CN114814261A - Automatic chemiluminescence immunoassay chip and detection method thereof - Google Patents

Abstract

The invention relates to an automatic chemiluminescence 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 processing layer and a reagent processing layer; the sample processing layer comprises a sample adding port, a sample separating pool, a first quantitative distribution unit, a quantitative area, a first micro-channel drainage unit and a reaction pool; the sample adding port, the sample separation pool, the first quantitative distribution unit and the quantitative region 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 both connected with the reaction pool; the first reagent pool and the second reagent pool are used for pre-packaging reagents required by the reaction. The automatic rapid detection chip is applied to high-sensitivity quantitative rapid detection and analysis of analytes in biological samples.

Description

An automated chemiluminescence immunoassay chip and its detection method

technical field

The invention relates to the field of microfluidic rapid detection, in particular to an automated chemiluminescence immunoassay chip and a detection method thereof, and particularly discloses a centrifugal automated rapid detection chip, which is used for high-sensitivity automated quantification of analytes in samples Rapid analysis detection.

Background technique

Point-of-care rapid detection is one of the general trends of modern detection technology. At present, most of the analytes in biological samples, including genes, proteins, sugars, elements, etc., can be analyzed and detected by large-scale instruments or complicated manual operations. However, due to their manufacturing costs High, professional and technical requirements, and high environmental requirements limit the promotion of these detection technologies, resulting in many analytes that cannot be detected and analyzed in time, which limits the development of science; many samples cannot be analyzed and tested on-site due to high storage conditions. As a result, the hidden dangers of biosafety cannot be detected in time, resulting in serious consequences; on the other hand, with the development of expensive detection technology, it also increases the economic burden of medical, public health, scientific research, patients and other systems or individuals.

Microfluidic analysis technology refers to a technology that integrates basic operation units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes into a micron-scale chip, and can automatically complete the entire analysis process. Compared with general detection technology, microfluidic chip has the advantages of high analysis efficiency, high accuracy, integration, flexible throughput, automation, energy saving and environmental protection. Microfluidic detection chips generally have the advantages of less sample consumption, fast detection speed, simple operation, multi-function integration, small size and easy portability, so they have great potential to reduce the overall detection cost, and are very suitable for rapid bedside detection. development of. In particular, the centrifugal microfluidic chip uses centrifugal force to control the fluid, which can simplify the instrument and equipment, and at the same time design various valve control structures in the disc chip. However, the fine micro-valve structure is prone to batch-to-batch differences in batch processing and manufacturing, which affects repeatability. More importantly, the existence of Coriolis and Euler forces in centrifugal microfluidic chips will cause deviations in the direction of fluid drainage and affect the final detection structure.

Therefore, most of the microfluidic chip applications for bedside rapid detection are still in the stage of theoretical research and patent application, and there are even fewer commercialized microfluidic detection chips. The main challenges of current microfluidic detection chips are as follows:

1. Realize the sequential release of reagents in the chip;

2. The complex structure of the chip requires local modification, which increases the cost and difficulty of chip processing and reduces the stability of the chip;

3. The simultaneous quantitative detection of multiple samples and multiple targets in a single chip requires a more ingenious chip design. At present, it is still in the rapid detection of single sample multiple targets or single targets, and at the same time, the distribution and quantification of samples cannot be well completed, so it is difficult to achieve Quantitative detection of analytes;

4. It is difficult to quantitatively distribute multiple reagents at the same time, and it is difficult to achieve sufficient mixing of the well-distributed reagents on the microfluidic channel;

5. During the drainage process of the centrifugal chip, the Coriolis force and the Euler force have deviations in the direction of the fluid.

SUMMARY OF THE INVENTION

Based on this, the present invention discloses a microfluidic automated rapid detection chip, which has the advantages of simple processing and manufacturing, can realize the sequential release of reagents required for detection, and simultaneously completes multi-target quantitative rapid detection of multiple samples in a single chip. The process is completely automated and does not require professional and technical personnel to operate. The supporting instruments are portable and simple, which is very suitable for rapid bedside detection.

