CN114814261B - Automatic chemiluminescent immunoassay chip and detection method thereof - Google Patents
Automatic chemiluminescent immunoassay chip and detection method thereof Download PDFInfo
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Abstract
The invention relates to an automatic chemiluminescent immunoassay chip and a detection method thereof, belonging to the technical field of microfluidic rapid detection. The chip comprises at least one detection unit, wherein each detection unit comprises a sample treatment layer and a reagent treatment layer; the sample treatment layer comprises a sample adding port, a sample separation tank, a first quantitative distribution unit, a quantitative region, a first micro-channel drainage unit and a reaction tank; the sample adding port, the sample separating tank, the first quantitative distribution unit and the quantitative area are sequentially connected; the reagent treatment layer comprises a first reagent pool and a second reagent pool, and the first reagent pool and the second reagent pool are connected with the reaction pool; the first reagent tank and the second reagent tank are used for pre-packaging reagents required by the reaction. The automatic rapid detection chip is applied to high-sensitivity quantitative rapid detection analysis of analytes in biological samples.
Description
Technical Field
The invention relates to the field of microfluidic rapid detection, in particular to an automatic chemiluminescent immunoassay chip and a detection method thereof, and particularly discloses a centrifugal automatic rapid detection chip which is used for high-sensitivity automatic quantitative rapid analysis and detection of analytes in samples.
Background
Bedside rapid detection is one of the general trends of modern detection technologies, and at present, most analytes in biological samples including genes, proteins, sugar, elements and the like can be basically analyzed and detected by large-scale instruments or complicated manual operation, but because the manufacturing cost is high, the professional technical requirements are high, the popularization of the detection technologies is limited due to the high environmental requirements, and a plurality of analytes cannot be timely detected and analyzed, so that the development of science is limited; many samples cannot be subjected to on-site analysis and detection due to higher preservation conditions, so that the biological potential safety hazard cannot be found in time, and serious consequences are caused; on the other hand, with the development of expensive detection technology, the economic burden of systems or individuals such as medical treatment, public health, scientific research, patients and the like is increased.
Microfluidic analysis technology refers to a technology that integrates basic operation units such as sample preparation, reaction, separation, detection, etc. in biological, chemical, and medical analysis processes onto one micron-scale chip, and can automatically complete the whole analysis process. Compared with the common detection technology, the microfluidic chip has the advantages of high analysis efficiency, high accuracy, integration, flux activation, automation, energy conservation, environmental protection and the like. The microfluidic detection chip generally has the advantages of low sample consumption, high detection speed, simple and convenient operation, multifunctional integration, small size, portability and the like, so that the microfluidic detection chip has larger potential capability on reducing the overall detection cost, and is very suitable for the development of rapid bedside detection. In particular, the centrifugal microfluidic chip can well simplify the instrument and equipment by controlling the fluid by virtue of centrifugal force, and meanwhile, various valve control structures are designed in the disc-type chip. However, the fine micro valve structure is easy to generate batch-to-batch difference in batch processing and manufacturing, which affects repeatability, and more importantly, the existence of coriolis force and Europe pulling force in the centrifugal micro-fluidic chip can cause deviation of fluid drainage direction, which affects the final detection structure.
Therefore, most of the microfluidic chips are applied to bedside rapid detection at the theoretical research and patent application stage, and the number of commercial microfluidic detection chips is reduced. The main challenges of the current microfluidic detection chip are as follows:
1. The sequential release of the reagents is realized in the chip;
2. The chip structure is complex and needs to be modified locally, so that the processing cost and difficulty of the chip are increased, and meanwhile, the stability of the chip is reduced;
3. The simultaneous quantitative detection of multiple samples and multiple targets is realized in a single chip, more ingenious chip design is needed, the rapid detection of the multiple targets or the single targets of the single sample is still realized at present, and meanwhile, the distribution and the quantification of the samples cannot be well completed, so that the quantitative detection of analytes is difficult to realize;
4. The difficulty in simultaneous quantitative dispensing of multiple reagents and the difficulty in achieving adequate mixing of the dispensed, metered reagents on the microfluidic channel;
5. the coriolis force and euler force in the centrifugal chip drainage process deviate the direction of the fluid.
Disclosure of Invention
Based on the detection, the invention discloses a microfluidic automatic rapid detection chip which has the advantages that the processing and the manufacturing are simple, the sequential release of reagents required by detection can be realized, meanwhile, the multi-target quantitative rapid detection of a plurality of samples is finished in a single chip, the detection process is fully and automatically finished, the operation of professional technicians is not needed, and the matched instrument is portable and simple and is very suitable for rapid bedside detection.
