An integrated shrimp molting inhibitory hormone detection kit

Abstract

This invention relates to the field of hormone detection technology, specifically to an integrated shrimp molting inhibitor hormone detection kit. The integrated shrimp molting inhibitor hormone detection kit includes a shell and a detection component disposed inside the shell. The detection component, arranged sequentially along the length of the shell, comprises a sample pad, a conjugate pad, a reaction membrane, and an absorbent pad. The conjugate pad includes an upper half and a lower half. The upper half is equipped with a micropillar filter, the inner surface of which is provided with a laminar flow plate to generate swirling and shearing forces on the flowing shrimp hemolymph mixture droplets, thereby breaking down large molecular aggregates. The lower half has parallel channels, a confluence zone, and a flat channel on its surface. The multiple parallel channels have the same channel size, and their outflow ends are connected to the flat channel through the confluence zone. Capillary traction actively anchors and horizontally flattens the liquid flow front, thereby outputting a liquid film of uniform thickness, with a straight front and consistent velocity, covering the reaction membrane.

Description

Technical Field

[0001] This invention relates to the field of hormone detection technology, and more specifically, to an integrated shrimp molting inhibition hormone detection kit. Background Technology

[0002] Ecdysin-inhibiting hormone (MIH) is a neuropeptide secreted by the X organ-sinus gland complex of crustaceans' eyestalks. Belonging to the hyperglycemic hormone family, it regulates the molting cycle by inhibiting the synthesis of Y organ ecdysin (EH). This hormone exhibits a sharp decline in gene expression during the pre-molting phase, forming a reverse regulatory mechanism with the ecdysin receptor (EcR) gene.

[0003] Test kits are typically used to detect the biochemical properties of a sample, thereby determining its biological or medical type, such as for detecting the type of virus. Existing kits generally consist of two interlocking housings and a test strip located between them. The test strip typically includes a sampling section and a detection section. An injection well is located on the upper housing corresponding to the sampling section, and an observation window is located on the upper housing corresponding to the detection section. A sample, such as blood, is dripped into the injection well and falls onto the sampling section of the test strip. The sample reaches the detection section via capillary action in the sampling section. The detection section displays the sample type through color development or color change, and the operator obtains this information through the observation window.

[0004] In shrimp farming and research, accurate monitoring of molting inhibitory hormones in shrimp hemolymph is crucial for scientific feeding and health management. While immunochromatographic test strips can be used for on-site detection, practical application has revealed several limitations. Shrimp hemolymph has high viscosity and a complex matrix. Current immunochromatographic test strips rely on a flat-laying method on a nitrocellulose membrane for chromatography. When high-viscosity shrimp hemolymph samples containing large molecular aggregates flow through the membrane pores, these aggregates can cause localized blockage or flux differences, resulting in inconsistent flow velocities across the membrane width and severe distortion of the chromatography front. Furthermore, this non-uniform flow leads to uneven distribution of the target antigen within the detection area, causing not only a mottled background but also a deterioration in the quantitative signal-to-noise ratio and poor repeatability, failing to meet the practical requirements for accurate detection. Summary of the Invention

[0005] This invention provides an integrated shrimp molting inhibitor hormone detection kit. It reduces sample concentration through a microcolumn filter and laminar flow plate in the upper part of the pad, and then uses a hydrophilic / hydrophobic distinguishing channel in the lower part to guide mixed droplets and enrich targeted antigens, forming a smooth liquid flow front covering the reaction membrane. This eliminates problems such as irregular chromatographic fronts and inconsistent widths of the colorimetric strips caused by liquid flow distortion and uneven flow rates, thus solving the problems mentioned in the background art. In existing technologies, when using immunochromatographic test strips to detect shrimp hemolymph molting inhibitory hormones, there is a lack of flow field control, which fails to guide the sample to spread evenly, resulting in inconsistent flow rates across the membrane width when the sample enters the reaction membrane.

[0006] To achieve the above objectives, the integrated shrimp molting inhibitory hormone detection kit includes a shell and a detection component disposed inside the shell. The detection component is provided with a sample pad, a conjugate pad, a reaction membrane and an absorbent pad arranged sequentially along the length of the shell. The detection component is disposed above a liner on the inner surface of the shell. The mating pad is internally divided into an upper and lower section by an inner cavity, and the upper and lower sections are connected. The surface of the lower section is provided with parallel flow channels, a confluence area, and paved flow channels, wherein: Multiple parallel channels are provided, and the channel size from the start to the end of the multiple parallel channels is the same. The ends of the multiple parallel channels are all connected and converge at the front end of the confluence area. The rear end of the confluence area is connected to the front end of the flat channel, and the rear end of the flat channel is connected to the upper surface of the reaction membrane.

