, an average pore size of 0.8 μm, a porosity of 90.8%, and a through-porosity of 83%.

[0096] Example 5

[0097] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0098] (1) Preparation of raw materials:

[0099] Polymer: Polyvinyl alcohol;

[0100] Solvent: Dimethyl sulfoxide;

[0101] (2) Dissolve polyvinyl alcohol in dimethyl sulfoxide to obtain a homogeneous solution, i.e. a spinning solution with a concentration of 15 wt%.

[0102] (3) After preparing a nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonally oriented nanofiber membrane grafted with aldehyde groups.

[0103] The electrospinning process parameters are as follows: spinning voltage 16kV, spinning solution flow rate 2.5mL / h, humidity 50%, temperature 35℃, distance between spinneret and receiving device 25cm, receiving speed 1800rpm, receiving method is auxiliary electrode receiving, with the first layer of fiber arrangement direction as 0° as a reference, the second layer of fiber arrangement direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time of each fiber layer is 3.2min.

[0104] The orthogonally oriented nanofiber membrane has 200 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.983. The orthogonally oriented nanofiber membrane is a disc with a diameter of 17 mm, a thickness of 0.2 μm, an average pore size of 1.2 μm, a porosity of 91%, and a through-porosity of 84.5%.

[0105] Example 6

[0106] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0107] (1) Preparation of raw materials:

[0108] Polymer: Polyethersulfone;

[0109] Solvent: N,N-dimethylformamide;

[0110] (2) A homogeneous solution obtained by dissolving polyethersulfone in N,N-dimethylformamide, i.e. a spinning solution with a concentration of 17.5 wt%;

[0111] (3) After preparing a nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonal oriented nanofiber membrane grafted with carboxyl groups.

[0112] The electrospinning process parameters are as follows: spinning voltage 18kV, spinning solution flow rate 3mL / h, humidity 55%, temperature 38℃, distance between spinneret and receiving device 30cm, receiving speed 2000rpm, receiving method is additional magnetic field reception, with the first layer of fiber alignment direction as 0° as a reference, the second layer of fiber alignment direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time for each layer of fiber is 3.6min.

[0113] The orthogonally oriented nanofiber membrane has 220 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.985. The orthogonally oriented nanofiber membrane is a disc with a diameter of 19 mm, a thickness of 0.22 μm, an average pore size of 1.8 μm, a porosity of 91.3%, and a through-porosity of 84.8%.

[0114] Example 7

[0115] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0116] (1) Preparation of raw materials:

[0117] Polymer: Polylactic acid;

[0118] Solvent: dichloromethane;

[0119] (2) Dissolve polylactic acid in dichloromethane to obtain a homogeneous solution, i.e. a spinning solution with a concentration of 20 wt%;

[0120] (3) After preparing nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonal oriented nanofiber membrane grafted with NHS ester groups.

[0121] The electrospinning process parameters are as follows: spinning voltage 20kV, spinning solution flow rate 3.5mL / h, humidity 60%, temperature 40℃, distance between spinneret and receiving device 5cm, receiving speed 2200rpm, receiving method is high-speed roller receiving, with the first layer of fiber arrangement direction as 0° as a reference, the second layer of fiber arrangement direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time of each layer of fiber is 4min.

[0122] The orthogonally oriented nanofiber membrane has 240 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.986. The orthogonally oriented nanofiber membrane is a disc with a diameter of 21 mm, a thickness of 0.24 μm, an average pore size of 2.4 μm, a porosity of 91.5%, and a through-porosity of 85%.

[0123] Example 8

[0124] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0125] (1) Preparation of raw materials:

[0126] Polymer: Nylon 6;

[0127] Solvent: Formic acid;

[0128] (2) A homogeneous solution obtained by dissolving nylon 6 in formic acid, i.e. a spinning solution with a concentration of 22.5 wt%;

[0129] (3) After preparing a nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonally oriented nanofiber membrane grafted with amino groups.

[0130] The electrospinning process parameters are as follows: spinning voltage 25kV, spinning solution flow rate 4mL / h, humidity 65%, temperature 25℃, distance between spinneret and receiving device 10cm, receiving speed 2400rpm, receiving method is auxiliary electrode receiving, with the first layer of fiber arrangement direction as 0° as a reference, the second layer of fiber arrangement direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time of each fiber layer is 4.2min.

[0131] The orthogonally oriented nanofiber membrane has 260 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.988. The orthogonally oriented nanofiber membrane is a disc with a diameter of 23 mm, a thickness of 0.26 μm, an average pore size of 2.8 μm, a porosity of 92%, and a through-porosity of 85.4%.