According to a first aspect of the present invention, there is provided an automated chemiluminescent immunoassay chip comprising at least one detection cell, each of said detection cells comprising a sample handling layer and a reagent handling layer;
the sample treatment layer comprises a sample adding port, a sample separation tank, a first quantitative distribution unit, a quantitative area, a first micro-channel drainage unit and a reaction tank; the sample adding port, the sample separating tank, the first quantitative distribution unit and the quantitative region are connected in sequence, and the first micro-channel drainage unit is used for draining liquid in the quantitative region into the reaction tank; the first micro-channel drainage unit is provided with a cavity, and the cavity is used for embedding substances required by reaction;
The reagent treatment layer comprises a first reagent pool and a second reagent pool, and the first reagent pool and the second reagent pool are connected with the reaction pool; the first reagent pool and the second reagent pool are used for pre-packaging reagents required by the reaction.
Preferably, the first reagent pool and the second reagent pool are respectively connected with a first quantitative chamber and a second quantitative chamber through a second quantitative distribution unit, the first quantitative chamber and the second quantitative chamber are respectively connected with a second micro-channel drainage unit and a third micro-channel drainage unit, the second micro-channel drainage unit is connected with the third micro-channel drainage unit through the second quantitative chamber, and the third micro-channel drainage unit is connected with the reaction pool; the first reagent pool and the second reagent pool are used for pre-packaging reagents required by the reaction.
Preferably, the first reagent pool is connected with a first quantitative chamber through a second quantitative distribution unit, the first quantitative chamber is connected with a second micro-channel drainage unit, and the second micro-channel drainage unit is connected with the reaction pool; the second reagent tank is directly connected with the reaction tank; the first reagent pool and the second reagent pool are used for pre-packaging reagents required by the reaction.
Preferably, the detection unit further comprises a valve, a waste liquid pool and an air hole; the valve is respectively connected with the reaction tank and the waste liquid tank; the waste liquid pool is connected with the first quantitative distribution unit and the second quantitative distribution unit; the waste liquid pool is used for collecting waste liquid in the reaction pool, the first quantitative distribution unit and the second quantitative distribution unit; the air hole is connected with the waste liquid pool and is used for balancing the air pressure inside the chip.
Preferably, the first microchannel drainage unit has a serpentine microchannel combination for increasing the mixing effect of the liquid and controlling the fluid flow rate by means of the effect of coriolis force and euler force on fluid deflection during chip centrifugation.
According to another aspect of the present invention, there is provided a method for detecting any one of the automated chemiluminescent immunoassay chips, comprising the steps of:
S1: embedding the target antibody marked by the signal molecule in a cavity on the first micro-channel drainage unit, and embedding the target antibody marked by the magnetic particles in a reaction tank;
S2: adding a sample to be detected at a sample adding port, and precipitating impurities in a sample separating tank under the action of centrifugal force; then, the sample solution enters a first quantitative distribution unit for distribution, enters a first micro-channel drainage unit through a quantitative region, and redissolves the target antibody marked by the signal molecule, wherein the target protein in the sample solution and the target antibody marked by the signal molecule form a first compound, and the first compound enters a reaction tank and reacts with the target antibody marked by the magnetic particle to form a second compound;
s3: starting magnet adsorption, fixing the second compound in a reaction tank, opening a valve, starting centrifugation, discharging the target antibody marked by the signal molecule which does not form the second compound into a waste liquid tank, and closing the valve;
S4: removing magnet adsorption, releasing reagents in the first reagent pool and the second reagent pool to enter a reaction pool, enabling luminescent molecules captured in the second compound to react and emit light, detecting a light signal, and calculating to obtain the content of target protein in a sample to be detected.
Preferably, the signal molecule is an acridinium ester, an acridone or horseradish peroxidase.
Preferably, the target protein is an acute aging reaction protein, procalcitonin or interleukin-6; the target antibody is an acute aging reaction protein antibody, a procalcitonin antibody or an interleukin-6 antibody.
Preferably, the reagents in the first reagent tank and the second reagent tank are respectively a pre-excitation liquid and an excitation liquid.
In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) The automatic rapid detection chip can be combined with detection technologies such as biochemistry, immunity, chemiluminescence and the like, is applied to high-sensitivity quantitative rapid detection and analysis of analytes in biological samples, and has huge potential application value in the fields such as biomedicine, biochemistry, bedside rapid detection and the like.
(2) The automatic rapid detection chip can complete high-flux distribution and quantification of various reagents, realize step-by-step full mixing reaction of the various reagents and improve detection sensitivity and accuracy.
(3) The automatic rapid detection chip can realize sequential mixing of the excitation liquid and the pre-excitation liquid in the chemiluminescent detection process, and can control the sequence of the excitation liquid entering the reaction chamber, and accurately collect and analyze portable signals.
(4) The automatic rapid detection chip can overcome the influence of Coriolis force and Euler force on fluid deflection through the drainage channel, and realize quantitative distribution and sequential mixing reaction of various reagents; and meanwhile, through the combination of the winding micro-channels perpendicular to or parallel to the circle center extension line, the liquid mixing degree can be improved by controlling the deflection of the fluid by means of the Coriolis force and the Euler force.