According to the first aspect of the present invention, an automated chemiluminescence immunoassay chip is provided, comprising at least one detection unit, each of which includes a sample processing layer and a reagent processing layer;

The sample processing layer includes a sample injection port, a sample separation pool, a first quantitative distribution unit, a quantitative area, a first microchannel drainage unit and a reaction pool; the sample injection port, the sample separation pool, and the first quantitative distribution unit Connected to the quantitative area in sequence, the first microchannel drainage unit is used to drain the liquid in the quantitative area into the reaction tank; the first microchannel drainage unit has a chamber, and the chamber is used for embedding the reaction required substance;

The reagent treatment layer includes a first reagent pool and a second reagent pool, both of which are connected to the reaction pool; the first reagent pool and the second reagent pool are used for pre-packaging the reaction chamber. Reagents are required.

Preferably, the first reagent pool and the second reagent pool are respectively connected to the first quantitative chamber and the second quantitative chamber through a second quantitative distribution unit, and the first quantitative chamber and the second quantitative chamber are respectively connected with the second microchannel drainage unit and the third microchannel drainage unit, the second microchannel drainage unit is connected with the third microchannel drainage unit through the second quantitative chamber, and the third microchannel drainage unit is connected with the reaction Pool connection; the first reagent pool and the second reagent pool are used for pre-packaged reagents required for the reaction.

Preferably, the first reagent pool is connected to a first quantitative chamber through a second quantitative distribution unit, the first quantitative chamber is connected to a second microchannel drainage unit, and the second microchannel drainage unit is connected to The reaction pool is connected; the second reagent pool is directly connected to the reaction pool; the first reagent pool and the second reagent pool are used for pre-packaged reagents required for 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 pool and the waste liquid pool; the waste liquid pool is connected with the first quantitative distribution unit and the second quantitative distribution unit; The waste liquid pool is used to collect waste liquid in the reaction pool, the first quantitative distribution unit and the second quantitative distribution unit; the air hole is connected to the waste liquid pool and is used to balance the air pressure inside the chip.

Preferably, the first microchannel drainage unit has a serpentine microchannel combination, and the serpentine microchannel combination is used to increase the mixing effect of the liquid by means of the effect of Coriolis force and Euler force on fluid deflection during the chip centrifugation process and control fluid flow rate.

According to another aspect of the present invention, there is provided any of the automated chemiluminescence immunoassay chip detection methods, comprising the following steps:

S1: pre-embed the target antibody labeled with signal molecules in the chamber on the first microchannel drainage unit, and pre-embed the target antibody labeled with magnetic particles in the reaction pool;

S2: Add the sample to be tested at the sample injection port. Under the action of centrifugal force, the sample to be tested precipitates impurities in the sample separation tank; then, the sample solution enters the first quantitative distribution unit for distribution, and enters the first microchannel through the quantitative area In the drainage unit, the target antibody labeled with the signal molecule is reconstituted, and the target protein in the sample solution and the target antibody labeled with the signal molecule form a first complex, and the first complex enters the reaction pool and is combined with the magnetic particle-labeled target. The antibody reacts to form a second complex;

S3: start the magnet adsorption to fix the second complex in the reaction tank, then open the valve, start centrifugation, and discharge the target antibody labeled with the signal molecule that does not form the second complex into the waste liquid tank, and then close the valve;

S4: remove the magnet adsorption, release the reagents in the first reagent pool and the second reagent pool into the reaction pool, make the luminescent molecules captured in the second complex react to emit light, detect the light signal, and calculate the to-be-detected The amount of target protein in the sample.

Preferably, the signal molecule is acridinium ester, acridone or horseradish peroxidase.

Preferably, the target protein is acute response protein, procalcitonin or interleukin-6; the target antibody is acute response protein antibody, procalcitonin antibody or interleukin-6 antibody.

Preferably, the reagents in the first reagent pool and the second reagent pool are pre-excitation liquid and excitation liquid, respectively.

In general, compared with the prior art, the above technical solutions conceived by the present invention mainly have the following technical advantages:

(1) The automated rapid detection chip of the present invention can be combined with detection technologies such as biochemistry, immunity, and chemiluminescence, and is applied to the high-sensitivity, quantitative and rapid detection and analysis of analytes in biological samples. Detection and other fields have huge potential application value.

(2) The automated rapid detection chip of the present invention can complete the high-throughput distribution and quantification of various reagents, and realize the step-by-step and full mixing reaction of various reagents, thereby improving the detection sensitivity and accuracy.