[0132] Example 9

[0133] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0134] (1) Preparation of raw materials:

[0135] Polymer: Chitosan;

[0136] Solvent: Acetic acid;

[0137] (2) Dissolve chitosan in acetic acid to obtain a homogeneous solution, i.e. a spinning solution with a concentration of 25 wt%;

[0138] (3) After preparing a nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonally oriented nanofiber membrane grafted with aldehyde groups.

[0139] The electrospinning process parameters are as follows: spinning voltage 28kV, spinning solution flow rate 4.5mL / h, humidity 70%, temperature 27℃, distance between spinneret and receiving device 15cm, receiving speed 2600rpm, receiving method is additional magnetic field reception, with the first layer of fiber alignment direction as 0° as a reference, the second layer of fiber alignment direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time for each layer of fiber is 4.6min.

[0140] The orthogonally oriented nanofiber membrane has 280 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.989. The orthogonally oriented nanofiber membrane is a disc with a diameter of 23 mm, a thickness of 0.28 μm, an average pore size of 3.6 μm, a porosity of 92.3%, and a through-porosity of 85.6%.

[0141] Example 10

[0142] A method for preparing an orthogonally oriented nanofiber membrane, comprising the following specific steps:

[0143] (1) Preparation of raw materials:

[0144] Polymer: Polyacrylonitrile;

[0145] Solvent: N,N-dimethylformamide;

[0146] (2) A homogeneous solution obtained by dissolving polyacrylonitrile in N,N-dimethylformamide, i.e. a spinning solution with a concentration of 30 wt%;

[0147] (3) After preparing a nanofiber membrane by electrospinning the spinning solution in step (2), the membrane is functionalized and then cut to obtain a circular orthogonal oriented nanofiber membrane grafted with carboxyl groups.

[0148] The electrospinning process parameters are as follows: spinning voltage 30kV, spinning solution flow rate 5mL / h, humidity 70%, temperature 30℃, distance between spinneret and receiving device 20cm, receiving speed 3000rpm, receiving method is high-speed roller receiving, with the first layer of fiber arrangement direction as 0° as a reference, the second layer of fiber arrangement direction as 90°, and the 0° and 90° layers are deposited sequentially; the deposition time of each layer of fiber is 5min.

[0149] The orthogonally oriented nanofiber membrane has 300 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.989. The orthogonally oriented nanofiber membrane is a disc with a diameter of 25 mm, a thickness of 0.3 μm, an average pore size of 4 μm, a porosity of 93%, and a through-porosity of 86%.

[0150] Example 11

[0151] A method for preparing an orthogonally oriented nanofiber membrane is basically the same as in Example 1, except that the polymer is polyvinyl alcohol and the solvent is dimethyl sulfoxide.

[0152] The orthogonally oriented nanofiber membrane has 100 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.976. The orthogonally oriented nanofiber membrane is a disc with a diameter of 13 mm, a thickness of 0.1 μm, an average pore size of 0.5 μm, a porosity of 90.7%, and a through-porosity of 82.4%.

[0153] Example 12

[0154] A method for preparing an orthogonally oriented nanofiber membrane is basically the same as in Example 1, except that the polymer is polyethylene glycol and the solvent is acetone.

[0155] The orthogonally oriented nanofiber membrane has 180 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.983. The orthogonally oriented nanofiber membrane is a disc with a diameter of 15 mm, a thickness of 0.16 μm, an average pore size of 2.2 μm, a porosity of 92.8%, and a through-porosity of 88.2%.

[0156] Example 13

[0157] A method for preparing an orthogonally oriented nanofiber membrane is basically the same as in Example 1, except that the polymer is polyaniline and the solvent is formic acid;

[0158] The orthogonally oriented nanofiber membrane has 210 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.981. The orthogonally oriented nanofiber membrane is a disc with a diameter of 17 mm, a thickness of 0.18 μm, an average pore size of 0.65 μm, a porosity of 93.2%, and a through-porosity of 88.6%.

[0159] Example 14

[0160] A method for preparing an orthogonally oriented nanofiber membrane is basically the same as in Example 1, except that the polymer is polycaprolactone and the solvent is N,N-dimethylformamide.

[0161] The orthogonally oriented nanofiber membrane has 300 layers of oriented nanofibers. Since the fabrication process is the same, only the vertical and horizontal orientations of the fibers are changed, so the unidirectional orientation parameters of each layer of the fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.986. The orthogonally oriented nanofiber membrane is a disc with a diameter of 21 mm, a thickness of 0.3 μm, an average pore size of 3.2 μm, a porosity of 92.7%, and a through-porosity of 89.3%.

