Magnetic particle up-converting chemiluminescence detection system

Abstract

The present invention relates to a magnetic particle up-converting chemiluminescence detection system. A sample carrying module for inputting, pre-loading and outputting several samples is arranged on a working face; a grabbing module is configured to grab a reaction cup and a pipette tip from a previous working position to the next working position; a liquid taking and sample adding module is configured to suction-in a sample and reagents and put same into the reactor cup, wherein the reagents at least comprise a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent marked by an up-converting luminescent material; a detection preparation module is configured to uniformly mix the sample and the reagents in the reaction cup once or multiple times, incubate same, and clean and separate same multiple times; and a light measurement module is configured to excite an up-converting fluorescent marker and measure the strength of a fluorescence signal. In the present invention, excitation can be repeated multiple times, and the excitation energy of high-power laser is far higher than the bioenergy of bioluminescence, such that the strength of a fluorescence signal is greatly improved; in addition to an up-converting material, fluorescence of other wavelengths generally can no longer be excited from other consumables and optical device materials that are used in a detection reaction, such that the background fluorescence signal is small and the detection sensitivity is high.

Description

A magnetic microparticle upconversion chemiluminescence detection system Technical Field

[0001] The present invention relates to the technical field of testing or analyzing materials by means of determining the chemical or physical properties of the materials, and in particular to a magnetic particle upconversion chemiluminescence detection system. Background Art

[0002] The magnetic particle chemiluminescence method is the most important immunoassay technology in the current in vitro diagnostic (IVD) field. The most important detection equipment in the hospital laboratory is the magnetic particle chemiluminescence fully automatic detection device. The market demand for reagents and instruments is huge and growing year by year.

[0003] Currently, there are three main methodologies for magnetic microparticle chemiluminescence: electrochemiluminescence, acridinium direct chemiluminescence, and enzymatic indirect chemiluminescence. Electrochemiluminescence is Roche's patented technology. It uses electrical energy to excite a marker to a high-energy unstable state, which then undergoes electron transition to the ground state to emit fluorescence. The problem with this methodology is that the electrode applying the electrical energy must be in contact with the sample solution, which places extremely high demands on electrode cleanliness and easily introduces cross-interference. Consequently, few manufacturers are currently developing electrochemiluminescence technology. Both acridinium direct chemiluminescence and enzymatic indirect chemiluminescence utilize separate reaction cups to isolate each sample from cross-interference, eliminating the risk of cross-contamination. The excitation solution catalyzes acridinium or alkaline phosphatase (horseradish peroxidase)-catalyzed substrate chemical reactions, causing the outer electrons of the luminescent marker to transition to a high-energy unstable state and then to the ground state to emit fluorescence. However, these luminescence methods all generate excitation energy through biochemical reactions, which is typically weak, resulting in low-intensity fluorescence signals. Furthermore, bioluminescence is a one-time emission and cannot be repeatedly excited and detected.

[0004] Summary of the Invention

[0005] The present invention solves the problems existing in the prior art and provides a magnetic particle upconversion chemiluminescence detection system.

[0006] The technical concept of the present invention is that a material composed of certain rare earth metal elements doped in the crystal lattice has the phenomenon of upconversion luminescence. There are three main components in this material: host matrix The host matrix consists of three main components: absorber and emitter. The host matrix consists of three main components: absorber and emitter. The main components are oxysulfides (such as Y2O2S, GdO2S, La2O2S, etc.), fluorides (such as YF3, GdF3, LaF3, etc.), gallates (such as YGaO3, Y3Ga5O12, etc.) and silicates (such as YSi2O5, YSi3O7, etc.). The rare earth metal ions commonly used as absorbers include ytterbium ion (Yb3+), erbium ion (Er3+), samarium ion (Sm3+), etc. The rare earth metal ions commonly used as emitters include erbium ion (Er3+), holmium ion (Ho3+), thulium ion (Tm3+), terbium ion (Tb3+), etc. The appropriate spatial orientation and distance of the absorber and emitter ion pair in the host matrix lattice are the basis for the generation of upconversion luminescence. The generation of upconversion luminescence is an optical process involving multiple photons (at least two). In this process, the absorber (such as Yb3+) in the upconversion luminescent material must absorb at least two low-energy photons (infrared light region, such as 980nm), and then undergo a series of internal energy conversions, in a non-radiative form (A1→A2, A2→A3), the energy of these two photons is continuously transferred to the emitter (such as Er3+), so that it is in an excited state (A3). The latter then undergoes a transition back to the ground state energy level, releasing a high-energy photon (visible light region, such as 525nm or 540nm) to complete the energy upconversion.

[0007] As a marker for magnetic microparticle chemiluminescence, upconversion luminescent material (UCP) has the advantage of being able to be excited repeatedly compared to traditional bioluminescence technology, and the excitation energy of high-power lasers is much higher than the biological energy of bioluminescence, which greatly improves the intensity of the fluorescence signal; the wavelength of the excitation light source is 980nm, and in addition to the upconversion material, it generally cannot excite fluorescence of other wavelengths for other consumables and optical device materials used in the detection reaction, so the background fluorescence signal is very clean, which is conducive to improving the sensitivity of detection. Compared with traditional downconversion fluorescent markers (such as quantum dots or Cy5 fluorescent dyes), upconversion luminescent material (UCP) markers have the advantage of clean background fluorescence signals. Usually, consumables such as reaction cups are stained with trace amounts of downconversion fluorescent materials, which will contaminate the background signal of fluorescence detection and lead to inaccurate detection. However, in upconversion fluorescence detection, the excitation light source is infrared excitation, which will not excite other fluorescent signals, further improving the sensitivity.

