Chemiluminescence immunodetection analyzer for in-situ luminescence determination and detection method
Technical Field
The invention relates to rapid detection and analysis of hormones, infection markers, cardiac markers, tumor markers and drugs, in particular to a chemiluminescence immunoassay analyzer and a detection method for in-situ luminescence determination.
Background
The immunological detection is a method for detecting mainly by utilizing a specific reaction between an antigen and an antibody, and can amplify and display a detection signal by using an isotope, an enzyme, a chemiluminescent substance, or the like, and is used for detecting a trace amount of a substance such as a protein, a hormone, or the like.
Chemiluminescence immunoassay is a novel labeling immunoassay technology which combines a chemiluminescence substrate with immunoreaction and is used for detecting trace antigens or antibodies.
The principle of the chemiluminescence reaction is that a luminescent substance or an enzyme is labeled on an antigen (or an antibody), and the luminescent substance is excited to emit light by oxidizing a substrate or the substrate is acted on by the enzyme to enable the substrate to emit light.
The core device in the chemiluminescence immune analyzer is a single photon counter, which is a vacuum electronic device capable of converting weak light signals into electric signals, the electric signals are detected by a single photon detector and transmitted to an amplifier, high-voltage current is added for amplification, the amplifier converts analog signals into digital signals, and the digital signals are transmitted to a computer for calculation to obtain final data. The chemiluminescence immunodetection is generally accepted in clinic due to the characteristics of environmental protection, rapidness, accuracy and the like, and becomes a main means of clinical immunodiagnosis at present.
POCT (Point-of-Care Testing) refers to clinical Testing conducted at the bedside of a patient, and in the POCT Testing technology, the common methods of POCT projects of immunoassay in the market at present are immunoturbidimetry and immunochromatography. Both methods have the limitations of measurement, the detection sensitivity is low, the full quantitative detection cannot be realized, and compared with the mainstream instrument which is a different platform, the detection result cannot be transversely compared. A small chemiluminescence determinator is needed at present, and can perform detection on the same platform as a mainstream large chemiluminescence determinator, so that transverse comparison can be realized.
The main stream of chemiluminescence immune instruments in the market at present is full-automatic, high-flux and multi-sample, which is more suitable for central laboratories of large hospitals, and is not suitable for the instant detection of clinical departments, and from the trends of market development and national promotion of graded diagnosis and treatment, the large-scale immune instrument test is not suitable for the small-sample detection of primary hospitals, and the market further needs a small-sized chemiluminescence analyzer capable of single-item detection.
Disclosure of Invention
The invention aims at the defects in the prior art and provides a chemiluminescence immunoassay analyzer and a chemiluminescence immunoassay detection method for in-situ luminescence determination.
In order to achieve the purpose, the invention adopts the following technical scheme that the device comprises a platform; it is characterized in that the platform is divided into four areas: a reagent tube storage area, a sample area to be detected, a reaction and cleaning area and an optical detection area.
The reagent tube storage area comprises a reagent tube rack and a first manipulator.
The sample area to be tested comprises a sample adding device, a sample pipe frame for placing a sample pipe and a sampling needle cleaning device for cleaning a sampling needle.
The sample adding device comprises a group of two-dimensional moving sliding tables and sampling needles arranged on the two-dimensional moving sliding tables; the two-dimensional moving sliding table drives the sampling needle to move to the sample tube to absorb a sample; the sampling needle cleaning device comprises a waste liquid tank and a sampling needle cleaning device arranged above the waste liquid tank.
The reaction and cleaning area comprises a semi-closed box body; an incubation reaction track is arranged in the semi-closed box body; the incubation reaction track comprises a synchronous belt transmission mechanism, a plurality of cylindrical teeth are arranged on the periphery (track) of a synchronous belt of the synchronous belt transmission mechanism at equal intervals, and the cross section of each cylindrical tooth is hourglass-shaped; the adjacent cylindrical teeth form a reagent tube clamping part (or called an independent test tube rack) for clamping a reagent tube.
The optical detection area comprises a darkroom, an exciting liquid adding device and a second manipulator.
The darkroom is a semi-closed structure with an opening at the upper end, the lower end of the darkroom is connected with the single photon counter, and after the reagent tube is put into the darkroom from the opening at the upper end, the upper cover of the darkroom is closed to form a light-proof darkroom.