[0162] Example 15

[0163] A method for preparing an orthogonally oriented nanofiber membrane is basically the same as in Example 1, except that the polymer is chitosan and the solvent is N,N-dimethylformamide.

[0164] The orthogonally oriented nanofiber membrane has 270 layers of oriented nanofibers. Since the fiber membrane preparation process is the same, only the vertical and horizontal directions of the fibers are changed, so the unidirectional orientation parameters of each fiber membrane are the same. The orientation level of the uppermost fiber membrane can be used to reflect the unidirectional orientation parameters of the overall fiber membrane. The unidirectional orientation parameter S of the overall fiber membrane is 0.985. The orthogonally oriented nanofiber membrane is a disc with a diameter of 25 mm, a thickness of 0.25 μm, an average pore size of 4 μm, a porosity of 90.2%, and a through-porosity of 83.4%.

[0165] Example 16: A heterogeneous immunoassay device based on orthogonally oriented nanofibers, such as... Figures 1-2As shown, the assembly includes a circular orthogonally oriented nanofiber membrane 5, a micro-injection pump 1, a sterile screw-in syringe 2, and a micro-analytical membrane replacement filter 3. The circular orthogonally oriented nanofiber membrane 5 has a first identification substance immobilized on it, which can react with the target substance in the test solution. The micro-analytical membrane replacement filter 3 has a clamp composed of two O-ring silicone seals 4, which holds the circular orthogonally oriented nanofiber membrane 5 within it. The outer diameter of the O-ring silicone seals 4 is equal to the diameter of the circular orthogonally oriented nanofiber membrane 5. The sterile screw-in syringe 2 is connected to the micro-analytical membrane replacement filter 3 via a threaded connection valve. The assembly consisting of the circular orthogonally oriented nanofiber membrane 5, the sterile screw-in syringe 2, and the micro-analytical membrane replacement filter 3 is assembled onto the micro-injection pump. The steps for heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonal oriented nanofibers are as follows: (1) The target substance to be tested is determined to be chloramphenicol, and the immobilized region consists of two dots. The first dot is 0.5 μL of 100 μM of the first recognition substance 5'-AGA-GGA-CTA-CGG-CCC-CAG-CGG-AGG-CGG-CAT-CCG-TTA-TTG-biotin-3', which serves as the detection group; the second dot is 0.5 μL of 100 μM of 5'-TTT-TTT-TTT-biotin-3', which serves as the control group.

[0166] (2) The colorimetric label was combined with the second recognition substance. The colorimetric label was colloidal gold, and the second recognition substance was 5'-SH-AAA-AAA-AAA-CAA-TAA-CGG-ATG-CCG-CCT–CCG-CTG-GGG-CCG-TAG-TCC-TCT-3'. The combination method was as follows: 1 mL of colloidal gold solution was incubated with 5 μL of 100 μM second recognition substance. After incubation, 50 μL of the incubated solution was added to 5 mL of the test solution (0.01 M, pH=7.4 PBST solution, Tween-20 content of 0.5%, v / v) and incubated for 4 h. Then, the incubated test solution was transferred to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0167] (3) Based on the size of the labeled second identifier, the average pore size of the solid-phase carrier was selected to be 0.5 μm (i.e., the circular orthogonally oriented nanofiber membrane of Example 1 was selected). The flow rate of the sterile screw-type syringe was controlled at 0.5 mL / h, allowing the test solution to complete the immunoreaction through the solid-phase carrier. The reaction time was 3 min. After washing, high-sensitivity detection was achieved through signal acquisition. The detection limit was 0.1 ng / mL, the sensitivity was 90%, and the false positive rate and false negative rate were both 4.8%.

[0168] Example 17 A heterogeneous immunoassay device based on orthogonal oriented nanofibers, comprising a circular orthogonal oriented nanofiber membrane, a micro-injection pump, a sterile screw-type syringe, and a micro-analytical membrane replacement filter;

[0169] A circular orthogonally oriented nanofiber membrane is immobilized with a first recognition substance that can react with the target substance in the test solution. A microanalytical membrane filter has a clamp composed of two O-ring silicone seals that holds the circular orthogonally oriented nanofiber membrane within it. The outer diameter of the O-ring silicone seals is equal to the diameter of the circular orthogonally oriented nanofiber membrane. A sterile screw-in syringe is connected to the microanalytical membrane filter via a threaded valve. The assembly consisting of the circular orthogonally oriented nanofiber membrane, the sterile screw-in syringe, and the microanalytical membrane filter is assembled onto a microinjection pump. The steps for performing heterogeneous immunoassay using the above-described heterogeneous immunoassay device based on orthogonally oriented nanofibers are as follows:

[0170] (1) The target substance was identified as microcystin LR (MC-LR), with two bands immobilized. The first band, representing the first recognition substance MC-LR-BSA, served as the detection group. The synthesis method was as follows: 1 mg MC-LR, 1 mg NHS, and 1 mg EDC were mixed in 200 μL of N,N-dimethylformamide and reacted at room temperature for 5 h to form a mixed solution. 10 mg BSA was dissolved in 2 mL of PBS buffer (0.01 M, pH = 7.4) and added to the mixed solution to form the LR-BSA complex. 0.7 μL of the complex was then immobilized on a nanofiber membrane disc to form a 0.8 cm long band. The second band, representing 0.7 μL of 10 ng / mL goat anti-mouse IgG antibody, served as the control group and was 0.8 cm long.