[0008] The technical solution adopted by the present invention is a magnetic microparticle upconversion chemiluminescence detection system, the system includes a working surface, and the working surface is provided with:

[0009] A sample carrier module, used for inputting, preloading and outputting a number of samples;

[0010] A grabbing module, used to grab the reaction cup and the pipette head from the previous working position to the next working position;

[0011] A liquid sampling module is used to draw samples and reagents and place them in a reaction cup, wherein the reagents include at least a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent labeled with an upconverting luminescent material;

[0012] A detection preparation module, used to perform one or more mixing, incubation, and multiple washing and separation of the sample and reagent placed in the reaction cup;

[0013] A light measurement module is used to excite the upconverted fluorescent marker and detect the intensity of the fluorescent signal.

[0014] Preferably, the sample transport module includes a sample loading channel and a sample output channel; a sample inlet for placing a sample rack is provided in conjunction with the sample loading channel, a sample outlet for outputting the sample rack is provided in conjunction with the sample output channel, and a traction unit is provided in conjunction with the sample loading channel and the sample output channel of the sample rack;

[0015] The ends of the sample loading channel and the sample output channel are matched with the sample rack and provided with a movable frame, and matched with the movable frame and provided with a movable traction unit;

[0016] A cover removal position is provided between the sample loading channel and the sample output channel, and a cover removal mechanism is provided corresponding to the cover removal position;

[0017] A roller tangential to the sample tube is provided in conjunction with the sample loading channel, and a barcode scanning mechanism for the sample tube is provided on the side corresponding to the capping position;

[0018] A sampling position is provided in conjunction with the sample loading channel, and the sampling position is provided in conjunction with the liquid sampling and adding module.

[0019] Preferably, a ferry transport auxiliary module is provided for assisting transport between the grabbing module and the liquid sampling and adding module;

[0020] The ferrying and transporting auxiliary module includes a moving part with a plurality of holes, and the moving part is arranged on a track, and the track is arranged between the grabbing module and the liquid sampling and adding module.

[0021] Preferably, the grabbing module includes a three-axis motion mechanism stacked above the working surface, a grabbing mechanism is provided at the lower part of the Z-axis of the three-axis motion mechanism, and the grabbing mechanism is arranged in conjunction with the ferrying and transporting auxiliary module; the working surface cooperating with the grabbing mechanism is respectively provided with a well plate and a detection preparation module for placing a pipette head (Tip head) and a reaction cup.

[0022] Preferably, the liquid sampling and loading module includes a Y-axis and Z-axis biaxial motion mechanism arranged above the working surface, and a first liquid sampling mechanism for picking up the pipette head and sucking the sample liquid from the sampling position of the sample loading channel into the reaction cup and a second liquid sampling mechanism for sucking the reagent into the reaction cup are provided in conjunction with the biaxial motion mechanism. The first liquid sampling mechanism and the second liquid sampling mechanism are arranged in conjunction with the ferrying and transporting auxiliary module, and a reagent storage chamber is provided in conjunction with the second liquid sampling mechanism.

[0023] Preferably, the reagent storage chamber includes a storage chamber cavity, a rotating mechanism is provided at the bottom of the storage chamber cavity, and several reagent links are divergently provided in the storage chamber cavity; any reagent link includes a reagent tube respectively provided with a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent marked with an upconverted luminescent material.

[0024] Preferably, the detection preparation module includes an incubation unit, a vibration mixing unit and a magnetic particle cleaning and separation unit, wherein the incubation unit is generally a constant temperature incubation; the magnetic particle cleaning and separation unit includes a turntable, which is provided with position 0 and at least 3 groups of cleaning positions, any group of cleaning positions includes a liquid extraction position and a liquid injection position, and the last liquid injection position is a pre-excitation position; a high-strength permanent magnet is provided on the outer wall of the turntable.

[0025] Preferably, the light measurement module includes a detection position, which is arranged in cooperation with the measurement chamber through an absorbent motion drawer. The absorbent motion drawer corresponding to the lower part of the reaction cup is provided with a hollow, and a light detector and an excitation light source are provided in the corresponding hollowed measurement chamber. A dichroic mirror is provided between the light detector and the hollow space, and the incident surface of the dichroic mirror faces the excitation light source, the reflective surface faces the hollow, and the transmission surface faces the light detector.

[0026] Preferably, the detection method of the system comprises the following steps:

[0027] Step 1: The sample is placed in the sample rack and enters the system through the sample carrier module;

[0028] Step 2: Scan the number of the first unprocessed sample tube, download the test items according to the sample tube number, and automatically remove the cap of the sample tube. During this process, the gripper module picks up the reaction cup and the pipette tip to the preset position;

[0029] Step 3: The liquid sampling module is loaded with a pipette head, which absorbs a quantitative sample and adds it to the reaction cup. The liquid sampling module absorbs the monoclonal antibody reagent bound to the magnetic particles and the monoclonal antibody reagent labeled with the upconversion luminescent material according to the obtained test item information, and adds them to the reaction cup with the existing sample to perform a double antibody sandwich immunoreaction or a competitive immunoreaction.

[0030] Step 4: The liquid sampling module waits for the next sample tube to be loaded; the grabbing module picks up the reaction cup that has been added with the sample and reagent, transfers it to the detection preparation module, shakes and mixes it, and incubates it at a constant temperature for a preset time for cleaning and separation; during cleaning and separation, the magnet adsorbs the magnetic particles to the inner wall of the reaction cup, and the unbound monoclonal antibody reagent labeled with the upconversion luminescent material is washed away, until only the combination of the monoclonal antibody reagent bound to the magnetic particles, the sample antigen, and the monoclonal antibody reagent labeled with the upconversion luminescent material remains in the reaction cup;

[0031] Step 5: Transfer the cuvette to the optical measurement module, which uses an excitation light source to excite the upconverted fluorescence, detects the fluorescence signal at a preset wavelength, and calculates the concentration of the analyte based on light intensity analysis.