The excitation liquid adding device comprises a second longitudinally moving sliding table, a darkroom upper cover connected with the second sliding table and an excitation liquid needle; the upper cover is connected with the sliding table sliding block of the sliding table II through a hollow tube, the liquid needle (device) is excited to be fixed on the sliding table sliding block, and the liquid needle body is excited to penetrate out of the hollow tube.
Further, first manipulator is located reagent pipe support top, just the reagent pipe support is used for bearing the reagent pipe of freeze-drying magnetic particle chemiluminescence reagent, first manipulator adopts miniature electric claw, and this miniature electric claw is driven by a set of three-dimensional slip table device.
Further, the sample tube rack comprises a sample plate and a sample plate driving motor; the motor shaft of the sample disc driving motor drives the sample disc to rotate, and sample tube placing grooves for placing sample tubes are arranged on the sample disc at equal intervals in the circumferential direction.
Further, the sampling needle cleaning device is positioned between the sample tube and the reaction position; the sampling needle cleaning device consists of a cleaning nozzle and a waste liquid tank and is used for cleaning the sampling needle; the sample adding device further comprises a diluent bottle containing diluent and a cleaning liquid bottle containing cleaning liquid.
Furthermore, the semi-closed box body is provided with a heating device and a temperature controller.
Furthermore, the synchronous belt transmission mechanism comprises a driving belt wheel, a driven belt wheel and a synchronous belt in transmission connection with the driving belt wheel and the driven belt wheel; the driving belt wheel is driven by a synchronous belt motor; the upper surface of the box body is provided with three openings: a sample loading position, a cleaning position and a removing position.
Furthermore, the reaction and cleaning area also comprises a magnetic bead cleaning device and a magnetic separation device.
The magnetic bead cleaning device is positioned on one side of the track cleaning position and comprises a first longitudinal moving sliding table and a cleaning needle which is connected with the first longitudinal moving sliding table and is driven to move by the first longitudinal moving sliding table.
The magnetic separation devices are positioned on two sides of the track cleaning position and comprise a magnet fixing frame, two permanent magnets arranged on the magnet fixing frame and a magnet fixing frame driving motor for driving the magnet fixing frame to move linearly; the two permanent magnets are arranged oppositely, and a gap is reserved between the two permanent magnets; when the device is used, the track cleaning position is positioned between the two permanent magnets, and the distance between the two permanent magnets is larger than the diameter of the reagent tube in the cleaning position; the magnet fixing frame driving motor is a linear motor, and a motor shaft extends out or retracts to drive the magnet fixing frame connected with the motor shaft to move forwards or backwards.
Further, the second manipulator comprises a miniature electric claw, and the miniature electric claw is driven by a group of two-dimensional sliding table devices.
Furthermore, the reagent tube storage area, the sample area to be detected, the reaction and cleaning area and the driving part of the optical detection area are all connected with the control system.
The chemiluminescence immunoassay method for in-situ luminescence determination comprises the following steps:
the reagent tube is placed in a reagent tube rack.
After the ID of the sample tube to be detected is input into the control system, the sample tube to be detected is placed at the designated position of the sample tray, and the sample tray rotates to enable the sample tube to be detected to rotate to the sampling position.
The system selects an item to be detected according to the ID information of the sample tube, the first manipulator grabs the reagent tube of the item to be detected and moves to a sample adding position (a first station) of a track in a box body of the reaction and cleaning area, and the first manipulator resets.
The sampling needle moves to a position to be sampled to absorb a sample, and then the sampling needle moves to a sample adding position of the reaction and cleaning area to add the sample; and adding a diluent into the reagent tube at the sample adding position by the sampling needle, and diluting the mixed solution of the sample and the freeze-dried reagent in the reagent tube.
The sampling needle repeatedly sucks and spits three times in the reagent tube to uniformly mix the solution.
And after the sampling needle is moved to the sampling needle cleaning device to clean the inner wall and the outer wall, the sampling needle is reset.
Starting timing after a sample is added into a reagent tube at a sample adding position (a first station), and moving a station from the sample adding position to a cleaning position (a ninth station) along a track at intervals of 90 seconds; the temperature controller controlled the temperature of the rail at 37 ℃.