[0171] (2) The chromogenic label was combined with the second recognition substance. The chromogenic label was fluorescent microspheres (carboxylated yellow-green fluorescent latex PS microspheres), and the second recognition substance was microcystin monoclonal antibody. The combination method was as follows: 15 μL of 2% w / v fluorescent microspheres were suspended in 1 mL of MES buffer (0.05 M, pH = 6), and 1 mg EDC and 1 mg NHS were added for activation. 20 μL of 0.2 mg / mL microcystin monoclonal antibody was added to the activated fluorescent microspheres, and the mixture was stirred for 30 min. Then, 20 μL of 20% w / v BSA solution was added in MES buffer (0.05 M, pH = 6), and the mixture was stirred for 15 min. Finally, the mixture was centrifuged at 10000 rpm for 10 min to obtain a precipitate. The precipitate was washed twice with 1 mL of PBS buffer (0.01 M, pH = 7.4) (by centrifuging twice in the same manner), and then washed with 200 μL of PBS buffer. The sample was resuspended in PBS and sonicated to form a conjugate. Then, 50 μL of the conjugate was added to 5 mL of the test solution (0.01 M, pH 7.4 PBST solution, Tween-20 content 0.5%, v / v) and incubated for 4 h. The incubated test solution was then transferred to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0172] (3) Select an average pore size of 0.55 μm for the solid-phase carrier based on the size of the second identifier being labeled (i.e., the circular orthogonal oriented nanofiber membrane of Example 2), control the flow rate of the sterile screw syringe to 1 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier, the reaction time is 3 min, and after washing, high-sensitivity detection is achieved through signal acquisition.

[0173] The detection limit of the test was 0.5 ng / mL, the sensitivity was 90.4%, and the false positive rate and false negative rate were both 4.6%.

[0174] Example 18

[0175] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0176] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0177] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0178] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0179] The steps for performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers are as follows:

[0180] (1) The target substance to be tested was determined to be botulinum neurotoxin A, and the immobilization region was two bands. The first band was 3 μL of 4.5 mg / mL of the first recognition substance anti-botulinum neurotoxin A antibody, which served as the detection group. The second band was 3 μL of 4.5 mg / mL of goat anti-mouse IgG, which served as the control group.

[0181] (2) The chromogenic label was combined with the second recognition substance. The chromogenic label was magnetic nanoparticles (carboxylated magnetic beads, superparamagnetic Fe3O4 particles), and the second recognition substance was polyclonal anti-botulinum neurotoxin A antibody. The combination method was as follows: 300 μg of magnetic nanoparticles were first washed with deionized water, and then the magnetic nanoparticles were added to 0.8 mL of MES buffer (0.1 M, pH = 5.0) and sonicated for 10 min. Then, 80 mg of EDC and 40 mg of Sulfo-NHS were added to the buffer after sonication and incubated for 20 min to obtain a magnetic nanoparticle solution. 10 μL of 3.5 mg / mL polyclonal anti-botulinum neurotoxin A antibody solution was added to 80 μL of borate buffer (0.1 M, pH = 8.6), and then added to the magnetic nanoparticle solution for coupling for 1 h to obtain the conjugate. Then, 50 μL of the conjugate was added to 5 mL of the test solution (0.01 M, pH = 5.0). Incubate the solution in 7.4% PBST solution (Tween-20 content 0.5%, v / v) for 6 hours, and then transfer the incubated test solution to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0182] (3) Select an average pore size of 0.6 μm for the solid-phase carrier based on the size of the second identifier being labeled (i.e., the circular orthogonal oriented nanofiber membrane of Example 3), control the flow rate of the sterile screw-type syringe to 1.5 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier. The reaction time is 4 min. After washing, high-sensitivity detection is achieved through signal acquisition.

[0183] The detection limit of the test was 0.7 ng / mL, the sensitivity was 90.6%, and the false positive rate and false negative rate were both 4.2%.