[0032] Preferably, after several rounds of washing and separation by the magnetic microparticles, a preset amount of diluent is added to the reaction cup after the last separation, and the initial mixture containing the magnetic beads and the upconverted luminescent marker enters the optical detection module. The fluorescence signal detected by laser excitation is recorded as X1, and the concentration is calculated using the standard curve to obtain the target concentration Y1;

[0033] Pump a preset amount of buffer into the reaction cup to dilute the reaction system to concentration a1, measure the fluorescence signal X2 again, and calculate the target concentration Y2;

[0034] Pump the preset amount of buffer into the reaction cuvette again to dilute the reaction system to the original concentration a2, measure the fluorescence signal X3 again, and calculate the target concentration Y3;

[0035] Pump the preset amount of buffer into the reaction cuvette again to dilute the reaction system to the original concentration a3, measure the fluorescence signal X4 again, and calculate the target concentration Y4;

[0036] Pump the preset amount of buffer into the reaction cuvette again to dilute the reaction system to the original concentration a4, measure the fluorescence signal X5 again, and calculate the target concentration Y5;

[0037] Given the target concentration value Y, establish the least squares formula, Z = (Y-Y1) 2 +(a1*Y-Y2) 2 +(a2*Y-Y3) 2 +(a3*Y-Y4) 2 +(a4*Y-Y5) 2 ;

[0038] The Y value is obtained by the least squares method to minimize the Z value. Y is the optimal target concentration value of the analyte after eliminating the shielding effect of the magnetic beads.

[0039] The present invention relates to a magnetic particle upconversion chemiluminescence detection system. A sample transport module for inputting, preloading, and outputting a plurality of samples is provided on a working surface. A grabbing module is used to grab a reaction cup and a liquid pipette head from a previous working position to a next working position. A liquid sampling module is used to aspirate samples and reagents and place them in the reaction cup. The reagents here include at least a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent labeled with an upconversion luminescent material. A detection preparation module is used to mix, incubate, and wash and separate the sample and reagent placed in the reaction cup once or multiple times. An optical measurement module is used to excite the upconversion fluorescent marker and detect the intensity of the fluorescent signal.

[0040] The beneficial effects of the present invention are:

[0041] (1) It can be excited repeatedly. The excitation energy of high-power laser is much higher than the biological energy of bioluminescence, and the fluorescence signal intensity is greatly improved;

[0042] (2) In addition to the up-conversion material, the excitation light source generally cannot excite other wavelengths of fluorescence for other consumables and optical device materials used in the detection reaction, so the background fluorescence signal is small and the detection sensitivity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG1 is a schematic diagram of the front view structure of the present invention;

[0044] FIG2 is a schematic perspective structural diagram of the present invention with part of the housing removed;

[0045] FIG3 is a schematic diagram of the structure from a top view of FIG2 ;

[0046] FIG4 is a schematic diagram of the structure of FIG2 from a side view;

[0047] FIG5 is a schematic structural diagram of the optical measurement module of the present invention;

[0048] FIG6 is a schematic structural diagram of a cross-sectional view taken along line AA of FIG5 . DETAILED DESCRIPTION

[0049] The present invention is further described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0050] The present invention relates to a magnetic microparticle upconversion chemiluminescence detection system. It should be noted that the mechanisms involving X-axis and Y-axis translation and Z-axis vertical movement in the following embodiments are generally implemented by a structure in which a motor outputs power to a driving wheel, and the driving wheel drives a driven wheel through a synchronous belt. Those skilled in the art can also implement it using existing transmission mechanisms, which is a conventional technology in this field.

[0051] The system of the present invention is described in detail below. It is agreed that the main working surface is the XOY coordinate plane, the left and right direction is the X-axis direction, and the front and back direction is the Y-axis direction. The structural setting in the present invention is adjustable and is not limited to the X-axis and Y-axis directions in the embodiment.

[0052] Example 1

[0053] The system includes a working surface provided with:

[0054] A sample carrier module 1, used for inputting, preloading and outputting a number of samples;

[0055] A grabbing module 2, used to realize the grabbing of the reaction cup and the pipette head from the previous working position to the next working position; obviously, a well plate 3 for placing the reaction cup and the pipette head is provided on the working surface that matches the reaction cup and the pipette head;

[0056] A liquid sampling module 4 is used to absorb samples and reagents and place them in a reaction cup, wherein the reagents include at least a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent labeled with an upconverting luminescent material;

[0057] A detection preparation module, used to perform one or more mixing, incubation, and multiple washing and separation of the sample and reagent placed in the reaction cup;

[0058] A light measurement module 5 is used to excite the upconverted fluorescent marker and detect the intensity of the fluorescent signal.

[0059] In the present invention, all modules of the system are controlled by a controller and work together. Those skilled in the art can set the controller according to their needs. The working process is as follows: the sample is loaded through the sample carrier module 1, the grabbing module 2 grabs the reaction cup and the pipette head, the liquid sampling and adding module 4 attaches the pipette head and adds the sample and reagents, the detection preparation module completes mixing, constant temperature incubation, magnetic particle separation and cleaning, and finally transfers to the light measurement module 5. The concentration of the sample to be tested is obtained by detecting the intensity of the excited fluorescence.

[0060] In the present invention, it is obvious that the working surface and the modules thereon are provided with a body shell 6, and the front side of the shell is provided with a control panel 7 and a display screen to facilitate operation by the operator; at the same time, the sample carrying module 1 is set as an open area to facilitate loading and unloading of the sample rack 9.

[0061] In the present invention, upconversion luminescent material (UCP) is used as a fluorescent marker. In an independent reaction cup, magnetic microparticle solid-phase labeled antibody binding and separation technology is used to clean the unbound fluorescent marker, and the UCP material is excited by a high-power near-infrared laser to emit fluorescence, thereby achieving high-precision detection.

