An integrated reagent detection tube and its detection method

Abstract

This invention discloses an integrated reagent detection tube and its usage method, comprising: a mounting bracket with several mounting holes; a solid microtube disposed within the bracket, the solid microtube including an upper part and a lower part, and a bottom part disposed on the inner bottom surface of the lower part, the lower part gradually narrowing from the upper part downwards, and the bottom part gradually narrowing from the inner bottom surface of the lower part upwards; several reagent tubes equipped with markers, the reagent tubes being placed within the mounting holes; and a sealing film covering the top of the solid microtube and the reagent tubes, which continuously narrows the lower part and bottom of the solid microtube, thereby significantly increasing the specific surface area of ​​the microtube reaction region and the total surface area of ​​the lower part of the microtube projecting upwards without affecting signal generation, increasing the light signal formed by the upward projection of the reaction products by the microtube, thereby improving the detection sensitivity and accelerating the reaction speed, which is beneficial for quickly establishing the reaction container and reaction medium of the detection reagent.

Description

Technical Field

[0001] This invention relates to a detection device, and more particularly to an integrated reagent detection tube and its detection method. Background Technology

[0002] To date, immunoassay technology has evolved towards full automation, high sensitivity, high and low throughput, and ease of operation.

[0003] Fully automated chemiluminescence immunoassay technology occupies a core position in modern clinical immunological detection methods due to its high sensitivity, good stability, and versatile and flexible operation on automated equipment. It can be used to achieve trace quantitative detection of bioactive substances or disease markers related to human physiology and pathology, as well as foreign antigens, for disease diagnosis, efficacy evaluation, and prognosis.

[0004] Due to the multi-component nature of the reagents used in fully automated chemiluminescence immunoassay analyzers, current fully automated detection systems involve multiple stages of liquid transfer and processing. Furthermore, because multiple samples are used in the system, the corresponding sample injection and reagent addition devices need to be cleaned repeatedly, making the instruments large, complex, difficult to manufacture, and with a high failure rate. Moreover, due to space and sample processing requirements, they are limited to use in central laboratories, resulting in a long turnaround time (TAT).

[0005] Recently, there has been an increasing trend of automated chemiluminescence immunoassay reagents based on multi-component integrated systems. While these reagents simplify the instrument structure to some extent, they also have drawbacks such as insufficient detection throughput, high reagent cost, and the need for cryogenic storage. Summary of the Invention

[0006] This invention provides an integrated reagent detection tube and its detection method. By utilizing a solid-phase microtube as both a reaction vessel and reaction medium, it enables the formation of an integrated reagent that can be stored, transported, and used entirely dry at room temperature. This simplifies the reagent structure, reduces costs, and facilitates use. In particular, when combined with time-resolved fluorescent microspheres and quantum dot fluorescent microspheres, it can create an integrated reagent system capable of being stored, transported, and used entirely dry at room temperature, greatly simplifying user experience and solving the aforementioned problems.

[0007] This invention is implemented as follows:

[0008] An integrated reagent detection tube, comprising:

[0009] A mounting bracket with several mounting holes;

[0010] The solid microtube disposed within the support includes an upper part and a lower part, and a bottom part disposed on the inner bottom surface of the lower part. The lower part gradually narrows as it extends downward from the upper part, and the bottom part gradually narrows as it extends upward from the inner bottom surface of the lower part.

[0011] Several reagent tubes, each equipped with a marker, are placed within the mounting hole;

[0012] A sealing film covering the top of the solid microtube and the reagent tube.

[0013] As a further improvement, the reagent tube includes an upper tube and a lower tube connected to the lower end of the upper tube, wherein the upper tube and the top surface of the upper part of the solid microtube are at the same horizontal plane.

[0014] As a further improvement, the upper part includes a cylindrical, square, or rectangular structure.

[0015] As a further improvement, the lower part includes cylindrical, square, rectangular, inwardly curved, obliquely tapering frustum, inwardly tapering trapezoid, inverted spherical band, and trumpet-shaped tubular structure.

[0016] As a further improvement, the bottom includes a platform, a rotating body protruding upward from the cavity, and a pyramid.