[0007] In the above technical solution, when the mixed droplets enter the conjugate pad through the sample pad, they are first effectively broken down by a microcolumn filter to reduce the concentration of the mixed droplets. Then, they are efficiently guided and pre-enriched with targeted antigens through hydrophilic-hydrophobic differentiation channels, ultimately forming a smooth liquid flow front that covers the reaction membrane. This ensures that when the liquid flows through the detection line and the control line, the captured antibodies of both lines have the same probability of contacting the antigen per unit time, eliminating problems such as irregular chromatography fronts and inconsistent width of the color development strips.

[0008] Meanwhile, the micropillar filter is a cylinder with a diameter of 80-120 micrometers, and the spacing between adjacent micropillar filters is 40-60 micrometers, thereby maintaining the high density of the micropillar filter.

[0009] Furthermore, the laminar flow plate includes alternating inclined plates and transverse baffles. The angle (L1) between the inclined plates and the horizontal plane is 30°-45°, the center-to-center distance between adjacent inclined plates is 80-120 micrometers, and the center-to-center distance between adjacent transverse baffles is 30-50 micrometers. The defined angle allows the mixed droplets to smoothly form a swirling flow as they flow down. At the same time, the density of the transverse baffles creates a shear force on the mixed droplets, reducing the concentration of the mixed droplets.

[0010] Furthermore, the width of the parallel channels is 100-300 micrometers, the depth is 50-150 micrometers, and the number of parallel channels is 5-10; this divides the received mixed droplets into multiple stable microflows to eliminate local flow differences and lay the foundation for subsequent uniform confluence and spreading.

[0011] Based on this, the width of the confluence zone is 200-600 micrometers; the paved channel is bell-shaped, and its width extends outward from the connection with the confluence zone; the confluence zone and the paved channel work together to smoothly decelerate and laterally expand the merged liquid flow through their gradually changing width structure, so as to form a liquid flow front with sufficient width and uniform thickness before entering the detection area.

[0012] Based on this, the front end of the sample pad is placed on the rear end of the binding pad; the reaction membrane is placed above the liner, and the front end of the binding pad and the rear end of the absorbent pad are respectively placed on the two upper surfaces of the reaction membrane. It should be further noted that the upper part of the conjugate pad is also freeze-dried with a marker, which is prepared by covalently coupling an anti-shrimp molting inhibitor monoclonal antibody to fluorescent microspheres via the carbodiimide method. The fluorescent microspheres have a particle size of 190-210 nm, an excitation wavelength of 355-375 nm, and an emission wavelength of 600-620 nm. As the source of the detection signal, the marker functions to rehydrate the freeze-dried fluorescent microspheres and specifically recognize and bind to the shrimp molting inhibitor antigen in the sample when the mixed droplets flow through the upper part of the conjugate pad, forming a detectable antigen-antibody complex for subsequent qualitative or quantitative analysis.

[0013] Based on this, the detection line of the reaction membrane is immobilized with a paired anti-shrimp molting inhibitor monoclonal antibody, and the control line is immobilized with a goat anti-mouse IgG antibody. The detection line is used to specifically capture and enrich antigen-antibody complexes with fluorescent microspheres, thereby achieving qualitative or quantitative analysis of the target analyte through fluorescence signals. The control line is used to capture excess labeled antibody to indicate whether the detection process has been reliably completed.

[0014] In addition, the kit also includes a diluent, which is either a phosphate buffer or a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.0-7.8, containing 1%-5% by weight of a stabilizer and 0.5%-2% by weight of a protein protectant.