When the reagent tube moves to the cleaning position, the magnet fixing frame drives the motor to drive the magnet fixing frame to enable the permanent magnet to be attached to one side of the reaction tube, magnetic particles are adsorbed on the wall of the reagent tube, the cleaning needle enters the reagent tube for cleaning, and waste liquid is discharged; then, the cleaning needle is withdrawn from the reagent tube, the magnet fixing frame drives the motor to drive the magnet fixing frame to make the permanent magnet on the other side movably attached to the other side of the reagent tube, and cleaning is carried out again; after three washes, the reagent tube is moved to the removal position (tenth station) along with the incubation track.
And the second manipulator moves to a moving-out position, the reagent tube is placed in the optical detection area darkroom, the sliding table II is moved longitudinally to drive the darkroom upper cover to move downwards, the darkroom is sealed, the exciting liquid needle injects exciting liquid into the reagent tube in the darkroom, the single photon counter detects the number of photons generated in the reagent tube, forwards the data to the control system, and calculates the concentration of the sample.
And after the detection is finished, the second longitudinally moving sliding table of the excitation liquid adding device moves upwards, and the second mechanical arm clamps the reagent tube and discards the reagent tube in a waste box to be treated as medical waste.
The above processes are repeated in a circulating way, and the detection is completed every 90 seconds. (40 samples can be tested every hour thereafter, except for 12 minutes for the first test).
Compared with the prior art, the invention has the beneficial effects.
The POCT full-automatic chemiluminescence immunoassay analyzer for in-situ detection can complete a series of operations such as reaction, incubation, cleaning, detection, data reading and the like in a chemiluminescence immunoassay process by automatically adding a sample to be detected into a special freeze-dried magnetic particle chemiluminescence reagent tube.
The method adopts acridinium ester as a substrate of the luminescent reagent to directly emit light, after the exciting liquid is added, the reagent does not need to move, the in-situ detection is directly carried out, the detection time is short, the detection is completed within 1 second, and the method has high luminescent efficiency, good repeatability and high sensitivity.
Drawings
The invention is further described with reference to the following figures and detailed description. The scope of the invention is not limited to the following expressions.
Fig. 1 is a schematic view of the overall structure of an embodiment of the present invention.
FIG. 2 is a schematic view of a reagent tube rack apparatus according to an embodiment of the present invention.
FIG. 3 is a schematic view of a sample tube holder apparatus according to one embodiment of the present invention.
FIG. 4 is a schematic view of a magnetic bead washing apparatus according to an embodiment of the present invention.
FIG. 5 is a schematic view of a track arrangement in the tank of the reaction and cleaning zone in accordance with one embodiment of the present invention.
FIG. 6 is a schematic view of a magnetic separation apparatus according to an embodiment of the present invention.
FIG. 7 is a schematic view of an exciting liquid adding apparatus according to an embodiment of the present invention.
Fig. 8 is a schematic view of a first robot according to an embodiment of the present invention.
Figure 9 is a schematic view of a second robot in accordance with an embodiment of the invention.
FIG. 10 is a schematic view of a sampling needle cleaning device in accordance with one embodiment of the present invention.
In the figure, 101 is a reagent tube, 102 is a reagent tube rack, 103 is a first manipulator, 201 is a sample tube, 202 is a sample tray, 203 is a sampling position, 204 is a sampling needle, 205 is a sampling needle cleaning device, 301 is a track, 302 is a sample adding position, 303 is a cleaning position, 304 is a magnet fixing frame driving motor, 305 is a magnet fixing frame, 306 is a permanent magnet, 307 is a cleaning needle, 308 is a removal position, 309 is a box body, 401 is a second manipulator, 402 is a dark room, 403 is a second longitudinally moving slide table, 404 is an upper cover, 405 is an excitation liquid needle, and 406 is a single photon counter.
Detailed Description
As shown in fig. 1-10, the present invention includes a platform; it is characterized in that the platform is divided into four areas: a reagent tube storage area, a sample area to be detected, a reaction and cleaning area and an optical detection area.
The reagent vessel storage section includes a reagent vessel rack 102 and a first robot 103. The reagent tube racks 102 are used for bearing reagent tubes 101 of the freeze-dried magnetic particle chemiluminescence reagent, and each reagent tube rack 102 bears 8-16 reagent tubes 101; the reagent storage area consists of 1-20 reagent racks 102.