[0017] A method of using an integrated reagent detection tube, comprising the aforementioned integrated reagent detection tube, specifically including:

[0018] S1: Solidify the antigen or antibody corresponding to the target analyte in the solid microtube cavity to match it with the marker in the reagent tube;

[0019] S2: Dispense the marker into a reagent tube, freeze-dry or in liquid form, and place the reagent tube into the mounting hole in the holder;

[0020] S3: Seal the top openings of the solid microtubes and reagent tubes with a membrane;

[0021] S4: Manually or automatically load the integrated reagent detection tube onto the detection track and remove the covering film;

[0022] S5: Transfer and mix the labels in different reagent tubes at least twice, and then transfer the mixed labels from the reagent tubes to solid microtubes and allow them to stand for reaction.

[0023] S6: Use an automatic washing device to fully extract and wash the reaction residue in the solid microtubes, and thoroughly clean them;

[0024] S7: Detect the fluorescence intensity in solid microtubes, or add a specified volume of luminescent substrate into solid microtubes and react for a certain time to measure the luminescence value. Based on the measured fluorescence intensity or luminescence value, establish a standard curve to calculate the concentration of the target substance in the target microtube and report the detection results.

[0025] In S4, the process of removing the coating further includes: if the marker in the reagent tube is a lyophilized marker, purified water needs to be added; if the marker in the reagent tube is a liquid marker, purified water does not need to be added.

[0026] The beneficial effects of this invention are:

[0027] This invention continuously narrows the lower part and bottom of the solid microtube, which significantly increases the specific surface area of ​​the microtube reaction region and the total surface area of ​​the lower part of the microtube projecting upwards without affecting signal generation. This increases the light signal formed by the reaction products projected upwards by the microtube, thereby improving the detection sensitivity and accelerating the reaction speed. It is also beneficial for establishing reaction containers and reaction media for rapid detection reagents.

[0028] This invention optimizes the combination of the lower part and bottom of solid microtubes of different shapes, which can generate microtube reaction zone specific surface area and total surface area of ​​microtubes projected upward from the lower part, and can select different configurations according to the user's needs, thus expanding the scope of application.

[0029] The markers of this invention, after being freeze-dried and filled into solid-phase microtubes, can form a fully dry integrated reagent detection tube. This tube can be stored, transported, and used at room temperature, achieving high-performance detection while providing convenience for users and greatly expanding the application scenarios.

[0030] This invention directly utilizes solid microtubes as reaction containers and reaction media, simplifying reagent structures and reducing costs. It eliminates the need to find separate containers and media, and since the microtubes and reagent tubes are housed in the same support, the integration is high. Experimenters do not need to move between multiple locations during experiments, making the experiment easier to conduct.

[0031] This invention integrates a scaffold, a solid-phase microtube, and several reagent tubes, making it compatible with chemiluminescence, time-resolved fluorescent microspheres, quantum dot fluorescent microspheres, or other fluorescently labeled immunoassays. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0033] Figure 1This is a schematic diagram of the structure of an integrated reagent detection tube provided in an embodiment of the present invention.

[0034] Figure 2 This is a schematic diagram illustrating the combination of an integrated reagent detection tube and a coating provided in an embodiment of the present invention.

[0035] Figure 3 This is a schematic diagram of the structure of a reagent tube provided in an embodiment of the present invention.

[0036] Figure 4 This is a schematic diagram of an integrated reagent detection tube, consisting of a square or rectangular solid-phase microtube and various bottom combination structures, provided by an embodiment of the present invention.

[0037] Figure 5 This is a schematic diagram of an integrated reagent detection tube cylindrical solid microtube and various bottom combination structures provided in Embodiment 1 of the present invention.

[0038] Figure 6 This is a schematic diagram of a solid microtube with an inverted frustum or inverted trapezoidal bottom section, which is an integrated reagent detection tube provided by an embodiment of the present invention, combined with a bottom structure of various rotating bodies.

[0039] Figure 7 This is a schematic diagram of an integrated reagent detection tube provided in Embodiment 2 of the present invention, which has an inverted spherical band at the bottom, a trumpet-shaped solid microtube, and a combination structure of various rotating bodies at the bottom. Detailed Implementation

[0040] All embodiments of the present invention are intended to fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0041] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating that the purpose, technical solution, and advantages of the method are clearer. The technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort indicate or imply the relative importance of the indicated technical features. Therefore, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0042] Reference Figure 1-7 As shown, an integrated reagent detection tube includes a mounting bracket 4 with several mounting holes 2; a solid microtube 1 disposed within the bracket 4; several reagent tubes 3 equipped with markers, the reagent tubes 3 being placed within the mounting holes 2; and a sealing film 6 covering the top of the solid microtube 1 and the reagent tubes 3.