[0015] In another technical solution, the stabilizer is either sucrose or trehalose, and the protein protectant is either bovine serum albumin, casein, or gelatin.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: By incorporating a laminar flow plate within the micropillar filter in the upper half of the pad, the shrimp hemolymph mixture droplets generate swirling and shearing forces, effectively trapping and homogenizing large molecular aggregates in the liquid flow. The liquid then flows into parallel channels, which divide the liquid flow into multiple independent microflows to eliminate initial flow inertia and homogenize local velocity differences. The hydrophilic walls of the channels and the solid-liquid interface effect of the fluid generate continuous capillary adsorption forces, driving the migration and adsorption of antigen molecules in the fluid towards the walls, achieving pre-enrichment of the target analyte. Subsequently, these liquid flows converge in the confluence zone, undergoing laminar fusion to achieve internal... When the pressure reaches a lateral equilibrium, a homogeneous liquid flow body is formed. This liquid flow body then enters a bell-shaped, hydrophilic, flat channel. The gradual expansion of the channel width slows the flow, while the channel sidewalls actively anchor and horizontally flatten the flow front through strong capillary traction. This results in a uniformly thick, straight-fronted, and consistently velocity liquid film covering the reaction membrane, fundamentally eliminating the distortion of the chromatographic front and uneven color development bands caused by local flow velocity differences. This ensures the uniform distribution and specific binding of the target antigen within the detection area, ultimately achieving pure background color development. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of the test kit of the present invention; Figure 2 This is a schematic diagram of the internal structure of the detection kit of the present invention; Figure 3 This is a schematic diagram of the internal cross-sectional structure of the detection kit of the present invention; Figure 4 For the present invention Figure 3 Schematic diagram of the structure at point A in the middle; Figure 5 This is a top view schematic diagram of the inner cavity structure of the pad in this invention; Figure 6 This is a schematic diagram of the cross-sectional structure of the micropillar filter screen of the present invention; Figure 7 This is a front view schematic diagram of the micropillar filter structure of the present invention.

[0018] The meanings of the labels in the diagram are as follows: 100. Outer shell; 110. Sample port; 111. Detection window; 200. Detection component; 210. Sample pad; 220. Binding pad; 221. Micropillar filter; 222. Laminar flow plate; 223. Inner cavity; 224. Parallel flow channel; 225. Merging area; 226. Paved flow channel; 230. Reaction membrane; 240. Absorbent pad; 250. Liner. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] To address the problem in existing technologies using immunochromatographic test strips to detect shrimp hemolymph molting inhibitory hormones that lack flow field control, fail to guide uniform sample spreading, and result in inconsistent flow velocities across the membrane width upon entry, this invention provides an integrated shrimp molting inhibitory hormone detection kit, wherein, as... Figures 1-7 As shown, the integrated shrimp molting inhibitory hormone detection kit includes a shell 100 and a detection component 200 disposed inside the shell 100. The detection component 200 is provided with a sample pad 210, a conjugate pad 220, a reaction membrane 230 and an absorbent pad 240 in sequence along the length of the shell 100. The detection component 200 is disposed above the liner 250 on the inner surface of the shell 100. The inner cavity 223 of the mat 220 divides the interior into an upper part and a lower part, which are connected. The surface of the lower part is provided with parallel flow channels 224, a confluence area 225, and paved flow channels 226, wherein: Multiple parallel channels 224 are provided. The channel size from the start to the end of the multiple parallel channels 224 is the same, and the ends of the multiple parallel channels 224 are all connected and converge at the front end of the confluence area 225. The rear end of the confluence area 225 is connected to the front end of the flat channel 226. The rear end of the flat channel 226 is connected to the upper surface of the reaction membrane 230.