(3) The automatic rapid detection chip of the present invention can realize the sequential mixing of the excitation liquid and the pre-excitation liquid in the chemiluminescence 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 of the present invention can overcome the influence of Coriolis force and Euler force on fluid deflection through the drainage channel, and realize the quantitative distribution and sequential mixing reaction of various reagents; The combination of meandering microchannels can control the deflection of the fluid by the Coriolis force and the Euler force, and improve the mixing degree of the liquid.

Description of drawings

Fig. 1 is the overall structure diagram of the automatic rapid detection chip of the present invention;

2 is a schematic diagram of a layered structure of an automated rapid detection chip of the present invention;

Fig. 3 is a schematic diagram of a detection unit of the automatic rapid detection chip of the present invention;

4 is a schematic diagram of a sample processing layer of a detection unit of an automated rapid detection chip of the present invention;

5 is a schematic diagram of a reagent processing layer of a detection unit of the automated rapid detection chip of the present invention;

6 is a schematic diagram of an embodiment of a reagent processing layer of a detection unit of an automated rapid detection chip of the present invention;

Fig. 7 is the partial structural schematic diagram of the automatic rapid detection chip of the present invention;

Fig. 8 is the effect that the microfluidic channel drainage unit of the present invention overcomes the influence of Coriolis force and Euler force in the centrifugal drainage process;

Fig. 9 is another form of the overall structure diagram of the automatic rapid detection chip of the present invention;

10 is a schematic diagram of another form of layered structure of the overall structure diagram of the automated rapid detection chip of the present invention;

11 is a schematic diagram of another form of a detection unit of the automated rapid detection chip of the present invention;

12 is a partial structural diagram of another form of a detection unit of an automated rapid detection chip of the present invention;

Figure 13 is a diagram of mechanical analysis during centrifugal drainage.

In all drawings, the same reference numerals are used to represent the same elements or structures, wherein: 200-detection unit, 51-sample processing layer, 52-reagent processing layer, 11-sample injection port, 12-sample separation pool , 13-first quantitative distribution unit, 14-quantitative area, 15-first microchannel drainage unit, 16-reaction pool, 17-chamber, 21-first reagent pool, 22-second reagent pool, 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 pool , 53 - middle layer, 54 - upper cover, 55 - lower cover.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

An automated rapid detection chip 400 of the present invention includes at least one detection unit 200, and each of the detection units includes a sample processing layer 51 and a reagent processing layer 52;

FIG. 4 is a schematic diagram of the sample processing layer of the automated rapid detection chip of the present invention. The sample processing layer 51 of the present invention includes a sample introduction port 11, a sample separation tank 12, a first quantitative distribution unit 13, a quantitative area 14, a first microchannel drainage unit 15 and a reaction tank 16; the sample introduction port 11, the sample separation The tank 12, the first quantitative distribution unit 13 and the quantitative area 14 are connected in sequence, and the first microchannel drainage unit 15 is used to drain the liquid in the quantitative area 14 into the reaction tank 16; the first microchannel drainage unit There is a chamber 17 on the 15, and the chamber 17 is used to embed the substances required for the reaction;

The reagent processing layer 52 includes a first reagent pool 21 and a second reagent pool 22, and the first reagent pool 21 and the second reagent pool 22 pass through the second quantitative distribution unit 23 and the first quantitative chamber 24 and the first quantitative chamber 24 respectively. Two quantitative chambers 25 are connected, the first quantitative chamber 24 and the second quantitative chamber 25 are respectively connected with the second microchannel drainage unit 26 and the third microchannel drainage unit 27, the second microchannel drainage unit 26 is connected to the third microchannel drainage unit 27 through the second quantitative chamber 25, and the third microchannel drainage unit 27 is connected to the reaction pool 16; the first reagent pool 21 and the second reagent pool 22 are used for pre-packaging Reagents required for the reaction.

The detection unit 200 further includes a valve 31 and a waste liquid pool 32; the valve 31 is respectively connected with the reaction pool 16 and the waste liquid pool 32; the waste liquid pool 32 is connected with the first quantitative distribution unit 13 and the second quantitative distribution The unit 23 is connected; the waste liquid pool 32 is used to collect the 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 detection unit of the automatic rapid detection chip of the present invention.