Drawings
FIG. 1 is a block diagram of an automated rapid test chip according to the present invention;
FIG. 2 is a schematic diagram of the layered structure of an automated rapid test chip according to the present invention;
FIG. 3 is a schematic diagram of a test unit of the automated rapid test chip of the present invention;
FIG. 4 is a schematic diagram of a sample processing layer of a detection unit of the automated rapid detection chip of the present invention;
FIG. 5 is a schematic diagram of a reagent processing layer of a detection unit of the automated rapid detection chip of the present invention;
FIG. 6 is a schematic diagram of an embodiment of a reagent processing layer of a detection unit of an automated rapid detection chip according to the present invention;
FIG. 7 is a schematic diagram of a portion of an automated rapid test chip according to the present invention;
FIG. 8 is a graph showing the effect of the microfluidic channel drainage unit of the present invention on overcoming the effects of Coriolis force and Euler force during centrifugation drainage;
FIG. 9 is another form of an overall block diagram of an automated rapid test chip according to the present invention;
FIG. 10 is a schematic view of a layered structure of another form of the overall architecture of the automated rapid test chip of the present invention;
FIG. 11 is a schematic diagram of another form of a test cell of the automated rapid test chip of the present invention;
FIG. 12 is a partial block diagram of another form of a test cell of the automated rapid test chip of the present invention;
Fig. 13 is a graph of mechanical analysis during centrifugation drainage.
The same reference numbers are used throughout the drawings to reference like elements or structures, wherein: 200-detection unit, 51-sample processing layer, 52-reagent processing layer, 11-sample addition port, 12-sample separation cell, 13-first quantitative distribution unit, 14-quantitative region, 15-first microchannel drainage unit, 16-reaction cell, 17-chamber, 21-first reagent cell, 22-second reagent cell, 23-second quantitative distribution unit, 24-first quantitative chamber, 25-second quantitative chamber, 26-second microchannel drainage unit, 27-third microchannel drainage unit, 31-valve, 32-waste liquid pool, 53-intermediate layer, 54-upper cover, 55-lower cover.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention relates to an automatic rapid detection chip 400, which comprises at least one detection unit 200, wherein each detection unit comprises a sample processing layer 51 and a reagent processing layer 52;
FIG. 4 is a schematic diagram of a sample processing layer of an automated rapid test chip according to the present invention. The sample processing layer 51 comprises a sample adding port 11, a sample separating tank 12, a first quantitative distribution unit 13, a quantitative region 14, a first micro-channel drainage unit 15 and a reaction tank 16; the sample adding port 11, the sample separating tank 12, the first quantitative distribution unit 13 and the quantitative region 14 are sequentially connected, and the first micro-channel drainage unit 15 is used for draining the liquid in the quantitative region 14 to the reaction tank 16; a chamber 17 is arranged on the first micro-channel drainage unit 15, and the chamber 17 is used for embedding substances required by reaction;
The reagent processing layer 52 comprises a first reagent pool 21 and a second reagent pool 22, the first reagent pool 21 and the second reagent pool 22 are respectively connected with a first quantitative chamber 24 and a second quantitative chamber 25 through a second quantitative distribution unit 23, the first quantitative chamber 24 and the second quantitative chamber 25 are respectively connected with a second micro-channel drainage unit 26 and a third micro-channel drainage unit 27, the second micro-channel drainage unit 26 is connected with the third micro-channel drainage unit 27 through the second quantitative chamber 25, and the third micro-channel drainage unit 27 is connected with the reaction tank 16; the first reagent reservoir 21 and the second reagent reservoir 22 are used for pre-packaging reagents required for the reaction.
The detection unit 200 further comprises a valve 31 and a waste liquid pool 32; the valve 31 is respectively connected with the reaction tank 16 and the waste liquid tank 32; the waste liquid pool 32 is connected with the first quantitative distribution unit 13 and the second quantitative distribution unit 23; the waste liquid pool 32 is used for collecting waste liquid in the reaction pool 16, the first quantitative distribution unit 13 and the second quantitative distribution unit 23. FIG. 3 is a schematic diagram of a test unit of the automated rapid test chip of the present invention.
The first micro-channel drainage unit 15, the second micro-channel drainage unit 26 and the third micro-channel drainage unit 27 have the functions of mixing and micro-valves, and the automatic rapid detection chip has a rotation center.