[0062] Example 2

[0063] Based on Example 1, the sample transport module 1 includes a sample loading channel 10 and a sample output channel 11; a sample inlet for inserting a sample rack 9 is provided in conjunction with the sample loading channel 10, and a sample outlet for outputting the sample rack 9 is provided in conjunction with the sample output channel 11; and a traction unit is provided in conjunction with the sample loading channel 10 and the sample output channel 11 of the sample rack 9;

[0064] The ends of the sample loading channel 10 and the sample output channel 11 are provided with a moving frame 12 in cooperation with the sample rack 9, and a moving traction unit is provided in cooperation with the moving frame 12;

[0065] A capping position is provided between the sample loading channel 10 and the sample output channel 11, and a capping mechanism 14 is provided corresponding to the capping position; a roller 13 is provided in contact with the sample tube 8 in cooperation with the sample loading channel 10, and a barcode scanning mechanism (not shown in the figure) is provided on the side corresponding to the capping position for cooperating with the sample tube 8;

[0066] A sampling position 15 is provided in conjunction with the sample loading channel 10 , and the sampling position is provided in conjunction with the liquid sampling and loading module 4 .

[0067] In the present invention, the sample transport module 1 realizes the transportation of samples and simultaneously matches functions such as barcode recognition and lid opening; the sample loading channel 10 and the sample output channel 11 of the sample transport module 1 are respectively arranged on both sides of the front end of the working surface and placed along the X-axis; in actual applications, the sample loading channel 10 can load up to 5 sample racks 9 at a time. Taking 10 samples placed in each sample rack 9 as an example, a total of 50 samples; the volume of each sample tube 8 can be 200uL-2000uL.

[0068] In the present invention, taking the sample loading channel 10 as an example, it is sequentially arranged as an input conveyor along the X-axis, an input queue area along the Y-axis, and a traction area of ​​the moving frame 12. The traction area of ​​the moving frame 12 matches the sample output channel 11 and is used to move the sample to the sample output channel 11 for output. The sample output channel 11 is symmetrically arranged with the sample loading channel 10.

[0069] The input conveyor is a conventional technology in the art, and those skilled in the art can design it according to their needs, or can omit it. When the input conveyor is set, the sample rack 9 is preset to stop when it is aligned with the input queue area.

[0070] The input queue area is provided with a panel corresponding to the input queue area along the Y axis. The panel is provided with at least one long hole for passing the traction unit. The traction unit here includes a traction buckle that passes through the long hole respectively. The traction buckle is buckled with the bottom of the sample rack 9 to drive the sample rack 9 to move; the traction buckle is connected to the synchronous belt of the synchronous transmission mechanism through a connecting block. The synchronous transmission mechanism is a conventional technology in this field, including a motor, a driving wheel provided at the output end of the motor, a driven wheel cooperating with the driving wheel, and a synchronization provided outside the driving wheel and the driven wheel;

[0071] The traction area of ​​the moving frame 12 is located at the end of the input queue area. When the sample rack 9 reaches the traction area of ​​the moving frame 12, it will directly enter the position of the moving frame 12. The moving traction unit is also configured as a conveying mechanism in the X-axis direction. Through the conveying mechanism, the moving frame 12 drives the sample rack 9 forward within the X-axis range to the capping position (also the scanning position), and then retreats to the sample sampling position 15. After processing all samples in the current sample rack 9, the entire sample rack 9 is driven to the sample output channel 11.

[0072] The structure of the sample output channel 11 is symmetrical with that of the sample loading channel 10, and is sequentially arranged as a traction area of ​​the moving frame 12, an output queue area along the Y axis, and an output conveyor belt along the X axis, wherein the output conveyor belt along the X axis moves in an opposite direction to the input conveyor belt along the X axis, and the output queue area along the Y axis moves in an opposite direction to the input queue area along the Y axis.

[0073] In the present invention, generally speaking, the capping position and the scanning position are consistent. When the sample rack 9 is moving forward, the roller 13 is arranged tangentially to the sample tube 8, which is used to drive the sample tube 8 to roll in a circle, and the roller 13 is used to move the barcode toward the barcode scanning mechanism; the barcode scanning mechanism scans the rolling sample tube 8 in real time to obtain sample information, so that the sample tube 8 can be placed in a disorderly order and can be scanned; while scanning to obtain the sample information, the capping mechanism 14 is used to remove the cap, and the capping mechanism 14 includes a downward component and a grasping component, such as a robot arm. This is content that can be easily understood by those skilled in the art, and those skilled in the art can set it according to their needs.

[0074] In the present invention, in order to facilitate the internal setting of the system, the sampling position 15 cooperates with the liquid sampling and loading module 4. The cooperation here means that the liquid sampling and loading module 4 moves on the Y axis in the XOY coordinate system, so the sampling position 15 is located directly in front of the liquid sampling and loading module 4.

[0075] Example 3

[0076] On the basis of Example 1, a ferry transport auxiliary module is provided in conjunction with the grabbing module 2 and the liquid sampling and adding module 4, for assisting transport between the grabbing module 2 and the liquid sampling and adding module 4;

[0077] The ferrying and transporting auxiliary module includes a moving part 26 with a plurality of holes. The moving part 26 is arranged on a track 16 . The track 16 is arranged between the grabbing module 2 and the liquid sampling and adding module 4 .

[0078] In the present invention, in order to reasonably arrange the structure within the system, as mentioned above, the liquid sampling and adding module 4 moves on the Y axis in the XOY coordinate system, and the grabbing module 2 is arranged in parallel with the liquid sampling and adding module 4 and moves on the X axis and Y axis in the XOY coordinate system, and can meet the movement of the grabbing mechanism on the Z axis. At the same time, since it involves the coordination of multiple actions, in order to arrange the mechanism more reasonably, a ferrying and transporting auxiliary module is set in the grabbing module 2 and the liquid sampling and adding module 4. Specifically, the moving part 26 is in the form of a track 16. The track 16 is arranged along the X axis direction, and in order to facilitate the grabbing module 2 and the liquid sampling and adding module For the operation of the sample module 4, a certain height difference can be set on the top of the track 16 along the X-axis direction, and a number of holes are set on the movable part 26, generally three, which are used to place two reaction cups and one pipette head (Tip head) respectively; the side of the movable part 26 is equipped with a transmission mechanism 18 through a vertical slide bar 17, and the transmission mechanism 18 performs X-axis transmission. The slide bar 17 can move along the X-axis direction, while satisfying the movement of the movable part 26 in the X-axis direction on the track 16 under its traction. By setting a vertical hole matching the movable part 26 on the slide bar 17, the freedom of the movable part 26 on the Y-axis is realized.