[0021] Figure 3 and Figure 4 In this embodiment, the front end of the sample pad 210 is placed on the rear end of the binding pad 220, and the reaction membrane 230 is placed above the liner 250. The front end of the binding pad 220 and the rear end of the absorbent pad 240 are respectively placed on the two upper surfaces of the reaction membrane 230. When the mixed droplet drips along the sample port 110 onto the surface of the sample pad 210, the sample pad 210 is preferably made of cellulose material in this embodiment. The mixed droplet can capillarily penetrate downward along the surface of the sample pad 210 until it migrates to the surface of the binding pad 220. The binding pad 220 is made of glass fiber and is hollow inside, forming an inner cavity 223. Due to the presence of the inner cavity 223, the binding pad 220 is divided into upper and lower parts, with the upper part connected to the upper and lower parts. Specifically, multiple micropillar filters 221 are provided at the position where the upper part of the binding pad 220 contacts the sample pad 210. Multiple laminar flow plates 222 are provided on the inner surface of the micropillar filters 221. When shrimp hemolymph mixed droplets with high viscosity and total protein content flow in, the laminar flow plates 222 generate swirling flow as the mixed droplets flow through. Combined with the shear force formed by the laminar flow plates 222, the large molecular aggregates and clumps in the mixed droplets are effectively physically broken up. This reduces the apparent viscosity of the mixed droplets while ensuring the passage of shrimp molting inhibitory hormones in the shrimp hemolymph, providing a basis for subsequent reactions. It is necessary to further explain here that, such as Figure 5 As shown, the lower half of the bonding pad 220, i.e., the position where the mixed droplets contact after flowing down along the micropillar filter 221, has parallel channels 224, a confluence area 225, and a paved channel 226 along its length. Multiple parallel channels 224 are provided, with the same channel size from start to end. The ends of all parallel channels 224 converge at the front end of the confluence area 225. The rear end of the confluence area 225 is connected to the front end of the paved channel 226, and the rear end of the paved channel 226 connects to the upper surface of the reaction membrane 230. The portion of the channel used for guiding the flow is a hydrophilic wall surface to guide the mixed droplets, while the remaining portion is hydrophobic (i.e., using inkjet printing hydrophobic ink or coating the surface with PDMS, fluorocarbon resin, etc.) to prevent circumferential diffusion of the mixed droplets. The parallel channels 224 divide the mixed droplets into multiple micro-flows, and the flow behavior of these micro-flows is determined by inertia. The flow pattern shifts from being dominated by viscous and capillary forces to being dominated by viscous forces, thereby suppressing unstable flow and jet phenomena caused by uneven initial kinetic energy. Secondly, multiple microflows flow in independent channels with consistent channel size and surface properties, and their hydraulic resistance is homogenized and standardized. This ensures that any local velocity fluctuations caused by slight differences in the permeability of the sample matrix or filter upstream are attenuated and isolated by the efficient resistance of each parallel channel 224, preventing them from being transmitted and amplified downstream. This achieves homogenization of the flow velocity of each microflow before entering the confluence zone 225. After multiple microflows flow through the confluence zone 225, they enter the bell-shaped and hydrophilic flat channel 226. The gradual expansion of the channel width slows down the liquid flow, while the sidewall of the channel actively anchors and horizontally flattens the liquid flow front through strong capillary traction, thereby outputting a liquid film with uniform thickness, straight front, and consistent velocity to cover the reaction membrane 230.

[0022] Furthermore, after the target antigen is dissociated by the micropillar filter 221, the released antigen is then carried by the liquid flow into the parallel channel 224 with an extended hydrophilic wall. The parallel channel 224 interacts continuously with the mixed droplet through its large specific surface area. The interfacial adsorption generated by this interaction at the microscale can preferentially capture and fix the antigen molecules in the mixed droplet, thereby forming a continuous mass transfer concentration gradient between the antigen solid phase interface and the mixed droplet bulk phase. This drives more antigen molecules in the mixed droplet to migrate to the hydrophilic wall and be adsorbed. This process is repeated, which is equivalent to the continuous capture and enrichment of the target antigen inside the macroscopic mixed droplet, thereby increasing the effective local concentration of the antigen in the mixed droplet and the probability of it binding to the subsequently immobilized antibody.

[0023] Multiple parallel channels 224 guide the mixed droplets through the confluence area 225 to the surface of the spreading channel 226. The hydrophilic walls of the spreading channel 226 allow the mixed droplets to spread out through capillary action into a thin liquid film with excellent frontal flatness and uniform thickness, covering the reaction membrane 230 at a consistent speed. The front end of the sample pad 210 overlaps the rear end of the binding pad 220. The reaction membrane 230 is positioned above the liner 250, and the front end of the binding pad 220 and the rear end of the absorbent pad 240 overlap respectively. The reaction membrane 230 is located on two upper surfaces. The reaction membrane 230 is made of nitrocellulose and has a detection line (T line) and a control line (C line) fixed on it using a spotting technique. The detection line is fixed with another monoclonal antibody (paired with the labeled antibody) against shrimp molting inhibitory hormone, and the control line is fixed with goat anti-mouse IgG antibody (used to capture the remaining labeled fluorescent microspheres). When the target antigen passes through the mixed droplets, the detection line will undergo a specific immune reaction and show color or generate a signal. The absorbent pad 240 is made of highly absorbent cellulose paper. Its core function is to provide continuous capillary traction force. Through the material's strong water absorption capacity, it pulls the mixed droplets through the sample pad 210, the binding pad 220 and the reaction membrane 230 in sequence, ensuring that the entire chromatography process can be carried out completely and quickly.