The sample area to be tested comprises a sample adding device, a sample pipe frame for placing a sample pipe and a sampling needle cleaning device for cleaning a sampling needle;
the sample adding device comprises a group of two-dimensional moving sliding tables and sampling needles 204 arranged on the two-dimensional moving sliding tables; the two-dimensional moving sliding table drives the sampling needle to move to the sample tube to absorb a sample; the sampling needle cleaning device comprises a waste liquid tank and a sampling needle cleaning device arranged above the waste liquid tank.
The reaction and wash zone comprises a semi-enclosed tank 309; an incubation reaction track 301 is arranged in the semi-closed box body 309; the incubation reaction track comprises a synchronous belt transmission mechanism, a plurality of cylindrical teeth are arranged on the periphery (track) of a synchronous belt of the synchronous belt transmission mechanism at equal intervals, and the cross section of each cylindrical tooth is hourglass-shaped; the adjacent cylindrical teeth form a reagent tube clamping part (or called an independent test tube rack) for clamping a reagent tube. The track moves in the box, the upper part of the box is provided with three openings which are a sample adding position 302, a cleaning position 303 and a removing position 308 respectively, the sample adding position 302 is positioned at the first station, the cleaning position 303 is positioned at the ninth station, and the removing position 308 is positioned at the tenth station.
The optical detection area comprises a darkroom, an exciting liquid adding device and a second manipulator 401.
The darkroom is a semi-closed structure with an opening at the upper end, the lower end of the darkroom is connected with the single photon counter 406, and after the reagent tube is put into the darkroom from the opening at the upper end, the upper cover of the darkroom is closed to form a light-proof darkroom.
The exciting liquid adding device comprises a second longitudinally moving sliding table 403, a darkroom upper cover connected with the second sliding table 403, an exciting liquid needle 405, an exciting liquid A (bottle) and an exciting liquid B (bottle). The upper cover is connected with the sliding table sliding block of the second sliding table 403 through a hollow tube, the excitation liquid needle (device) is fixed on the sliding table sliding block, and the excitation liquid needle body penetrates out of the hollow tube. Specifically, the darkroom upper cover is connected with the sliding table sliding block through a hollow tube, and the liquid needle is excited to penetrate out through the hollow tube; when the sliding table moves downwards, the upper cover of the dark room closes the dark room, and the exciting liquid needle is inserted into the reagent tube and added with exciting liquid.
Preferably, the first manipulator 103 is located above the reagent tube rack 102, and the reagent tube rack 102 is used for carrying the reagent tubes 101 of the lyophilized magnetic particle chemiluminescence reagent, and the first manipulator 103 adopts a micro electric claw which is driven by a set of three-dimensional sliding table devices.
Preferably, the sample tube rack comprises a sample plate and a sample plate driving motor; the motor shaft of the sample disc driving motor drives the sample disc to rotate, and sample tube placing grooves for placing sample tubes are arranged on the sample disc at equal intervals in the circumferential direction.
Preferably, the sampling needle cleaning device is located between the sample tube 201 and the reaction site; the device consists of a cleaning nozzle and a waste liquid tank and is used for cleaning the sampling needle; the sample adding device further comprises a diluent bottle containing diluent and a cleaning liquid bottle containing cleaning liquid.
Preferably, the semi-closed box 309 is provided with a heating device and a temperature controller. The heating device is used for heating the track and can adopt an electric heating wire or electric heating oil and the like. In particular to the conventional technology in the field, and is not described in detail.
Preferably, the synchronous belt transmission mechanism comprises a driving pulley, a driven pulley and a synchronous belt in transmission connection with the driving pulley and the driven pulley; the driving belt wheel is driven by a synchronous belt motor; the upper surface of the box body is provided with three openings: a loading position 302, a washing position 303 and a removing position 308.
Preferably, the reaction and washing area further comprises a magnetic bead washing device and a magnetic separation device.
The magnetic bead cleaning device is positioned on one side of the track cleaning position and comprises a first longitudinal moving sliding table and a cleaning needle 307 which is connected with the first longitudinal moving sliding table and is driven by the first longitudinal moving sliding table to move; also comprises cleaning solution (bottle) and waste liquid (bottle).