[0079] Example 4

[0080] On the basis of Example 3, the grabbing module 2 includes a three-axis motion mechanism stacked above the working surface, a grabbing mechanism is provided at the lower part of the Z-axis of the three-axis motion mechanism, and the grabbing mechanism is arranged in conjunction with the ferrying and transport auxiliary module; the working surface of the grabbing mechanism is respectively provided with a well plate 3 for placing the pipette head and the reaction cup and a detection preparation module.

[0081] The liquid sampling and loading module 4 includes a Y-axis and Z-axis biaxial motion mechanism arranged above the working surface. In conjunction with the biaxial motion mechanism, a first liquid sampling mechanism 19 for picking up the pipette head and sucking the sample liquid from the sampling position 15 of the sample loading channel 10 into the reaction cup and a second liquid sampling mechanism 20 for sucking the reagent into the reaction cup are provided. The first liquid sampling mechanism 19 and the second liquid sampling mechanism 20 are arranged in conjunction with the ferrying and transporting auxiliary module, and a reagent storage chamber 21 is provided in conjunction with the second liquid sampling mechanism 20.

[0082] The reagent storage chamber 21 includes a storage chamber cavity, and a rotating mechanism (not shown in the figure) is provided at the bottom of the storage chamber cavity. Several reagent links 22 are divergently provided in the storage chamber cavity; any reagent link 22 includes a reagent tube respectively provided with a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent marked with an upconverted luminescent material.

[0083] In the present invention, the liquid taking and sample adding module 4 uses a Y-axis and Z-axis biaxial motion mechanism. More precisely, the Y-axis motion mechanism realizes the transfer of the liquid taking mechanism, and the Z-axis motion mechanism realizes the liquid taking and sample adding of the liquid taking mechanism. The liquid taking mechanism includes a first liquid taking mechanism 19 and a second liquid taking mechanism 20, which are respectively used to pick up the liquid taking head and suck the sample liquid into the reaction cup from the sampling position 15 of the sample loading channel 10, and suck the reagent into the reaction cup; the liquid taking head (Tip head) is grabbed by the grabbing module 2 and placed in the hole position of the moving part 26. The liquid taking end of the first liquid taking mechanism 19 moves downward and connects with the liquid taking head to suck the sample liquid; the reagent storage chamber 21 cooperating with the second liquid taking mechanism 20 can rotate around the central axis. After rotating to the corresponding position, the liquid taking head of the second liquid taking mechanism 20 can take liquid and add it to the reaction cup. The reagent link 22 at least includes a monoclonal antibody reagent with magnetic particle binding and a monoclonal antibody reagent labeled with upconverted luminescent material;

[0084] Since the second liquid taking mechanism 20 needs to be cleaned after each reagent addition, the second liquid taking mechanism 20 is composed of a steel needle structure and a cleaning structure; the first liquid taking mechanism 19 adds samples through the Tip head to avoid cross-interference of samples. After each liquid is taken, the liquid taking end is separated from the Tip head and is ready to enter the next liquid taking;

[0085] During the process, the volume of each reaction cup is generally 1000uL, and each detection item requires an independent reaction cup. The sample is added using ADP and a disposable Tip pipette structure, and the sample volume is generally 50-100uL. The liquid pipette head is set in the form of a Tip head to effectively avoid carryover contamination between samples. The reagent is added using a reagent needle, and the steel needle controls the sample volume through a plunger pump. The reagent is added twice, and the sample volume each time is 150uL. Each time it is added, the inner and outer walls are cleaned through the cleaning hole to avoid reagent contamination; the reagent storage chamber 21 is generally provided with 12 slots for reagent links 22, and a rotating mechanism is provided at the bottom of the storage chamber cavity. The rotating mechanism drives the magnetic particle reagent tube of the reagent link 22 to rotate, ensuring that the magnetic bead liquid can remain suspended when the reagent link 22 is placed in the storage chamber cavity.

[0086] In the present invention, in actual application, a liquid level detection sensor is provided at the liquid taking end of the first liquid taking mechanism 19, which can automatically identify the liquid level and accurately add samples when the liquid level is detected to be below the liquid level.

[0087] In the present invention, the gripping module 2 uses a gripping mechanism, such as a gripper, to transport the reaction cup and the pipette tip, and returns to a fixed position after sample addition is completed, so that the gripper can transfer the reaction cup from the moving block, the vibration mixing unit 23, the incubation unit 24, the magnetic particle cleaning and separation unit 25, and the optical measurement module 5 in sequence. The structure of the bidirectional motion mechanism of the gripping module 2 and the gripping mechanism connected thereto, such as the gripper, is easily understood by those skilled in the art and can be configured by those skilled in the art according to their needs. The range of motion of the bidirectional motion mechanism needs to cover the well plate 3 on which the pipette tip and reaction cup are placed and the detection preparation module.

[0088] Example 5

[0089] Based on Example 1, the detection preparation module includes an incubation unit 24, a vibration mixing unit 23 and a magnetic particle cleaning and separation unit 25; the magnetic particle cleaning and separation unit 25 includes a turntable, which is provided with position 0 and at least 3 groups of cleaning positions, any group of cleaning positions includes a liquid extraction position and a liquid injection position, and the last liquid injection position is a pre-excitation position; a high-strength permanent magnet is provided on the outer wall of the turntable.

[0090] In the present invention, the incubation unit 24 allows the reaction cup loaded with samples and reagents to be incubated at a preset temperature, such as 37±0.1°C, according to experimental requirements. It includes a carrier with multiple wells, such as 60, which can support 60 reaction cups for incubation at the same time. The incubation time here can be controlled by software, and an alarm can be issued immediately when the temperature is abnormal.

[0091] In the present invention, the vibration mixing unit 23 is used to place the reaction cup therein (on it) for vibration mixing, including but not limited to before incubation, after incubation, after cleaning and separation, and after injection of the pre-excitation solution.