[0184] Example 19

[0185] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0186] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0187] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0188] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0189] The steps for performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers are as follows:

[0190] (1) The target substance to be tested was identified as juvenile ketone (ZEA), and the immobilization area consisted of two dots. The first dot contained 0.5 μL of 0.8 mg / mL ZEA-BSA, which served as the detection group; the second dot contained 0.5 μL of 0.6 mg / mL goat anti-mouse IgG, which served as the control group.

[0191] (2) The chromogenic label was combined with the second recognition substance, which was carbon nanoparticles and the second recognition substance was juvenile ketone monoclonal antibody. The combination method was as follows: 4 mg of carbon nanoparticles were suspended in 1 mL of borate buffer (0.01 M, pH = 8) and shaken at room temperature for 24 h. Then, 12 μL of 1 mg / mL juvenile ketone monoclonal antibody was added. Subsequently, 50 μL of 25% glutaraldehyde was added to the suspension and stirred for 2 h. Then, 100 μL of 20% w / t BSA solution was added in PBS buffer (0.01 M, pH = 7.4) and stirred for 30 min. After centrifugation at 10000g for 10 min, the precipitate was resuspended in 1 mL of PBS buffer containing 2% w / v BSA and 20% v / v glycerol to obtain the carbon nanoparticle-juvenile ketone monoclonal antibody conjugate. Then, 50 μL of the conjugate was added to 5 mL of the test solution (0.01 M, pH = 8). Incubate the solution in 7.4% PBST solution (Tween-20 content 0.5%, v / v) for 6 hours, and then transfer the incubated test solution to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0192] (3) Select an average pore size of 0.8 μm for the solid-phase carrier based on the size of the second identifier being labeled (i.e., the circular orthogonal oriented nanofiber membrane of Example 4), control the flow rate of the sterile screw-in syringe to 2 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier. The reaction time is 4 min. After washing, high-sensitivity detection is achieved through signal acquisition.

[0193] The detection limit of the test was 1.3 ng / mL, the sensitivity was 90.8%, and the false positive rate and false negative rate were both 4.2%.

[0194] Example 20

[0195] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0196] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0197] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0198] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0199] The steps for performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers are as follows:

[0200] (1) The target substance to be tested was identified as β-lactoglobulin, and the immobilization region consisted of two dots, with the first dot being 0.6 μL.

[0201] The first recognition substance, anti-β-lactoglobulin antibody, was 1 mg / mL, which served as the detection group; the second dot was 0.6 μL of 1 mg / mL recombinant endotoxin A, which served as the control group.

[0202] (2) The chromogenic label was combined with the second recognition substance, which was latex microspheres. The chromogenic label was β-lactoglobulin polyclonal antibody. The combination method was as follows: 200 μL of 1 mg / mL β-lactoglobulin polyclonal antibody was added to 800 μL of 70 nm red latex microspheres to obtain a final concentration of 0.2 mg / mL of β-lactoglobulin polyclonal antibody conjugate. The conjugate was incubated at room temperature with shaking at 12 rpm / min and a 90° angle for 2.5 h. Then, 30 μL of ethanolamine was added to obtain a mixture, which was incubated at room temperature for 30 min. The mixture was then centrifuged at 17000 g for 15 min, and the supernatant was discarded. Finally, add 1 mg BSA and incubate at room temperature for 2 h to form a conjugate; then take 50 μL of the conjugate and add it to 5 mL of the test solution (0.01 M, pH 7.4 PBST solution, Tween-20 content 0.5%, v / v) and incubate for 8 h. Then transfer the incubated test solution to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0203] (3) Select an average pore size of 1.2 μm for the solid-phase carrier based on the size of the second identifier being labeled (i.e., the circular orthogonal oriented nanofiber membrane of Example 5), control the flow rate of the sterile screw-type syringe to 2.5 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier. The reaction time is 5 min. After washing, high-sensitivity detection is achieved through signal acquisition.

[0204] The detection limit of the test was 2.8 ng / mL, the sensitivity was 91%, and the false positive rate and false negative rate were both 4%.

[0205] Example 21

[0206] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0207] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0208] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0209] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0210] The steps for performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers are as follows:

[0211] (1) The target substance to be tested was streptomycin (SM), and the immobilized region was two bands. The first band was 0.4 μL 1 mg / mL of the first recognition substance SM-OVA, which was used as the detection group; the second band was 0.4 μL 0.25 mg / mL of goat anti-mouse IgG, which was used as the control group.