Specifically, each detection unit 200 in the chip is mutually independent, and the structure of the chip can be consistent or inconsistent and can be adjusted according to the detection requirement;
Specifically, in one embodiment, the chip has a 5-layer structure and includes an upper cover 54, a lower cover 55, a sample processing layer 51, a reagent processing layer 52 and an intermediate layer 53, where the sample processing layer 51 and the reagent processing layer 52 are not in the same plane, and are only communicated with each other through the reaction tank 16;
In another form (FIG. 8), the sample processing layer is the same layer as the reagent processing layer (FIG. 9), and in one embodiment the chip is a 3-layer structure comprising an upper cover 54, a lower cover 55, and an intermediate layer 53 (FIG. 10). FIG. 11 is a schematic diagram of another form of a test cell of the automated rapid test chip of the present invention; FIG. 12 is a partial block diagram of another form of a test cell of the automated rapid test chip of the present invention. In another form of the invention, the first reagent reservoir 21 is connected to the first dosing chamber 24 by the second dosing unit 23, the first dosing chamber 24 is connected to the second microchannel drainage unit 26, and the second microchannel drainage unit 26 is connected to the reaction reservoir 16; the second reagent reservoir 22 is directly connected to the reaction reservoir 16; the first reagent tank 21 and the second reagent tank 22 are used for pre-packaging reagents required for the reaction.
Specifically, the sample adding port 11 is communicated with the sample separating tank 12, and samples are added from the sample adding port 11, and the samples can be various samples such as whole blood, plasma, serum, urine and the like;
Specifically, the sample separation tank 12 is communicated with the first quantitative distribution unit 13, the connection port is designed as the middle part of the sample separation tank 12, when the chip is centrifuged, solid-phase impurities in the sample can be preferentially settled at the bottom of the sample separation tank 12 due to the centrifugal force and the high centrifugal speed, the supernatant can enter the first quantitative distribution unit 13 along with the continuous centrifugal force, and the connection port can be variously designed to prevent the impurities from entering the first quantitative distribution unit 13, such as a micro column, a biological film, a micro valve and the like;
Specifically, the first quantitative distribution unit 13 is in communication with the first microchannel drainage unit 15, the first quantitative distribution unit 13 includes a plurality of quantitative regions 14, when the chip is centrifuged at a low speed, the sample solution enters the quantitative regions 14, and along with the continuation of the centrifugal force, the excess sample solution is discharged into the waste liquid pond 32, so that the distribution and the quantification of the sample solution can be completed, and a certain volume of sample solution is provided for the subsequent multi-target quantitative detection;
Specifically, the micro channel of the first micro channel drainage unit 15 has a size of 10-100 μm wide and 10-100 μm high, but is not limited thereto;
Specifically, the first micro-channel drainage unit 15 includes three serpentine channels in one embodiment, the micro-channel near the quantifying area 14 is a hydrophobic area, the other channels may be modified with or without any modification, and the serpentine channels may control the flow rate of the fluid;
Specifically, when the chip is centrifuged at a high speed, the sample solution in the quantitative region 14 breaks through the hydrophobic resistance of the micro-channel, and enters the first serpentine channel of the first micro-channel unit 15, the serpentine channel can reduce the flow velocity of the sample solution to make full contact with the reagent pre-embedded in the first micro-channel unit 15, and then enters the second serpentine channel and the third serpentine channel to make full mixing reaction, meanwhile, the serpentine micro-channel is designed as a micro-channel perpendicular to or parallel to the outwards extending line of the center of the circle, and in the centrifugal drainage process, the mixing effect can be improved by the influence of the coriolis force and the euler force on the deflection of the fluid, and the mechanical analysis is shown in fig. 13 without considering the extrusion force of the fluid;
Specifically, the first serpentine channel is closer to the center of the circle than the second serpentine channel, the second serpentine channel is closer to the center of the circle than the third serpentine channel, the distance between the first serpentine channel and the second serpentine channel can be adjusted according to the detection requirement, and the structure is not limited to the first serpentine channel and the second serpentine channel;
specifically, after the chip is centrifuged at a high speed, the mixed solution of the sample and the reagent enters a reaction tank 16, and the reagent can be embedded in the reaction tank 16 in advance or subjected to surface modification to capture a detection target in the sample;
Specifically, the valve 31 is communicated with the reaction tank 16 and the waste liquid tank 32, the valve 31 is opened, high-speed centrifugation is started, redundant waste liquid in the reaction tank 16 can be completely discharged into the waste liquid tank 32, only the analyte required for detection remains in the reaction tank 16, and then the valve 31 is closed;
Specifically, the first reagent tank 21 and the second reagent tank 22 are used for storing solution reagents, the first reagent tank 21 and the second reagent tank 22 are respectively communicated with a second quantitative distribution unit 23 for distribution and quantification of the reagents, and the quantitative process is the same as the quantitative process of the sample;
Specifically, the first reagent tank 21 is communicated with the second micro-channel drainage unit 26, the second micro-channel drainage unit 26 is communicated with the first quantitative chamber 24, the third micro-channel drainage unit 27 is further away from the center of the circle than the second micro-channel drainage unit 26, meanwhile, the second micro-channel drainage unit 26 is provided with a serpentine channel, the time for the reagent to enter the reaction tank 16 can be controlled by controlling the resistance and the length of the micro-channel, finally, the reagent in the second reagent tank 22 enters the reaction tank 16 first, then the reagent at the first reagent tank 21 sequentially and rapidly enters the reaction tank 16 for reaction, finally, a detection signal in each reaction tank 16 is obtained, meanwhile, in another form, the solution can be injected into the reaction tank by pre-storing the final reaction reagent in the second reagent tank 22 (fig. 9), the signal acquisition is completed as required, and the strength of the detection signal is related to the content of the analyte in a certain solution volume, so that the analyte of the sample can be quantitatively detected.