[0092] In the present invention, the magnetic particle cleaning and separation unit 25 realizes the cleaning and separation of magnetic beads and the recovery of waste liquid. An independent cleaning liquid pump is responsible for the injection of the cleaning liquid (injection position). Three peristaltic pumps are connected to three waste liquid needles 35 and fixed on the lifting frame 27. They are responsible for extracting the waste liquid in the reaction cup (extraction level). Finally, the pre-excitation liquid in the reaction cup is injected by the pre-excitation liquid pump. During the process, the extraction level and the injection level are set alternately, and a high-strength permanent magnet is set on the outer wall of the turntable to ensure that the combination of the monoclonal antibody reagent bound to the magnetic particles, the antigen of the sample and the monoclonal antibody reagent marked with the up-converted luminescent material is on the cup wall of the reaction cup. The high-strength permanent magnet can be purchased by those skilled in the art according to their needs based on the measured value; the setting of the lifting frame 27 here is a well-known technology in the field, and those skilled in the art can set it according to their needs.

[0093] Example 6

[0094] Based on Example 1, the optical measurement module 5 includes a detection position 28, which is arranged in conjunction with a measurement chamber 29 via an absorbent motion drawer. The absorbent motion drawer corresponding to the lower portion of the reaction cup is provided with a hollow 30. A light detector 31 and an excitation light source 32 are provided in the measurement chamber 29 corresponding to the hollow 30. A dichroic beam splitter 33 is provided between the light detector 31 and the hollow 30. The incident surface of the dichroic beam splitter 33 faces the excitation light source 32, the reflective surface faces the hollow 30, and the transmissive surface faces the light detector 31.

[0095] In the present invention, the detection position 28 of the optical measurement module 5 is a retractable motion drawer structure, which cooperates with the measurement chamber 29 through the retractable motion drawer. This retractable motion drawer is realized by an electronic control mechanism. This is easy to understand for those skilled in the art, and those skilled in the art can configure it according to their needs.

[0096] After cleaning and mixing, the cuvette is transferred to the detection position 28, and the absorbing movable drawer is closed toward the measurement chamber 29; the excitation light source 32 is activated to excite the sample to be tested, such as a high-power 980nm near-infrared laser. After being emitted to the dichroic mirror 33, it is reflected toward the sample to be tested in the cuvette corresponding to the hollow 30, and excited fluorescence is obtained. After the excited fluorescence is transmitted through the dichroic mirror 33, the fluorescence signal of a specific wavelength, such as 540nm, is measured by the light detector 31. Here, a high-sensitivity PMT is used to detect the photon signal generated by chemiluminescence.

[0097] In the present invention, in order to further improve the detection effect, a combination 34 of a polarizing plate, an interference filter, a color filter, etc. can be set in the corresponding channel of the measurement chamber 29.

[0098] In the present invention, after the measurement is completed, the grabbing module 2 can transfer the reaction cup to the waste liquid recovery position, discard the reaction cup, or extract the waste liquid first and then discard the reaction cup.

[0099] Example 7

[0100] Based on Example 1, the detection method of the system includes the following steps:

[0101] Step 1: The sample is placed in the sample rack 9 and enters the system through the sample carrier module 1;

[0102] Step 2: Scan the number of the unprocessed sample tube 8 in the first position, download the test items according to the number of the sample tube 8, and automatically remove the cap of the sample tube 8. During this process, the gripping module 2 picks up the reaction cup and the pipette head to the preset position;

[0103] Step 3: The liquid sampling module 4 is loaded with a pipette head, which absorbs a quantitative sample and adds it to the reaction cup; the liquid sampling module 4 respectively absorbs the monoclonal antibody reagent bound to the magnetic particles and the monoclonal antibody reagent labeled with the upconverted luminescent material according to the obtained detection item information, and adds them to the reaction cup with the existing sample to perform a double antibody sandwich immunoreaction or a competitive immunoreaction;

[0104] Step 4: The liquid sampling module 4 waits for the next sample tube 8 to be loaded; the grabbing module 2 picks up the reaction cup with the sample and reagent added, transfers it to the detection preparation module, shakes and mixes it, and incubates it at a constant temperature for a preset time for cleaning and separation; during the cleaning and separation, the magnet adsorbs the magnetic particles to the inner wall of the reaction cup, and the unbound monoclonal antibody reagent labeled with the upconversion luminescent material is washed away, until only the combination of the monoclonal antibody reagent bound to the magnetic particles, the sample antigen, and the monoclonal antibody reagent labeled with the upconversion luminescent material remains in the reaction cup;

[0105] Step 5: The cuvette is transferred to the optical measurement module 5 . The optical measurement module 5 excites the upconverted fluorescence with the excitation light source 32 , detects the fluorescence signal of the preset wavelength, and calculates the concentration of the analyte based on the light intensity analysis.

[0106] During the implementation of the present invention, the sample in the blood collection tube is placed in the sample rack 9, passes through the input conveyor along the X axis and the input queue area along the Y axis, until it reaches the traction area of ​​the moving frame 12. The moving frame 12 drives the sample rack 9 to rub the sample tube 8 by the roller 13 and continuously scans the sample tube 8 number. The test item is downloaded according to the sample number, and the sample tube 8 is uncapped. The ADP module of the first liquid collection mechanism 19 is loaded with a liquid suction head, which is inserted below the liquid surface through automatic liquid level detection to absorb a quantitative sample and then added to the reaction cup. The sample tube 8 is covered with a lid and moved out of the sample loading position, waiting for the next sample tube 8 to be loaded; the second liquid collection mechanism 20 extracts the monoclonal antibody reagent bound to the magnetic particles and the upper transfer according to the acquired test item information. A monoclonal antibody reagent labeled with a luminescent material is added to a reaction cup containing a sample for a double antibody sandwich immunoreaction or a competitive immunoreaction. The reaction cup is then grabbed by the grabbing module 2 and transferred to the vibration mixing unit 23. After high-speed vibration, it is transferred to the incubation unit 24. After incubation at a constant temperature for 10 minutes, it enters the magnetic particle cleaning and separation unit 25. The magnet adsorbs the magnetic beads to the wall of the reaction cup tube, and the up-converted labeled antibody that is not bound to the magnetic particles is washed away, leaving only the complex to be tested in the reaction cup. After adding a certain diluent, the reaction cup is transferred to the optical measurement module 5. The optical measurement module 5 uses a 980nm high-power near-infrared laser to excite the up-converted fluorescence and detects the fluorescence signal with a wavelength of 540nm to analyze and calculate the concentration of the analyte.