The first microchannel drainage unit 15, the second microchannel drainage unit 26 and the third microchannel drainage unit 27 have the functions of mixing and micro-valve, and the automatic rapid detection chip has a center of rotation.

Specifically, each detection unit 200 in the chip of the present invention is independent of each other, the structure of the chip can be consistent or inconsistent, and can be adjusted according to the detection requirements;

Specifically, in one embodiment, the chip has a five-layer structure, 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 sample processing layer 51 and the reagent processing layer 52 are Not in the same plane, and only communicated with each other through the reaction tank 16;

In another form of the detection chip (Fig. 8), the sample processing layer and the reagent processing layer are located on the same layer (Fig. 9). In one embodiment, the chip has a 3-layer structure, including an upper cover 54 and a lower cover 55. and the intermediate layer 53 (FIG. 10). FIG. 11 is a schematic diagram of another form of a detection unit of an automatic rapid detection chip of the present invention; FIG. 12 is a partial structural diagram of another form of a detection unit of an automatic rapid detection chip of the present invention. In another form of the present invention, the first reagent pool 21 is connected to the first quantitative chamber 24 through the second quantitative distribution unit 23, the first quantitative chamber 24 is connected to the second microchannel drainage unit 26, and the second microchannel The channel drainage unit 26 is connected to the reaction cell 16; the second reagent cell 22 is directly connected to the reaction cell 16; the first reagent cell 21 and the second reagent cell 22 are used for pre-packaged reagents required for the reaction.

Specifically, the sample introduction port 11 is communicated with the sample separation tank 12, and the sample is added from the sample introduction port 11, and the sample can be various samples such as whole blood, plasma, serum, and urine;

Specifically, the sample separation tank 12 communicates with the first quantitative distribution unit 13, and the connection port is designed as the middle of the sample separation tank 12. When the chip is centrifuged, the solid-phase impurities in the sample will preferentially settle due to the high centrifugal force and centrifugal speed. At the bottom of the sample separation tank 12, the supernatant will enter the first quantitative distribution unit 13 with continuous centrifugal force, and the connection port can be designed in various ways to prevent impurities from entering the first quantitative distribution unit 13, such as microcolumns, biofilms, Micro valve, etc.;

Specifically, the first quantitative distribution unit 13 communicates with the first microchannel drainage unit 15, and the first quantitative distribution unit 13 includes a plurality of quantitative areas 14. When the chip is centrifuged at a low speed, the sample solution will enter the quantitative area. 14, and as the centrifugal force continues, the excess sample solution will be discharged into the waste liquid pool 32, so the distribution and quantification of the sample solution can be completed, and a certain volume of sample solution can be provided for the subsequent multi-target quantitative detection;

Specifically, the size of the microchannel of the first microchannel drainage unit 15 is 10-100 μm in width and 10-100 μm in height, but is not limited thereto;

Specifically, the first microchannel drainage unit 15 includes three meandering channels in one embodiment, the microchannel near the quantitative area 14 is a hydrophobic area, and the remaining channels can be modified or not modified in any way. The serpentine channel can control the flow rate of the fluid;

Specifically, when the chip is centrifuged at high speed, the sample solution in the quantitative area 14 will break through the hydrophobic resistance of the microchannel and enter the first meandering channel of the first microchannel unit 15. The meandering channel can reduce the flow rate of the sample solution to make it match the first meandering channel of the first microchannel unit 15. The reagents embedded in the microchannel unit 15 are fully contacted, and then enter the second meandering channel and the third meandering channel for sufficient mixing reaction. At the same time, this meandering microchannel is designed to extend outward from the center of the circle. For microchannels with vertical or parallel lines, in the process of centrifugal drainage, the effect of Coriolis force and Euler force on the deflection of fluid can be used to improve the mixing effect. Without considering the fluid extrusion force, the mechanical analysis is as follows: As shown in Figure 13;

Specifically, the first meandering channel is closer to the center of the circle than the second meandering channel, the second meandering channel is closer to the center of the circle than the third meandering channel, and the distance before the meandering channel can be adjusted according to detection requirements, The structure is not limited to this;