[0212] (2) The colorimetric label is combined with the second recognition substance. The colorimetric label is quantum dots, and the second recognition substance is anti-streptomycin antibody. The combination method is as follows: First, the anti-streptomycin antibody is dialyzed in PBS buffer (0.01M, pH=7.6). Take 300μL of the dialyzed anti-streptomycin antibody and 25μL of 8μM quantum dots, 50μL of 0.8mM EDC, and 50μL of 0.8mM Sulfo-NHS and stir at room temperature in the dark for 90min. Centrifuge at 10000g five times at room temperature for 15min each time using an ultrafiltration tube. Store the concentrated conjugate (i.e., the conjugate) at 4℃. Then, take 50μL of the conjugate and add it to 5mL of the test solution (0.01M, pH 7.4 PBST solution, Tween-20 content of 0.5%, v / v) and incubate for 8h. Then, transfer the incubated test solution to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0213] (3) Select an average pore size of 1.8 μm for the solid-phase carrier based on the size of the second identifier being labeled (i.e., the circular orthogonal oriented nanofiber membrane of Example 6), control the flow rate of the sterile screw-in syringe to 3 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier, the reaction time is 6 min, and after washing, high-sensitivity detection is achieved through signal acquisition.

[0214] The detection limit of the test was 4.5 ng / mL, the sensitivity was 91.4%, and the false positive rate and false negative rate were both 3.8%.

[0215] Example 22

[0216] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0217] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0218] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0219] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0220] The steps for performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers are as follows:

[0221] (1) The target substance to be tested was determined to be melamine (MEL), and the immobilization region consisted of two dots. The first dot was 0.5 μL of 1 mg / mL of the first recognition substance MEL-BSA, which served as the detection group; the second dot was 0.5 μL of 1 mg / mL of goat anti-mouse IgG, which served as the control group.

[0222] (2) The chromogenic label was combined with the second recognition substance, which was colloidal selenium and anti-melamine monoclonal antibody. The combination method was as follows: 10 μL of 1 mg / mL anti-melamine monoclonal antibody was incubated with 1 mL of 50 nm colloidal selenium (Se NPs) for 30 min, centrifuged at 10000 rpm for 15 min, and the supernatant was removed. The supernatant was resuspended in 1 mL of PBS (0.01 M, pH = 7.4, 0.5% w / v BSA, 0.1% v / v Tween-20) to obtain the final Se NPs-antibody conjugate (i.e., the conjugate). Then, 50 μL of the conjugate was added to 5 mL of the test solution (0.01 M, pH 7.4 PBST solution, Tween-20 content of 0.5%, v / v) and incubated for 4 h. The incubated test solution was then transferred to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0223] (3) Based on the size of the second identifier being labeled, the average pore size of the solid-phase carrier is selected to be 2.4 μm (i.e., the circular orthogonal oriented nanofiber membrane of Example 7). The flow rate of the sterile screw-type syringe is controlled to be 3.5 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier. The reaction time is 7 min. After washing, high-sensitivity detection is achieved by signal acquisition.

[0224] The detection limit of the test was 5.8 ng / mL, the sensitivity was 91.8%, and the false positive rate and false negative rate were both 3.6%.

[0225] Example 23

[0226] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0227] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0228] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0229] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0230] The steps for performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers are as follows:

[0231] (1) The target substance to be tested was determined to be malachite green, and the immobilized region was two strips. The first strip was 0.8 μL of 100 μM of the first recognition substance 5'-AGG-GCT-GAC-CTT-GTC-CAT-TGC-TTA-CCT-biotin-3', which served as the detection group; the second strip was 0.8 μL of 100 μM of 5'-TTT-TTT-TTT-biotin-3', which served as the control group.

[0232] (2) The colorimetric label was combined with the second recognition substance. The colorimetric label was colloidal gold, and the second recognition substance was 5'-SH-AAA-AAA-AAA-UCC-CGA-CUG-GAA-CAG-GUA-ACG-AAU-GGA-3'. The combination method was as follows: 1 mL of colloidal gold solution was incubated with 5 μL of 100 μM second recognition substance. Then, 50 μL of the incubated solution was added to 5 mL of the test solution (0.01 M, pH 7.4 PBST solution, Tween-20 content of 0.5 v / v%) and incubated for 4 h. The incubated test solution was then transferred to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0233] (3) Select an average pore size of 2.8 μm for the solid-phase carrier based on the size of the second identifier being labeled (i.e., the circular orthogonal oriented nanofiber membrane of Example 8), control the flow rate of the sterile screw-in syringe to 4 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier, the reaction time is 8 min, and after washing, high-sensitivity detection is achieved through signal acquisition.

[0234] The detection limit of the test was 0.2 ng / mL, the sensitivity was 92%, and the false positive rate and false negative rate were both 3.4%.