In the invention, the micro-channel drainage unit has the effects of mixing and drainage.
According to the invention, the micro-channel drainage unit can control the sequence and time of liquid entering the reaction tank through the length of the micro-channel and the resistance of the micro-channel.
In the invention, the quantitative distribution unit and the micro-channel drainage unit are communicated with each other but not on the same plane, the micro-channel size of the micro-channel drainage unit can be 10-100 μm wide and 10-300 μm high, but the quantitative distribution unit and the micro-channel drainage unit are not limited in the range, and are used for limiting liquid to enter the micro-channel when the chip is centrifuged at a low speed, and enabling the liquid to flow into the micro-channel for drainage and mixing when the chip is centrifuged at a high speed; the quantitative distribution unit can freely flow liquid during low-speed centrifugation of the chip, distribute and quantify the liquid, and redundant liquid enters a waste liquid pool communicated with the quantitative distribution unit.
In the invention, the reaction tank is communicated with the micro valve, when liquid is required to be stored, the valve is closed, and when waste liquid is required to be discharged, the reaction tank is closed.
According to the quantitative distribution unit, independent distribution and quantification of multiple liquids can be realized on the same horizontal plane, and the quantitative distribution unit is used for detecting multiple samples and multiple targets.
The chip of the present invention can be used for various biochemical tests, immunoassay tests, etc., but is not limited thereto.
The detection method comprises the following steps:
Sample liquid is put into a matched centrifugal detection instrument from the sample separation pool of the sample adding Kong Jiazhi;
The low-speed centrifugal chip can quickly settle impurities in a sample at the bottom of the sample separation tank due to large centrifugal force, supernatant enters the quantitative distribution unit due to continuous centrifugal force to distribute and quantify sample liquid, a foundation is provided for subsequent quantitative analysis and detection, and redundant liquid is discharged into a waste liquid tank;
The high-speed centrifugal chip injects quantitative sample solution into the micro-channel drainage unit, controls the flow rate of liquid by controlling the centrifugal speed and the micro-channel, redissolves the pre-buried reagent in the channel, fully mixes and reacts the sample and the reagent through the micro-channel drainage unit, and finally enters the reaction tank;
The pre-embedded reagent in the reaction tank can be fully mixed with the entered sample mixed solution to react to form a target compound, then a valve is opened, and the chip is centrifuged at a high speed to discharge redundant waste liquid into a waste liquid tank, and the required target compound still remains in the reaction tank;
releasing the reagents in all the reagent tanks, quantitatively distributing the reagents to the quantitative distribution unit by using a low-speed centrifugal chip, discharging redundant reagents into the waste liquid tanks, then introducing the quantitatively distributed reagents into the reaction tanks through a micro-channel drainage unit by using high-speed centrifugation, and sequentially entering the reaction tanks to react due to different lengths and resistances of micro-channels, and finally sequentially obtaining detection signals in each reaction tank.
The invention relates to a detection method of an automatic chemiluminescent immunoassay chip, which comprises the following steps:
s1: embedding a target antibody (primary antibody) marked by signal molecules in a cavity 17 on the first microchannel drainage unit 15, and embedding a target antibody (secondary antibody) marked by magnetic particles in a reaction tank 16;
s2: adding a sample to be detected at a sample adding port 11, and precipitating impurities in a sample separating tank 12 under the action of centrifugal force; subsequently, the sample solution enters a first quantitative distribution unit 13 for distribution, enters a first micro-channel drainage unit 15 through a quantitative region 14, and is subjected to redissolution of the target antibody (primary antibody) marked by the signal molecule, the target protein in the sample solution and the target antibody marked by the signal molecule form a first complex, and the first complex enters a reaction tank 16 and reacts with the target antibody (secondary antibody) marked by the magnetic particle to form a second complex;
S3: the magnet adsorption is started to fix the second complex in the reaction tank 16, then the valve 31 is opened, centrifugation is started, the target antibody (primary antibody) marked by the signal molecule which does not form the second complex is discharged into the waste liquid tank, and then the valve 31 is closed.
S4: removing the magnet adsorption, releasing the reagents in the first reagent pool 21 and the second reagent pool 22 to enter the reaction pool 16, enabling the luminescent molecules captured in the second compound to react and emit light, detecting the optical signal, and calculating to obtain the content of the target protein in the sample to be detected.