[0107] However, upconverter luminescence markers (UCPs) can be excited multiple times by external near-infrared 980nm lasers, and the stimulated emission of fluorescent signals is stable, without any phenomenon such as fluorescence signal bleaching or decline. However, in chemiluminescence systems, since the particle size of magnetic beads is generally much larger than the volume of antibody proteins and fluorescent labels, there is a certain degree of fluorescence signal masking, resulting in poor repeatability of signal detection. In particular, luminescence technology based on acridinium lipid biochemical reactions can only be excited once by the excitation solution. The masking effect of magnetic beads on the fluorescent signal is more random, making it difficult to improve the repeatability of multiple measurements.

[0108] Example 8

[0109] Based on Example 7, after multiple washing and separation of the magnetic particles, 300 μL of diluent was added to the reaction cup after the final separation. The initial mixture containing the magnetic beads and the upconverted luminescent marker entered the optical detection module. The fluorescence signal detected by laser excitation was recorded as X1, and the concentration was calculated using the standard curve to obtain the target concentration Y1.

[0110] Here, the standard curve generally converted from fluorescence signal to target concentration is a four-parameter fitting algorithm. The relevant parameters and fitting curve of the four-parameter fitting algorithm are obtained by preselecting the standard solution. Based on this,

[0111] Use the liquid addition plunger pump to pump 100uL of buffer into the reaction cup to dilute the reaction system to 3 / 4 of the original concentration, measure the fluorescence signal X2 again, and calculate the target concentration Y2;

[0112] Then, 100 μL of buffer solution is pumped into the reaction cup using the liquid addition plunger pump to dilute the reaction system to 3 / 5 of the original concentration. The fluorescence signal X3 is measured again, and the target concentration Y3 is calculated.

[0113] Furthermore, 100 μL of buffer solution is pumped into the reaction cup using a liquid adding plunger pump to dilute the reaction system to 1 / 2 of the original concentration. The fluorescence signal X4 is measured again, and the target concentration Y4 is calculated.

[0114] Finally, 200 μL of buffer solution was pumped into the reaction cup using a liquid addition plunger pump to dilute the reaction system to 3 / 8 of the original concentration. The fluorescence signal X5 was measured again, and the target concentration Y5 was calculated.

[0115] Given the target concentration value Y, establish the least squares formula:

[0116] Z=(Y-Y1) 2 +(0.75*Y-Y2) 2 +(0.6*Y-Y3) 2 +(0.5*Y-Y4) 2 +(0.375*Y-Y5) 2 ;

[0117] The Y value is obtained by the least squares method to minimize the Z value. Y is the optimal target concentration value of the analyte after eliminating the shielding effect of the magnetic beads.

[0118] During implementation, multiple additions of buffer can be made directly in the measurement area of ​​the optical measurement module 5. The present invention gradually dilutes the sample concentration by adding buffer multiple times in a cascade. The newly added buffer will disturb the orientation of the magnetic beads in the original system. Based on multiple detections, the fluorescence signal intensities at different dilution concentrations are obtained, and the optimal concentration value can be obtained using the least squares principle. Since the buffer added between multiple detections disturbs the orientation of the magnetic beads, the fluorescence signal values ​​obtained from multiple measurements have multi-directional signal characteristics, which can obtain more uniform fluorescence signal intensity, solve the problem of fluorescence signal shielding by the magnetic beads, and improve repeatability.

Claims

1. A magnetic microparticle upconversion chemiluminescence detection system, characterized in that: The system comprises a working surface, on which is provided: A sample carrier module, used for inputting, preloading and outputting a number of samples; A grabbing module, used to realize the grabbing of the reaction cup and the pipette head from the previous working position to the next working position; A liquid sampling module, used for taking samples and reagents and placing them in a reaction cup, wherein the reagents at least include a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent labeled with an upconversion luminescent material; A detection preparation module, used for mixing, incubating, washing and separating the sample and reagent placed in the reaction cup once or multiple times; A light measurement module is used to excite the upconverted fluorescent marker and detect the intensity of the fluorescent signal.

2. A magnetic microparticle upconversion chemiluminescence detection system according to claim 1, characterized in that: The sample transport module includes a sample loading channel and a sample output channel; A sample inlet for placing a sample rack is provided in cooperation with the sample loading channel, a sample outlet for outputting the sample rack is provided in cooperation with the sample output channel, and a traction unit is provided in cooperation with the sample loading channel and the sample output channel of the sample rack; The ends of the sample loading channel and the sample output channel are provided with a moving frame in cooperation with the sample rack, and a moving traction unit is provided in cooperation with the moving frame; A cover removal position is provided between the sample loading channel and the sample output channel, and a cover removal mechanism is provided corresponding to the cover removal position; A roller tangent to the sample tube is provided in cooperation with the sample loading channel, and a barcode scanning mechanism matching the sample tube is provided on the side corresponding to the cover removal position; A sampling position is provided in cooperation with the sample loading channel, and the sampling position is provided in cooperation with the liquid sampling and adding module.

3. A magnetic microparticle upconversion chemiluminescence detection system according to claim 1, characterized in that: A ferrying and transporting auxiliary module is provided in cooperation with the grabbing module and the liquid sampling and adding module, and is used to assist in transporting between the grabbing module and the liquid sampling and adding module; The ferrying and transporting auxiliary module comprises a moving part with a plurality of holes, wherein the moving part is arranged on a track, and the track is arranged between the grabbing module and the liquid sampling and adding module.