[0043] The solid microtube 1 and the mounting bracket 4 are integrally injection molded, while the reagent tube 3 is an independently injection molded cup-shaped structure. The integrated reagent detection tube is used for detection by top excitation and reading of fluorescence value during fluorescent labeling immunoassay. It can also adopt detection modes such as top excitation and sidewall fluorescence measurement, or sidewall excitation and top fluorescence measurement.

[0044] In order to facilitate the transfer of the solid microtubes 1 and reagent tubes 3 in the entire support 4 into the detection structure, a sealing film 6 is required to cover the solid microtubes 1 and reagent tubes 3 to keep them in a closed state, so that their interiors can be kept in a relatively dry state and to prevent them from scattering or being lost during the transfer process.

[0045] To facilitate the loading of markers, the reagent tube 3 includes an upper tube 31 and a lower tube 32 connected to the lower end of the upper tube 31. The upper tube 31 is at the same level as the top surface of the upper part 11 of the solid microtube 1. The diameter of the upper tube 31 is larger than that of the lower tube 32, which facilitates loading. The smaller diameter of the lower tube 32 can better prevent the markers from falling out when containing them.

[0046] In a conventional solid-phase microtube 1, the solid-phase microtube 1 includes an upper part 11 and a lower part 12, as well as a bottom part 13 disposed on the inner bottom surface of the lower part 12. If a conventional cylindrical or rectangular structure is adopted, its surface area ratio is relatively small. The problem caused by the small surface area ratio is that its reflective effect is poor, and correspondingly, the overall detection rate is greatly affected. Therefore, in this embodiment, the lower part 12 gradually narrows from the upper part 11 downwards, and the bottom part 13 gradually narrows from the inner bottom surface of the lower part 12 upwards.

[0047] A method of using an integrated reagent detection tube, comprising the aforementioned integrated reagent detection tube, specifically including:

[0048] S1: Solidify the antigen or antibody corresponding to the target analyte in the cavity of solid microtube 1 to match it with the marker in reagent tube 3;

[0049] S2: Dispense the marker into reagent tube 3, freeze-dry or in liquid state, and place reagent tube 3 into mounting hole 2 in bracket 4;

[0050] S3: Seal the top openings of solid microtube 1 and reagent tube 3 with a membrane;

[0051] S4: Manually or automatically load the integrated reagent detection tube onto the detection track and remove the covering film;

[0052] S5: Transfer and mix the labels in different reagent tubes 3 at least twice, and then transfer the mixed labels from reagent tubes 3 to solid microtubes 1 and let them stand to react.

[0053] S6: Use an automatic washing device to fully extract and wash the reaction residue in the solid microtubes, and thoroughly clean them;

[0054] S7: Detect the fluorescence intensity in solid microtube 1, or add a specified volume of luminescent substrate to solid microtube 1 and react for a certain time to measure the luminescence value. Based on the measured fluorescence intensity or luminescence value, calculate the concentration of the target substance in the target microtube using a standard curve and report the detection results.

[0055] In S4, the process of removing the coating also includes: if the marker in the reagent tube 3 is a lyophilized marker, purified water needs to be added; if the marker in the reagent tube 3 is a liquid marker, purified water does not need to be added.

[0056] It should be noted that in the prior art, a micropore for immunoassay with application number CN201510408362.9, although its lower part 12 and bottom part 13 also adopt a gradually narrowing shape, the increase in specific surface area is not significant. Therefore, in this embodiment, the upper part 11 includes a cylindrical, square, or rectangular structure, the lower part 12 includes a cylindrical, square, rectangular, inwardly arc-shaped, obliquely tapering frustum, inverted trapezoidal frustum, inverted spherical band, or trumpet-shaped cavity structure, and the bottom part 13 includes a platform, a rotating body protruding upward from the cavity, or a pyramid. The upper part 11, lower part 12, and bottom part 13 can be freely combined. Specific embodiments are as follows:

[0057] Example 1:

[0058] like Figure 1-3 An integrated reagent detection tube is shown, comprising: Figure 5 The solid microtube 1 is formed by the ball-shaped notch at the bottom of the rotating body 1123A, 1123B, or 1123C in the inner solid microtube 1A.

[0059] like Figure 5 As shown, the upper part 11 and the lower part 12 of the solid microtube 1A are both cylindrical structures, while the bottom 13 is formed by a spherical defect of 1123A, 1123B, or 1123C, which increases the surface area inside the cavity and has the function of upward illumination. When combined with the lower part 12, it can further reflect the light to the top detector.