Example 1
The embodiment provided by the invention is that the chip is applied to acridine ester chemiluminescence immunoassay, the immunoassay principle is that an immune sandwich principle is used for detecting C-reactive protein in whole blood, and the specific technical scheme is as follows:
1. Antibody modification
Combining acridinium ester with an anti-CRP antibody through an EDC and NHS system to prepare a signal labeled antibody complex, and similarly, combining magnetic particles with the anti-CRP monoclonal antibody through the EDC and NHS system to prepare a capture antibody complex;
2. Chip processing
The chip processing mode adopts CNC numerical control processing, each layer of the chip is processed independently, the sample processing layer and the reagent processing layer are processed on two sides, a micro-channel drainage unit is formed on the lower surface, the quantitative distribution unit is engraved and communicated on the upper surface at the connecting position, and the chip processing material is PMMA material;
It can be appreciated that the chip processing technology can be chip processing technologies such as laser engraving technology, CNC (computerized numerical control) technology, soft lithography technology and the like, but is not limited to the chip processing technologies;
It is understood that the processing material of the chip may be, but not limited to, organic glass or polymer such as PMMA, PC, PDMS.
3. Chip assembly
As shown in fig. 2, the chip has five layers in total, including an upper cover 54, a lower cover 55, a sample processing layer 51, a reagent processing layer 52 and an intermediate layer 53, the chip assembly is sealed and combined by a double-sided adhesive tape, before the combination, the acridinium ester marked CRP antibody is pre-embedded in the chamber 17 of the first microchannel drainage unit 15 (as shown in fig. 7), and the magnetic particle marked CRP monoclonal antibody is pre-embedded in the reaction tank 16; the excitation liquid (NaOH) reagent pack is pre-packaged in the first reagent tank 21 before the photograph is sealed by the upper cover 54, the pre-excitation liquid (H 2O2) is pre-packaged in the first reagent tank 22, and then chip sealing is performed, so that a complete detection chip is formed.
4. Sample detection
Specifically, 100-200 mu l of whole blood sample is added at the sample adding port 11, and the chip is put into a matched detection instrument in a sealing way;
Specifically, the chip is in the instrument, firstly, low-speed centrifugation is carried out, cells and impurities in a whole blood sample are precipitated at the bottom of the sample separation tank 12, the separation of blood plasma, blood cells and impurities in the whole blood is completed, the centrifugation speed is improved, the whole blood supernatant enters the first quantitative distribution unit 13 for distribution and determination under the continuous centrifugal force effect, the final volume of the sample is accurately quantified through the size of the quantitative region 14, redundant waste liquid enters the waste liquid tank 32, and then the centrifugation is finished;
Specifically, high-speed centrifugation of the chip is started, a certain amount of sample solution is injected into the first micro-channel drainage unit 13, the sample solution is redissolved with the CRP antigen of the CRP antibody marked by the acridine ester in the sample to react to form an acridine ester-marked CRP antibody-CRP compound, the control of the flow rate of the fluid can be realized through the serpentine micro-channel, meanwhile, the reaction is more sufficient, finally, under the action of continuous high-speed centrifugation, the combination of serpentine micro-channels which are vertical or parallel to the circle center extension line can improve the mixing effect by means of the deflection effect of the Coriolis force and the Euler force on the fluid, and finally, all the liquid is discharged into the reaction tank 16, and the valve 31 is in a closed state at the moment;
Specifically, the high-speed centrifugation stopping process can make the solution in the reaction tank 16 vibrate, and complete the reaction of the magnetic particle marked CRP monoclonal antibody and the acridinium ester marked CRP antibody-CRP complex, so that the reaction is quicker, the reaction time is shortened, the reaction forms the acridinium ester marked CRP antibody-CRP-magnetic particle marked CRP monoclonal antibody complex, at the moment, the magnet adsorption is started, the magnetic particles are fixed in the reaction tank 16, the valve 31 is opened, the chip centrifugation is started, all the liquid is discharged into the waste liquid tank 32, and the centrifugation is stopped; then the valve 31 is closed, the adsorption of the magnet to the magnetic particles is removed, the pre-excitation liquid and the excitation liquid in the first reagent tank 21 and the second reagent tank 22 are released, the low-speed centrifugation is started to accurately quantify the reagent, then the high-speed centrifugation is performed, the pre-excitation liquid preferentially and quickly enters the reaction tank 16 to resuspend the magnetic particles, then the excitation liquid passes through the third meandering micro-channel drainage unit 27, as shown in fig. 5 and 6, the excitation liquid simultaneously or sequentially and quickly enters the reaction tank 16 by controlling the length and the resistance of the micro-channel, the captured acridinium ester completes rapid reaction luminescence, the instrument detects an optical signal, and the accurate quantitative detection of the CRP content in the sample is completed.
It will be appreciated that, as shown in fig. 1, each chip can perform simultaneous detection of three different samples, each detection unit in the chip can perform analysis detection of the target detection object in 7, and meanwhile, the final detection unit can be sacrificed, and positive control detection is set to perform quality control of a single chip.