4. A magnetic microparticle upconversion chemiluminescence detection system according to claim 3, characterized in that: The grabbing module includes a three-axis motion mechanism stacked above the working surface, a grabbing mechanism is provided at the lower part of the Z-axis of the three-axis motion mechanism, and the grabbing mechanism is arranged in cooperation with the ferrying and transport auxiliary module; a well plate and a detection preparation module for placing a pipette head and a reaction cup are respectively provided on the working surface cooperating with the grabbing mechanism.

5. A magnetic microparticle upconversion chemiluminescence detection system according to claim 3, characterized in that: The liquid sampling and loading module includes a Y-axis and Z-axis biaxial motion mechanism arranged above the working surface, and a first liquid sampling mechanism for picking up a liquid pipette head and sucking sample liquid from a sampling position of a sample loading channel into a reaction cup and a second liquid sampling mechanism for sucking reagents into the reaction cup are provided in conjunction with the biaxial motion mechanism. The first liquid sampling mechanism and the second liquid sampling mechanism are arranged in conjunction with a ferrying and transporting auxiliary module, and a reagent storage chamber is provided in conjunction with the second liquid sampling mechanism.

6. A magnetic microparticle upconversion chemiluminescence detection system according to claim 5, characterized in that: The reagent storage chamber comprises a storage chamber cavity, a rotating mechanism is arranged at the bottom of the storage chamber cavity, and a plurality of reagent links are arranged in a divergent shape in the storage chamber cavity; any reagent link comprises a reagent tube respectively provided with a monoclonal antibody reagent bound to magnetic particles and a monoclonal antibody reagent marked with an upconverted luminescent material.

7. A magnetic microparticle upconversion chemiluminescence detection system according to claim 1, characterized in that: The detection preparation module includes an incubation unit, a vibration mixing unit and a magnetic particle cleaning and separation unit; the magnetic particle cleaning and separation unit includes a turntable, on which is provided a No. 0 position and at least 3 groups of cleaning positions, any group of cleaning positions includes a liquid extraction position and a liquid injection position, and the last liquid injection position is a pre-excitation position; a high-strength permanent magnet is provided in conjunction with the outer wall of the turntable.

8. The magnetic microparticle upconversion chemiluminescence detection system according to claim 1, characterized in that: The optical measurement module includes a detection position, which is arranged in cooperation with the measurement chamber through an absorption-type motion drawer. The absorption-type motion drawer corresponding to the lower part of the reaction cup is provided with a hollow, and a light detector and an excitation light source are provided in the corresponding hollowed-out measurement chamber. A dichroic mirror is provided between the light detector and the hollowed-out space, and an incident surface of the dichroic mirror faces the excitation light source, a reflection surface faces the hollow, and a transmission surface faces the light detector.

9. A magnetic microparticle upconversion chemiluminescence detection system according to claim 1, characterized in that: The detection method of the system comprises the following steps: Step 1: The sample is placed in the sample rack and enters the system through the sample transport module; Step 2: Scan the number of the unprocessed sample tube in the first position, download the test items according to the sample tube number, and automatically remove the cover of the sample tube. During the process, the gripping module picks up the reaction cup and the pipette head to the preset position; Step 3: The liquid sampling module is loaded with a liquid pipette head, which absorbs a quantitative sample and adds it to a reaction cup; the liquid sampling module absorbs the monoclonal antibody reagent bound to magnetic particles and the monoclonal antibody reagent labeled with an upconverted luminescent material according to the acquired detection item information, and adds them to the reaction cup with the existing sample to perform a double antibody sandwich immune reaction or a competitive immune reaction; Step 4: The liquid sampling module waits for the next sample tube to be sampled; the grabbing module picks up the reaction cup that has been added with the sample and reagent, transfers it to the detection preparation module, shakes and mixes it, and incubates it at a constant temperature for a preset time for cleaning and separation; during cleaning and separation, the magnet adsorbs the magnetic particles to the inner wall of the reaction cup, and the unbound monoclonal antibody reagent labeled with the upconversion luminescent material is washed, until only the monoclonal antibody reagent bound to the magnetic particles, the antigen of the sample, and the monoclonal antibody labeled with the upconversion luminescent material remain in the reaction cup. A combination of reagents; Step 5: Transfer the reaction cup to the optical measurement module, which excites the upconverted fluorescence with an excitation light source, detects the fluorescence signal of a preset wavelength, and calculates the concentration of the analyte based on light intensity analysis.

10. A magnetic microparticle upconversion chemiluminescence detection system according to claim 9, characterized in that: After several washing and separations are completed by the magnetic microparticles, a preset amount of diluent is added to the reaction cup after the last separation, and the initial mixed solution containing magnetic beads and upconverted luminescent markers enters the optical detection module, and the fluorescence signal detected by laser excitation is recorded as X1. The concentration is calculated by the standard curve to obtain the target concentration Y1; Pump a preset amount of buffer into the reaction cup to dilute the reaction system to the concentration a1, measure the fluorescence signal X2 again, and calculate the target concentration Y2; Pump a preset amount of buffer into the reaction cup again to dilute the reaction system to the original a2 concentration, measure the fluorescence signal X3 again, and calculate the target concentration Y3; Pump a preset amount of buffer into the reaction cup again to dilute the reaction system to the original concentration a3, measure the fluorescence signal X4 again, and calculate the target concentration Y4; Pump a preset amount of buffer into the reaction cup again to dilute the reaction system to the original a4 concentration, measure the fluorescence signal X5 again, and calculate the target concentration Y5; Given the target concentration value as Y, establish the least squares formula, Z = (Y-Y1) 2 +(a1*Y-Y2) 2 +(a2*Y-Y3) 2 +(a3*Y-Y4) 2 +(a4*Y-Y5) 2 ; The Y value is obtained by the least square method to minimize the Z value. Y is the optimal target concentration value of the analyte after eliminating the shielding effect of the magnetic beads.

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