[0024] It should be further explained that the capture antibody immobilized on the detection line (T line) and control line (C line) of reaction membrane 230 is a monoclonal antibody against shrimp molting inhibitor hormone, used to specifically bind to shrimp molting inhibitor hormone; the binding pad 220 contains a lyophilized label, which is prepared by covalently coupling the anti-shrimp molting inhibitor hormone monoclonal antibody to carboxylated fluorescent microspheres with a particle size of 200 nm using the carbodiimide method; the diluent used in the scheme is phosphate buffer or Tris-HCl buffer containing stabilizer and protein protectant, with a pH of 7.0-7.8; among which, sucrose or trehalose is preferably used as the stabilizer in this scheme, and inert proteins such as bovine serum albumin (BSA), casein or gelatin are used as the protein protectant; and the concentration range of the stabilizer is 1%-5%; the concentration range of the protein protectant is 0.5%-2%.

[0025] Additionally, see Figures 6-7 The upper part of the pad 220 is provided with a micropillar filter 221, and the inner surface of the micropillar filter 221 is provided with a laminar flow plate 222, which is used to generate swirling and shearing forces on the flowing shrimp blood-lymph mixture droplets to break up the large molecular aggregates therein. In this scheme, the micropillar filter 221 is preferably cylindrical with a diameter of 100 micrometers, and the spacing between each micropillar filter 221 is 50 micrometers. The laminar flow plate 222 is divided into alternating inclined plates and transverse baffles, both of which are 4-8 micrometers thick, and the spacing between each layer of inclined plates is 8 micrometers. The width of the parallel channel 224 is 100-300 micrometers, the depth is 50-150 micrometers, and the horizontal angle between the inclined plate and the liner 250 is L1, which is between 30° and 45°. The hydrophilic wall width of the parallel channel 224 is 100-300 micrometers, the depth is 50-150 micrometers, and the total number of parallel channels 224 is between 5 and 10. The total width of the confluence zone 225 is between 200-600 micrometers. The overall width of the flat channel 226 gradually increases from the confluence zone 225 outwards, and the whole is in the shape of a horizontal bell.

[0026] Example 1: This embodiment describes the preparation of an integrated shrimp molting inhibitory hormone detection kit, as detailed below: 1. Preparation of main materials and reagents: Reaction membrane 230 (nitrocellulose membrane), conjugation pad 220 (glass fiber pad), sample pad 210 (cellulose pad), absorbent pad 240 (superabsorbent cellulose paper), outer shell 100 and liner 250 (ABS engineering plastic), fluorescent microspheres (200nm, carboxylated polystyrene, excitation / emission wavelength: 365nm / 610nm), anti-shrimp molting inhibitory hormone monoclonal antibody, paired anti-shrimp molting inhibitory hormone monoclonal antibody, goat anti-mouse IgG antibody, bovine serum albumin, sucrose, and analytical grade.

[0027] 2. Preparation of labeled solutions: Take 1.0 mL of a 1% (w / v) suspension of carboxylated fluorescent microspheres and add 5.0 mg of a monoclonal antibody against shrimp molting inhibitory hormone activated by carbodiimide and N-hydroxysuccinimide under magnetic stirring. Adjust the volume of the mixture to 2 mL using 0.05 mol / L morpholine ethanesulfonate buffer (pH 6.0) and incubate at 25°C in the dark for 2 hours.

[0028] After the reaction was completed, 100 μL of 10% bovine serum albumin solution was added, and the reaction was continued for 1 hour.

[0029] Finally, the microspheres were washed three times by centrifugation with 0.01 mol phosphate buffer (pH 7.4, containing 1% bovine serum albumin and 2% sucrose) and resuspended to a final volume of 2 mL to obtain the label working solution, which was then stored at 4°C for later use.