Example 2
The other embodiment provided by the invention is that the chip is applied to luminol chemiluminescence immunoassay based on horseradish peroxidase (HRP), the immunoassay principle is that an immune sandwich principle is used for detecting Procalcitonin (PCT) in whole blood, and the specific technical scheme is as follows:
1. Antibody modification
Binding HRP to anti-PCT antibody by EDC and NHS system to prepare a signal-labeled antibody complex, and similarly, binding magnetic microparticles to anti-PCT monoclonal antibody by EDC and NHS system to prepare a capture antibody complex;
2. The chip processing mode is similar to that of the specific embodiment 1;
3. Chip assembly
As shown in fig. 10, the chip has 3 layers in total, including an upper cover 54, a lower cover 55 and an intermediate layer 53, the chip assembly is sealed and combined by adopting a double-sided adhesive manner, the HRP-labeled PCT antibody is pre-embedded at the initial end of the first microchannel drainage unit 15 (i.e. the end connected with the quantification unit 14) before the combination, and the magnetic particle-labeled PCT monoclonal antibody is pre-embedded in the reaction tank 16; the luminol reagent pack is pre-packaged in the first reagent tank 21 before the photograph is sealed by the upper cover 54, and the pre-hydrogen peroxide (H 2O2) is pre-packaged in the second reagent tank 22, followed by chip sealing, to form a complete detection chip.
4. Sample detection
Specifically, 100-200 mu l of whole blood sample is added at the sample adding port 11, and the chip is put into a matched detection instrument in a sealing way;
specifically, the chip is in the instrument, firstly, low-speed centrifugation is carried out, cells and impurities in a whole blood sample are precipitated at the bottom of the sample separation tank 12, the separation of blood plasma, blood cells and impurities in the whole blood is completed, the centrifugation speed is improved, the whole blood supernatant enters the first quantitative distribution unit 13 for distribution and quantification under the continuous centrifugal force effect, the final volume of the sample is accurately quantified through the size of the quantitative region 14, redundant waste liquid enters the waste liquid tank 32, and then the centrifugation is finished;
Specifically, the chip is started to perform high-speed centrifugation, a certain amount of sample solution is injected into the first microchannel drainage unit 15, the sample solution is re-dissolved with PCT antigen of PCT antibody marked by HRP in the sample to react to form PCT antibody-PCT compound marked by HRP, the flow rate of fluid can be controlled through the meandering microchannel, meanwhile, the reaction is more sufficient, finally, under the action of continuous high-speed centrifugation, the combination of meandering micro-channels which are vertical or parallel to the circle center extension line can improve the mixing effect by the influence of the coriolis force and the euler force on the deflection of the fluid, and finally, all liquid is discharged into the reaction tank 16, and the valve 31 is in a closed state;
Specifically, the high-speed centrifugation stopping process can shake the solution in the reaction tank 16, complete the reaction of the magnetic particle labeled PCT monoclonal antibody and the HRP labeled PCT antibody-PCT complex, make the reaction faster, shorten the reaction time, react to form the HRP labeled PCT antibody-PCT-magnetic particle labeled PCT monoclonal antibody complex, at the moment, start the magnet adsorption, fix the magnetic particles in the reaction tank 16, open the valve 31, start the chip centrifugation, discharge all the liquid into the waste liquid tank, and stop the centrifugation; then the valve 31 is closed, the magnet is removed to adsorb magnetic particles, luminol in the first reagent tank 21 is released, low-speed centrifugation is started to accurately quantify the reagent, then high-speed centrifugation is performed to enable the reagent to enter the reaction tank 16, then hydrogen peroxide solution in the second reagent tank is rapidly injected into the reaction tank 16 in a simultaneous or sequential pressing or injection mode, rapid reaction luminescence is completed with the captured HRP enzyme, an instrument detects an optical signal, and accurate quantitative detection of PCT content in a sample is completed.
It will be appreciated that, as shown in fig. 1 and 9, each chip can perform simultaneous detection of three different samples, each detection unit in the chip can perform analysis detection of 7 target detection objects, and meanwhile, a final detection unit can be sacrificed, and positive control detection is set to perform quality control of a single chip.