[0235] Example 24

[0236] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0237] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0238] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0239] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0240] The process of performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers is as follows:

[0241] (1) The target substance to be tested was determined to be kanamycin, and the immobilization area was two dots. The first dot was 0.5 μL of 100 μM first recognition substance 5'-CTCAACCCCCAAAAAAA-Biotin-3', which was used as the detection group; the second dot was 0.5 μL of 100 μM 5'-Biotin-GCTAAGCCGATTTTTTT-3', which was used as the control group.

[0242] (2) The colorimetric label was combined with the second recognition substance. The colorimetric label was colloidal gold and the second recognition substance was 5'-SH-TGGGGGTTGAGGCTAAGCCGA-3'. The combination method was as follows: 1 mL of colloidal gold solution was incubated with 5 μL of 100 μM second recognition substance. After incubation, 50 μL of the mixture was added to 5 mL of the test solution (0.01 M, pH 7.4 PBST solution, Tween-20 content of 0.5%, v / v) and incubated for 6 h. Then the test solution was transferred to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0243] (3) Based on the size of the second identifier being labeled, the average pore size of the solid-phase carrier is selected to be 3.6 μm (i.e., the circular orthogonal oriented nanofiber membrane of Example 9). The flow rate of the sterile screw-type syringe is controlled to be 4.5 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier. The reaction time is 9 min. After washing, high-sensitivity detection is achieved through signal acquisition.

[0244] The detection limit of the test was 8.3 ng / mL, the sensitivity was 92.5%, and the false positive rate and false negative rate were both 3.2%.

[0245] Example 25

[0246] A heterogeneous immunoassay device based on orthogonally oriented nanofibers includes a circular orthogonally oriented nanofiber membrane, a microinjection pump, a sterile screw-type syringe, and a microanalytical membrane filter.

[0247] A first recognition substance that can react with the target substance in the test solution is immobilized on a circular orthogonally oriented nanofiber membrane.

[0248] The microanalytical membrane filter has a clamp consisting of two O-ring silicone seals that holds a circular orthogonally oriented nanofiber membrane within it; the outer diameter of the O-ring silicone seal is equal to the diameter of the circular orthogonally oriented nanofiber membrane.

[0249] The sterile screw-in syringe and the microanalytical membrane filter are connected via a threaded connection valve; the assembly consisting of a circular orthogonally oriented nanofiber membrane, a sterile screw-in syringe, and a microanalytical membrane filter is assembled onto the microinjection pump.

[0250] The process of performing heterogeneous immunoassay using the above-mentioned heterogeneous immunoassay device based on orthogonally oriented nanofibers is as follows:

[0251] (1) The target substance to be tested was determined to be enrofloxacin (ENR), and the immobilization area was two dots. The first dot was 0.5 μL of 1.0 mg / ml of the first recognition substance ENR-BSA, which served as the detection group; the second dot was 0.5 μL of 1.0 mg / ml of goat anti-mouse IgG, which served as the control group.

[0252] (2) The chromogenic label was combined with the second recognition substance. The chromogenic label was colloidal gold, and the second recognition substance was anti-enrofloxacin monoclonal antibody. The combination method was as follows: 5 mL of colloidal gold solution was reacted with 20 μL of 1.5 μg / mL anti-enrofloxacin monoclonal antibody in a vortex mixer at room temperature for 30 min. Then, the AμNPs-anti-enrofloxacin monoclonal antibody solution was centrifuged at 6,000 rpm and 4℃ for 30 min to obtain a precipitate. The precipitate was then resuspended in borate buffer (0.02 M, pH = 9, 1% BSA, 5% sucrose) to 5 mL to obtain the conjugate. 50 μL of the conjugate was then added to 5 mL of the test solution (0.01 M, pH 7.4 PBST solution, Tween-20 content 0.5%, v / v) and incubated for 6 h. The incubated test solution was then transferred to a sterile screw-type syringe in the heterogeneous immunoassay analyzer.

[0253] (3) Select an average pore size of 4 μm for the solid-phase carrier based on the size of the second identifier being labeled (i.e., the circular orthogonal oriented nanofiber membrane of Example 10), control the flow rate of the sterile screw syringe to 5 mL / h, so that the test solution can complete the immune reaction through the solid-phase carrier, the reaction time is 10 min, and after washing, high-sensitivity detection is achieved through signal acquisition.

[0254] The detection limit of the test was 10 ng / mL, the sensitivity was 92.8%, and the false positive rate and false negative rate were both 3%.