[0030] 3. Assembly of the test kit: The components are assembled on the liner 250 in the following order: First, the sample pad 210 is placed, then the treated conjugate pad 220 (the conjugate pad 220 is laser-engraved with microstructures and hydrophilic-hydrophobic patterning as described in the specific embodiment, and then freeze-dried for later use), with its upper half overlapping the end of the sample pad 210 by 2 mm. Next, the reaction membrane 230 is placed, with its beginning overlapping the flat channel 226 at the lower end of the conjugate pad 220 by 2 mm. Finally, the absorbent pad 240 is placed, with its beginning overlapping the end of the reaction membrane 230 by 2 mm. Pressure-sensitive adhesive tape is used to firmly attach each component to the liner 250. Finally, the assembled liner 250 is inserted into the outer shell 100, ensuring that the sample pad 210 is aligned with the sample port 110 on the outer shell 100, and that the detection lines and control lines on the reaction membrane 230 are aligned with the detection window 111 on the outer shell 100, thus obtaining the integrated shrimp molting inhibitory hormone detection kit.

[0031] 4. Preparation of diluent: A 0.02 mol / L phosphate buffer solution with a pH of 7.4 was prepared, containing 3% sucrose as a stabilizer and 1% bovine serum albumin as a protein protectant. After preparation, the solution was sterilized by filtration through a 0.22 μm pore size membrane and then aliquoted for storage.

[0032] Example 2: Detection Performance Verification and Comparison Experiment 1. Samples and Experimental Methods 1.1 Sample preparation: Hemolymph was collected from healthy whiteleg shrimp and mixed with the diluent prepared in Example 1 at a volume ratio of 1:4 to prepare a negative sample matrix.

[0033] Shrimp molting inhibitory hormone standard was precisely added to the negative sample matrix to prepare simulated positive samples with concentrations of 0 ng / mL (as negative control), 1.0 ng / mL, 2.5 ng / mL, 5.0 ng / mL, and 10.0 ng / mL.

[0034] 1.2 Detection Procedure: Take 80 μL of each of the above concentration samples and add them to the sample ports 110 of the three test kits prepared in Example 1. Simultaneously, parallel experiments were conducted using existing chromatography test strips as comparative examples; the binding pad 220 of the comparative test strips was a glass fiber pad without any microstructure treatment, and the remaining components (including antibodies, labels, reaction membrane 230, etc.) were completely identical to those of the kit of this invention.

[0035] 1.3 Signal Reading and Data Analysis: After reacting for 15 minutes at room temperature (25°C), the fluorescence intensity values ​​of the test line and control line for each kit were read using a portable fluorescence reader. The fluorescence intensity ratio of the test line to the control line was calculated. Simultaneously, the chromatography completion time for each test strip was recorded, and the smoothness of the liquid front during chromatography was observed.

[0036] 2. Results Analysis: 2.1 Sensitivity and Linearity Range: The detection limit (calculated based on a signal-to-noise ratio greater than 3) of the shrimp molting inhibitory hormone in the kit of Example 1 was 0.3 ng / mL. Within the concentration range of 1.0 ng / mL to 10.0 ng / mL, the fluorescence intensity ratio of the detection line to the control line showed a good linear relationship with the hormone concentration, and the linear regression equation was Y = 0.198X + 0.025 (coefficient of determination R²). 2 =0.995). In contrast, the detection limit of the comparative test strip is only 1.5 nanograms / mL, and its linear relationship is poor within the same concentration range (coefficient of determination R0). 2 =0.912).

[0037] 2.2 Chromatographic Behavior and Effects: The chromatography completion time of the kit in Example 1 was stable at around 10 minutes (fluctuation range ±1 minute), and the liquid flow front was very flat, with uniform and consistent color development of the detection line. The chromatography time of the comparative test strip fluctuated between 15 and 20 minutes, and the liquid flow front showed a distinct arc shape, resulting in uneven color development of the detection line with varying shades at both ends.

[0038] 2.3 Validation of Antigen Enrichment Effect: To validate the enrichment effect of the parallel flow channel 224 on the target antigen, a sample with a concentration of 5.0 ng / mL was used for testing. The average absolute fluorescence intensity of the test line in the Example 1 kit was 2850 (fluctuation range ±150), while the average absolute fluorescence intensity of the test line in the comparative test strip was 1250 (fluctuation range ±320). This result clearly demonstrates that the design of the parallel flow channel 224, the confluence zone 225, and the flat flow channel 226 in Example 1 increased the local concentration of the target antigen in the reaction area, thereby improving the immunobinding efficiency.