[0060] The upper part 11, lower part 12, and bottom part 13 of the cylindrical structure are pre-solidified with solid microtubes 1 formed by spherical defects of 1123A, 1123B, or 1123C, together with the support 4 and the base 5, to form the target detection reaction components antigens or antibodies.

[0061] The bottom 1313 of this invention is formed by the combination of a spherical notch of 1123A, 1123B, or 1123C and the sidewall 12, which increases the solid-liquid reaction interface of the immunoreaction solution, accelerates the immunoreaction, shortens the detection time, and improves and ensures the detection sensitivity and improves the detection efficiency. The lower part 12 can reflect the light signal formed by the reaction of the spherical notch of 1123A, 1123B, or 1123C, so that it can be effectively focused. The photomultiplier tube (PMT) probe of the detection instrument receives more light signals from the solid-liquid interface, which improves and ensures the detection sensitivity.

[0062] It is particularly important to emphasize that the 1123C spherical notch adopts an inward concave design. Compared with the existing round notch, the inward concave part greatly increases the overall specific surface area. This increased surface area can accelerate the reaction speed and improve the detection efficiency.

[0063] Among them, the inward spherical groove in the 1123C spherical notch no longer serves only a reflective function. Due to its spherical effect, it can generate a light-focusing effect, which can first focus the light projected from the top and then reflect it. The amount of light signal converged is greater and the convergence speed is faster. This changes the commonly used reflection method in the field to focusing before reflection, which greatly improves the detection sensitivity.

[0064] exist Figure 5 The calculated data for the integrated reagent detection tube of the solid-phase microtube 1, formed by the upper part 11, the lower part 12, and the spherical cap 1123A of the cylindrical structure 1, are as follows:

[0065] Beneficial effects

[0066]

[0067] With an inner diameter of 6.64 mm at the top and 6.6 mm at the bottom of the cylinder, an inner cavity height of 11.1 mm, and a reaction solution of 60 μL, the changes in effective surface area and specific surface area of ​​the cylindrical solid-phase microtube 1 and pore compared to those of the solid-phase microtube 1 and pore of this invention were calculated. The results showed that the effective surface area increased by 36.73%, and the specific surface area reached 0.78, an increase of 36.84%. The increase in effective area and specific surface area will inevitably shorten the reaction time, improve signal intensity, and enhance detection sensitivity.

[0068] Example 2:

[0069] like Figures 1-3 An integrated reagent detection tube is shown, comprising: Figure 7The solid microtube 1 is composed of a 1123B or 1123C or 1122C rotating body, or 1121C or 1121D truncated cone, plus an upper cylindrical part 11 and a lower inverted truncated cone 1F.

[0070] like Figure 7 As shown, the solid-phase microtube 1 of the present invention has an upper cylindrical part 11 and a lower inverted frustum 1F structure 12. The bottom 13 is formed by a frustum 1123B, or 1123C, or 1122C rotating body, or 1121C, or 1121D, which increases the surface area inside the cavity and has the function of upward illumination. In conjunction with the lower part 12, it can further reflect light to the top detector, increase the solid-liquid reaction interface of the immune reaction solution, accelerate the immune reaction, shorten the detection time, and improve and ensure the detection sensitivity and improve the detection efficiency. The lower part 12 can reflect the light signal formed by the reaction of the frustum 1123B, or 1123C, or 1122C rotating body, or 1121C, or 1121D, so that it can be effectively converged. The photomultiplier tube (PMT) probe of the detection instrument receives more light signals from the solid-liquid interface, improving and ensuring the detection sensitivity.

[0071] It is particularly important to emphasize that this embodiment uses a pyramidal base 13 with a truncated pyramidal base and grooves. Compared with the existing structure, firstly, the multiple contact surfaces of the truncated pyramid can increase the adhesion area. Secondly, the method of slotting on the truncated pyramid increases the surface area more than the method of simply cutting off the rounded part of the truncated pyramid. Preferably, the depth of the slot can be set according to the requirements.

[0072] Furthermore, the aforementioned solids of revolution and frustums of prisms all have a segmented surface, and the height between the segmented surface and the ground below requires extensive experimentation. It is not simply a matter of cutting off a segmented surface directly from the solid of revolution or frustum. If the segmented surface is too large or too small, the overall surface area may actually decrease.

[0073] The upper part 11 of the cylinder, the 1F structure of the lower part 12 of the inverted truncated cone, and the bottom 13 are formed by solid-phase microtubes 1 of 1123B or 1123C or 1122C rotating bodies or 1121C or 1121D prisms, together with the support 4 and the base 5, pre-solidify the target detection substance reaction components antigen or antibody.