The magnetic separation devices are positioned on two sides of the track cleaning position and comprise a magnet fixing frame, two permanent magnets arranged on the magnet fixing frame and a magnet fixing frame driving motor for driving the magnet fixing frame to move linearly; the two permanent magnets are arranged oppositely, and a gap is reserved between the two permanent magnets; when the device is used, the track cleaning position is positioned between the two permanent magnets, and the distance between the two permanent magnets is larger than the diameter of the reagent tube in the cleaning position; the magnet fixing frame driving motor is a linear motor, and a motor shaft extends out or retracts to drive the magnet fixing frame connected with the motor shaft to move forwards or backwards. The motor drives the magnet to horizontally approach or be far away from one side or the other side of the reagent tube on the track, so that the magnetic particles in the reagent tube move to achieve the aim of cleaning.
Preferably, the second manipulator comprises a miniature electric claw, and the miniature electric claw is driven by a group of two-dimensional sliding table devices.
Preferably, the reagent tube storage area, the sample area to be tested, the reaction and cleaning area and the driving part of the optical detection area are all connected with the control system.
The chemiluminescence immunoassay method for in-situ luminescence determination comprises the following steps: after the system is powered on and started, all devices of the detection system are reset.
1. The reagent tubes 101 (see fig. 2) of the same specification are placed in one reagent tube rack 102, ten reagent tubes can be placed in each row of reagent tube racks, ten reagent tube racks can be placed in the reagent storage area, and the product information (name, batch number, position of the reagent area, and the like) is input into the system.
2. A sample tube 201 to be tested is selected (see fig. 3), the ID of the sample tube 201 is inputted into the system, the sample tube is placed at a designated position of the sample tray 202, and the motor drives the sample tray 202 to rotate the sample tube to the position 203 to be sampled.
3. The system selects an item to be tested based on the ID information of the sample tube, the first manipulator 103 moves the reagent tube 101 gripping the item to be tested to the sample loading position 302 (first station) of the track 301 (see fig. 5) in the reaction-wash-zone box 309, and the first manipulator 103 is reset.
4. The sampling needle 204 is moved to the position 203 to be sampled to suck the sample, and then the sampling needle 204 is moved to the sample application position 302 of the reaction washing area to apply the sample. The sampling needle adds a diluent into the reagent tube of the sample adding position 302, and dilutes the mixture of the sample and the freeze-dried reagent in the reagent tube.
5. The sampling needle 204 was repeatedly aspirated and spitted three times in the reagent tube to mix the solution.
6. After the sampling needle 204 moves to the sampling needle cleaning device 205 to clean the inner wall and the outer wall, the sampling needle is reset.
7. After the sample is loaded into the reagent tube at the loading position 302 (the first station), timing is started, and at intervals of 90 seconds, the sample is moved one station along the track in the direction from the loading position 302 to the cleaning position 303 (the ninth station). The temperature control device controls the temperature of the incubation track at 37 ℃.
8. When the reagent tube moves to the cleaning position 303, the motor 304 drives the magnet holder 305 (see fig. 6) to attach the magnet 306 to one side of the reaction tube, the magnetic particles are adsorbed on the wall of the reagent tube, the cleaning needle 307 enters the reagent tube for cleaning, and the waste liquid is discharged. Then, the cleaning needle 307 is withdrawn from the reagent tube, and the motor 304 drives the magnet holder 305 to move the other magnet to be attached to the other side of the reagent tube, and the reagent tube is cleaned again. After three washes, the reagent tube moves with the incubation track to the removal station 308 (tenth station).
9. The second manipulator 401 moves to the moving-out position 308, the reagent tube is placed in the optical detection area darkroom 402, the upper cover 404 of the darkroom is driven to move downwards by the longitudinal sliding table 403, the darkroom is sealed, the exciting liquid needle 405 injects exciting liquid into the reagent tube in the darkroom, the single photon counter 406 detects the number of photons generated in the reagent tube, and forwards the data to the software system, and the concentration of the sample is calculated.
10. After the detection is finished, the exciting liquid adding device 403 moves upwards, the second manipulator 401 clamps the reagent tube and discards the reagent tube in a waste box, and finally the reagent tube is treated as medical waste.
11. The above processes are repeated in a circulating way, and the detection is completed every 90 seconds. After that, 40 samples can be tested per hour, except for 12 minutes for the first test.
It should be understood that the detailed description of the present invention is only for illustrating the present invention and is not limited by the technical solutions described in the embodiments of the present invention, and those skilled in the art should understand that the present invention can be modified or substituted equally to achieve the same technical effects; as long as the use requirements are met, the method is within the protection scope of the invention.