[0039] Overall conclusion: The results of Example 2 fully demonstrate that the integrated shrimp molting inhibitory hormone detection kit provided by the present invention, through its unique microcolumn filter 221, laminar flow plate 222 and hydrophilic-hydrophobic patterned flow channel design in the binding pad 220, effectively solves the problem that high-viscosity shrimp hemolymph samples lack flow field control in traditional chromatography test strips, which cannot guide the sample to spread evenly and causes inconsistent flow velocity in the membrane width direction when the sample enters the reaction membrane 230.

[0040] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. An integrated shrimp molting inhibitory hormone detection kit, comprising a shell (100) and a detection component (200) disposed inside the shell (100), wherein the detection component (200) is provided with a sample pad (210), a conjugate pad (220), a reaction membrane (230) and an absorbent pad (240) sequentially arranged along the length of the shell (100), and the detection component (200) is disposed above a liner (250) on the inner surface of the shell (100); characterized in that: The bonding pad (220) is divided into an upper part and a lower part by an inner cavity (223), and the upper part and the lower part are connected; the surface of the lower part is provided with parallel flow channels (224), a confluence area (225) and a paved flow channel (226), wherein: Multiple parallel channels (224) are provided. The channel size from the start to the end of the multiple parallel channels (224) is the same, and the end points of the multiple parallel channels (224) are all connected and converge at the front end of the confluence area (225). The rear end of the confluence area (225) is connected to the front end of the flat channel (226), and the rear end of the flat channel (226) is connected to the upper surface of the reaction membrane (230).

2. The integrated shrimp molting inhibition hormone detection kit according to claim 1, characterized in that: The upper half of the bonding pad (220) is provided with a plurality of micropillar filters (221), and the inner surface of the micropillar filters (221) is provided with a laminar flow plate (222) for generating swirling and shearing forces on the flowing shrimp blood lymph mixed droplets to break up the large molecular aggregates therein. The micropillar filter (221) is a cylinder with a diameter of 80-120 micrometers, and the spacing between adjacent micropillar filters (221) is 40-60 micrometers.

3. The integrated shrimp molting inhibition hormone detection kit according to claim 2, characterized in that: The laminar flow plate (222) includes alternating inclined plates and transverse baffles. The angle (L1) between the inclined plates and the horizontal plane is 30°-45°. The center-to-center distance between adjacent inclined plates is 80-120 micrometers, and the center-to-center distance between adjacent transverse baffles is 30-50 micrometers.

4. The integrated shrimp molting inhibition hormone detection kit according to claim 1, characterized in that: The width of the parallel flow channel (224) is 100-300 micrometers, the depth is 50-150 micrometers, and the number of the parallel flow channels (224) is 5-10.

5. The integrated shrimp molting inhibition hormone detection kit according to claim 4, characterized in that: The width of the confluence area (225) is 200-600 micrometers; the paved channel (226) is bell-shaped in general, and its width extends outward from the connection with the confluence area (225).

6. The integrated shrimp molting inhibition hormone detection kit according to claim 1, characterized in that: The upper part of the conjugation pad (220) is also freeze-dried with a marker, which is prepared by covalently coupling a monoclonal antibody against shrimp molting inhibitory hormone to fluorescent microspheres via the carbodiimide method.

7. The integrated shrimp molting inhibition hormone detection kit according to claim 6, characterized in that: The front end of the sample pad (210) is placed on the rear end of the bonding pad (220); The reaction membrane (230) is positioned above the liner (250), and the front end of the bonding pad (220) and the rear end of the absorbent pad (240) are respectively placed on the two upper surfaces of the reaction membrane (230).

8. The integrated shrimp molting inhibition hormone detection kit according to claim 1, characterized in that: The detection line of the reaction membrane (230) is immobilized with a paired anti-shrimp molting inhibitor monoclonal antibody, and the control line is immobilized with a goat anti-mouse IgG antibody.

9. The integrated shrimp molting inhibition hormone detection kit according to claim 1, characterized in that: The kit also includes a diluent, which is either a phosphate buffer or a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer with a pH of 7.0-7.8, containing 1%-5% by weight of a stabilizer and 0.5%-2% by weight of a protein protectant.

10. The integrated shrimp molting inhibition hormone detection kit according to claim 9, characterized in that: The stabilizer is one of sucrose or trehalose, and the protein protectant is one of bovine serum albumin, casein, or gelatin.

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