[0074] Figure 7 The solid-phase microtube 1D structure consists of a rotating body 1122C, an upper cylinder 11, and an inverted frustum 1F 12, forming an integrated reagent detection tube. Let the radii of the upper and lower bases of the frustum be r and R, respectively, the height be h, and l be the generatrix = √[(Rr)]. 2 +h 2 (The value of l is equal to the length of the lateral surface of the frustum), according to the following formula:

[0075] Volume of sphere: V = (4 / 3)πr 3 Surface area of ​​a sphere: S = 4πr 2 .

[0076] Surface area of ​​a frustum: S = πr 2 +πR 2 +πRl+πrl=π(r 2 +R 2 +Rl+rl);

[0077] Lateral surface area of ​​a frustum: S = πRl + πrl = πl(R + r);

[0078] Volume of a frustum: V = 1 / 3πh (r 2 +R 2 +rR).

[0079] Figure 7 The calculated data for the integrated reagent detection tube of the solid-phase microtube 1, consisting of the rotating body 1122C in the solid-phase microtube 1D structure, plus the upper cylindrical part 11 and the lower inverted frustum 1F of the tube body, are as follows:

[0080] Beneficial effects

[0081]

[0082] Based on the above dimensions and a reaction solution of 60 μL, calculate... Figure 7 The calculation data for the solid-phase microtube 1 integrated reagent detection tube, consisting of the 1122C rotating body in the 1D structure of the internal solid-phase microtube, plus the upper cylinder 11 and the lower inverted frustum 1F, shows changes in the effective area and specific surface area. The effective surface area increased by 230.6%, and the specific surface area reached 1.88, an increase of 229.8%. This significant increase in effective area and specific surface area will undoubtedly greatly shorten the reaction time, improve signal intensity, and enhance detection sensitivity.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. An integrated reagent detection tube, characterized in that, include: A mounting bracket with several mounting holes; The solid microtube disposed within the support includes an upper part and a lower part, and a bottom part disposed on the inner bottom surface of the lower part. The lower part gradually narrows as it extends downward from the upper part, and the bottom part gradually narrows as it extends upward from the inner bottom surface of the lower part. The solid microtube (1) and the mounting bracket (4) are integrally injection molded, while the reagent tube (3) is an independently injection molded cup-shaped structure. Solid-phase microtubes serve as reaction vessels and reaction media; The upper part is a cylindrical structure; The lower part is an inverted frustum that curves inward; The bottom is a rotating body that protrudes upward from inside the cavity; The bottom (13) rotating body has a spherical notch, which is recessed inward; the shape of the recess is a spherical groove. Several reagent tubes, each equipped with a marker, are placed within the mounting hole; A sealing film covering the top of the solid microtube and the reagent tube.

2. The integrated reagent detection tube according to claim 1, characterized in that, The reagent tube includes an upper tube and a lower tube connected to the lower end of the upper tube. The upper tube and the top surface of the upper part of the solid microtube are at the same horizontal plane.

3. A method of using an integrated reagent detection tube, comprising the integrated reagent detection tube as described in any one of claims 1-2, characterized in that: S1: Solidify the antigen or antibody corresponding to the target analyte in the solid microtube cavity to match it with the marker in the reagent tube; S2: Dispense the marker into a reagent tube, freeze-dry or in liquid form, and place the reagent tube into the mounting hole in the holder; S3: Seal the top openings of the solid microtubes and reagent tubes with a membrane; S4: Manually or automatically load the integrated reagent detection tube onto the detection track and remove the covering film; S5: Transfer and mix the labels in different reagent tubes at least twice, and then transfer the mixed labels from the reagent tubes to solid microtubes and allow them to stand for reaction. S6: Use an automatic washing device to fully extract and wash the reaction residue in the solid microtubes, and thoroughly clean them; S7: Detect the fluorescence intensity in solid microtubes, or add a specified volume of luminescent substrate into solid microtubes and react for a certain time to measure the luminescence value. Based on the measured fluorescence intensity or luminescence value, establish a standard curve to calculate the concentration of the target substance in the target microtube and report the detection results.

4. The method of using an integrated reagent detection tube according to claim 3, characterized in that, In S4, the removal of the film also includes: If the label in the test tube is a lyophilized label, purified water needs to be added; if the label in the test tube is a liquid label, purified water does not need to be added.

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