Claims

1. A heterogeneous immunoassay device based on orthogonally oriented nanofibers, characterized in that: This includes orthogonally oriented nanofiber membranes, microinjection pumps, sterile screw-in syringes, and microanalytical membrane filters; The sterile screw-in syringe is connected to the microanalytical membrane filter via a threaded connection valve; the microanalytical membrane filter has a clamp composed of two O-ring silicone seals to hold the orthogonally oriented nanofiber membrane; The assembly consisting of an orthogonal oriented nanofiber membrane, a sterile screw-in syringe, and a micro-analytical membrane filter is assembled onto a micro-injection pump; the orthogonal oriented nanofiber membrane is circular in shape, and the outer diameter of the O-ring silicone seal is equal to the diameter of the orthogonal oriented nanofiber membrane; The orthogonal-oriented nanofiber membrane is a functionalized orthogonal-oriented nanofiber membrane; Orthogonally oriented nanofiber membranes are composed of multiple layers of fibers, with all fibers in each layer arranged in an orthogonal orientation, and the fiber orientations in any two adjacent layers being perpendicular to each other. An orthogonally oriented nanofiber membrane is immobilized with a first recognition substance that can react with the target substance in the test solution, and the first recognition substance is distributed on the fiber membrane in a vertical direction. The porosity of orthogonally oriented nanofiber membranes is >90%, and the open porosity is >80%. Heterogeneous immunoassay was performed using the orthogonally oriented nanofiber-based heterogeneous immunoassay device, with a detection limit ranging from 0.1 to 10 ng / mL.

2. The heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 1, characterized in that, In orthogonally oriented nanofiber membranes, the number of oriented nanofiber layers is 100 to 300, and the unidirectional orientation parameter S of each fiber layer is greater than 0.

97.

3. The heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 2, characterized in that, The orthogonal-oriented nanofiber membrane is a circular membrane with a diameter of 13-25 mm; the thickness of the orthogonal-oriented nanofiber membrane is 0.1-0.3 μm, and the average pore size is 0.5-4 μm.

4. The heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 3, characterized in that, The functional groups on orthogonally oriented nanofiber membranes are carboxyl, aldehyde, amino, sulfonic acid, secondary amine, tertiary amine, NHS ester, maleimide, carbodiimide, and imine ester.

5. A heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 4, characterized in that, Orthogonally oriented nanofiber membranes are obtained by preparing nanofiber membranes through electrospinning of a spinning solution, followed by functionalization treatment. The receiving methods include high-speed roller receiving, auxiliary electrode receiving, or receiving with an additional magnetic field. The alignment direction of the first layer of fibers is taken as 0° as a reference, the alignment direction of the second layer of fibers is taken as 90°, and so on. o 90 o Layer-by-layer deposition.

6. The heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 5, characterized in that, Spinning solution is a homogeneous solution obtained by dissolving a polymer in a solvent. The polymer is one or more of the following: polyvinylidene fluoride, poly(vinyl alcohol-co-ethylene), polycarbonate, polyvinyl acetate, polyethylene, polystyrene, polylactic acid, nylon 6, polyethylene oxide, polyacrylonitrile, polyvinyl alcohol, polyethylene glycol, polyaniline, polyurethane, polyhydroxybutyrate, polycaprolactone, polyethersulfone, chitosan, collagen, and cellulose. The solvent is methanol, ethanol, isopropanol, hexafluoroisopropanol, tetraethylene glycol, triethylene glycol, formic acid, acetic acid, phenol, methyl formate, or ethyl formate. One or more of the following: propyl formate, methyl n-acetone, diisopropanone, diisobutyl ketone, acetone, hexafluoroacetone, chloromethane, chloroethane, dichloromethane, chloroform, p-chlorotoluene, 1,1-dichloroethane, 1,2-dichloroethane, dichloropropane, trichloromethane, trichloroethane, dibromoethane, dibromopropane, bromomethane, bromoethane, bromopropane, acetic acid, benzene, toluene, hexane, cyclohexane, cyclohexanone, cyclopentane, o-xylene, p-xylene, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, pyridine, and water.

7. A heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 5, characterized in that, The concentration of the spinning solution is 5~30 wt%; the process parameters for electrospinning are: spinning voltage 8~30 kV, spinning solution flow rate 0.5~5 mL / h, humidity 30~70%, temperature 25~40 ℃, distance between the spinneret and the receiving device 5~30 cm, and receiving speed 1000~3000 rpm.

8. A heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 5, characterized in that, The deposition time for each fiber layer is 2-5 minutes.

9. A heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 1, characterized in that, The fixed load area consists of one or more dots or strips.

10. A heterogeneous immunoassay device based on orthogonally oriented nanofibers according to claim 1, characterized in that, Heterogeneous immunoassay using the orthogonally oriented nanofiber-based heterogeneous immunoassay device achieved a sensitivity of over 90%, with both false positive and false negative rates below 5%.

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