The verification of the effect of the drainage channel against the coriolis force and the euler force is shown in fig. 8: and designing drainage channels with different extension lengths, and finally verifying that the deflection of liquid is larger and larger from left to right, wherein when the channels extend to a certain length, the deflection of the liquid is not influenced by Coriolis force and Europe tension basically, so that the reliability and the repeatability of an analysis and detection structure are further ensured.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. An automated chemiluminescent immunoassay chip comprising at least one detection cell (200), each of said detection cells comprising a sample handling layer (51) and a reagent handling layer (52);
The sample treatment layer (51) comprises a sample adding port (11), a sample separation tank (12), a first quantitative distribution unit (13), a quantitative region (14), a first micro-channel drainage unit (15) and a reaction tank (16); the sample adding port (11), the sample separating tank (12), the first quantitative distribution unit (13) and the quantitative region (14) are sequentially connected, and the first micro-channel drainage unit (15) is used for draining liquid in the quantitative region (14) into the reaction tank (16); the first micro-channel drainage unit (15) is provided with a cavity (17), and the cavity (17) is used for embedding substances required by reaction;
The reagent treatment layer (52) comprises a first reagent tank (21) and a second reagent tank (22), and the first reagent tank (21) and the second reagent tank (22) are connected with the reaction tank (16); the first reagent pool (21) and the second reagent pool (22) are used for pre-packaging reagents required by the reaction;
The first reagent pool (21) and the second reagent pool (22) are respectively connected with a first quantitative chamber (24) and a second quantitative chamber (25) through a second quantitative distribution unit (23), the first quantitative chamber (24) and the second quantitative chamber (25) are respectively connected with a second micro-channel drainage unit (26) and a third micro-channel drainage unit (27), the second micro-channel drainage unit (26) is connected with the third micro-channel drainage unit (27) through the second quantitative chamber (25), and the third micro-channel drainage unit (27) is connected with the reaction pool (16); the first reagent pool (21) and the second reagent pool (22) are used for pre-packaging reagents required by the reaction.
2. The automated chemiluminescent immunoassay chip of claim 1, wherein the first reagent reservoir (21) is connected to a first dosing chamber (24) by a second dosing unit (23), the first dosing chamber (24) is connected to a second microchannel drainage unit (26), the second microchannel drainage unit (26) is connected to the reaction reservoir (16); the second reagent tank (22) is directly connected with the reaction tank (16); the first reagent pool (21) and the second reagent pool (22) are used for pre-packaging reagents required by the reaction.
3. The automated chemiluminescent immunoassay chip of claim 1, wherein the detection cell (200) further comprises a valve (31), a waste liquid reservoir (32), and an air vent (33); the valve (31) is respectively connected with the reaction tank (16) and the waste liquid tank (32); the waste liquid pool (32) is connected with the first quantitative distribution unit (13) and the second quantitative distribution unit (23); the waste liquid pool (32) is used for collecting waste liquid in the reaction pool (16), the first quantitative distribution unit (13) and the second quantitative distribution unit (23); the air hole (33) is connected with the waste liquid pool (32) and used for balancing the air pressure in the chip.
4. The automated chemiluminescent immunoassay chip of claim 1, wherein the first microchannel drainage unit (15) has a serpentine combination of microchannels for increasing the mixing effect of liquids and controlling fluid flow rate by means of the effect of coriolis and euler forces on fluid deflection during centrifugation of the chip.
5. The method for detecting an automated chemiluminescent immunoassay chip according to any one of claims 1-4 comprising the steps of:
S1: embedding a target antibody marked by a signal molecule in a cavity (17) on a first micro-channel drainage unit (15), and embedding a target antibody marked by magnetic particles in a reaction tank (16);
S2: adding a sample to be detected at a sample adding port (11), and precipitating impurities in a sample separating tank (12) under the action of centrifugal force; subsequently, the sample solution enters a first quantitative distribution unit (13) for distribution, enters a first micro-channel drainage unit (15) through a quantitative region (14) and redissolves the target antibody marked by the signal molecule, and the target protein in the sample solution and the target antibody marked by the signal molecule form a first complex, and the first complex enters a reaction tank (16) and reacts with the target antibody marked by the magnetic particle to form a second complex;
S3: starting magnet adsorption to fix the second compound in the reaction tank (16), then opening a valve (31), starting centrifugation, discharging target antibodies marked by signal molecules which do not form the second compound into a waste liquid tank, and then closing the valve (31);
S4: removing magnet adsorption, releasing reagents in the first reagent pool (21) and the second reagent pool (22) to enter the reaction pool (16), enabling luminescent molecules captured in the second compound to react and emit light, detecting a light signal, and calculating to obtain the content of target protein in a sample to be detected.
6. The method of claim 5, wherein the signal molecule is acridinium ester, acridone or horseradish peroxidase.
7. The method of claim 5 or 6, wherein the target protein is an acute aging response protein, procalcitonin or interleukin-6; the target antibody is an acute aging reaction protein antibody, a procalcitonin antibody or an interleukin-6 antibody.
8. The detection method according to claim 5, wherein the reagents in the first reagent reservoir (21) and the second reagent reservoir (22) are a pre-excitation liquid and an excitation liquid, respectively.
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| CN109870582A (en) * | 2019-02-27 | 2019-06-11 | 华中科技大学 | A kind of more target magnetic immunochemiluminescence micro-fluidic chip detection platforms and method |
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| CN106018784A (en) * | 2016-07-05 | 2016-10-12 | 深圳普门科技有限公司 | Small electrochemical luminescence immunoassay analyzer and analysis method thereof |
| CN109870582A (en) * | 2019-02-27 | 2019-06-11 | 华中科技大学 | A kind of more target magnetic immunochemiluminescence micro-fluidic chip detection platforms and method |
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