Specifically, after the chip is centrifuged at high speed, the mixture of the sample and the reagent will enter the reaction tank 16, and the reaction tank 16 can be pre-embedded with the reagent, or modified on the surface to capture the detection target in the sample;

Specifically, the valve 31 is communicated with the reaction tank 16 and the waste liquid tank 32, opening the valve 31 and starting high-speed centrifugation can completely discharge the excess waste liquid in the reaction tank 16 into the waste liquid tank 32, so that only the waste liquid in the reaction tank 16 is discharged. The analyte required for the detection is retained, and then the valve 31 is closed;

Specifically, the first reagent pool 21 and the second reagent pool 22 are used for solution reagent storage, and the first reagent pool 21 and the second reagent pool 22 are respectively connected to a second quantitative distribution unit 23 for quantitative distribution of reagents, The quantification process is the same as the sample quantification process described above;

Specifically, the first reagent pool 21 communicates with the second microchannel drainage unit 26, the second microchannel drainage unit 26 communicates with the first quantitative chamber 24, and the third microchannel drainage unit 27 is smaller than the second microchannel drainage unit 27. The channel drainage unit 26 is further away from the center of the circle, and at the same time, the second microchannel drainage unit 26 is provided with a meandering channel, and the time for the reagent to enter the reaction tank 16 can be controlled by controlling the resistance size and length of the microchannel, and finally the second reagent pool 22 After the reagents in the first reagent pool 21 first enter the reaction pool 16, the reagents at the first reagent pool 21 quickly enter the reaction pool 16 for reaction in an orderly manner, and finally obtain the detection signal in each reaction pool 16. At the same time, in another form, The final reaction reagent can be pre-stored in the second reagent cell 22 (Fig. 9), the solution can be injected into the reaction cell by pressing or injection, the signal acquisition can be completed as needed, and the strength of the signal and the analysis within a certain solution volume can be detected. The level of the analyte is correlated with the level of the analyte, so the quantitative detection of the analyte in the sample can be carried out.

In the present invention, the microchannel drainage unit has the effects of mixing and drainage.

In the present invention, the microchannel drainage unit can control the sequence and time duration of the liquid entering the reaction tank through the length of the microchannel and the resistance of the microchannel.

In the present invention, the quantitative distribution unit and the microchannel drainage unit are connected to each other but not on the same plane. When the chip is centrifuged at a low speed, the liquid is restricted from entering the microchannel. When the chip is centrifuged at a high speed, the liquid flows into the microchannel for drainage and mixing; the quantitative distribution unit can freely flow the liquid when the chip is centrifuged at a low speed, and distribute the liquid quantitatively, and the excess liquid It will enter the waste liquid pool connected with it, and when the chip is centrifuged at high speed, the quantitative liquid will be introduced into the corresponding reaction pool through the microchannel drainage unit.

In the present invention, the reaction tank is communicated with the microvalve, and the valve is closed when the liquid needs to be stored, and is closed when the waste liquid needs to be discharged.

In the present invention, the quantitative distribution unit can realize the independent distribution and quantification of multiple liquids on the same level, which is used for the detection of multiple samples and multiple targets.

The chip in the present invention can be used for various biochemical detection and immunoassay detection, etc., but is not limited thereto.

The detection method in the present invention comprises the following steps:

Add the sample solution from the sample addition hole to the sample separation pool, and put the chip into the matching centrifugal detection instrument;

In the low-speed centrifugation chip, the impurities in the sample will quickly settle at the bottom of the sample separation tank due to the large centrifugal force, and the supernatant will enter the quantitative distribution unit due to the continuous centrifugal force to distribute and quantify the sample liquid, providing a basis for subsequent quantitative analysis and detection. , the excess liquid will be discharged into the waste pool;

The high-speed centrifugal chip injects a quantitative sample solution into the microchannel drainage unit, controls the liquid flow rate by controlling the centrifugal speed and the microchannel, redissolves the pre-embedded reagent in the channel, and fully mixes and reacts the sample and the reagent through the microchannel drainage unit , and finally enter the reaction tank;

The pre-embedded reagent in the reaction tank will be fully mixed with the incoming sample mixture, and the target complex formed after the reaction will then open the valve and centrifuge the chip at a high speed, so that the excess waste liquid is discharged into the waste liquid tank, while the The desired target complex remains in the reaction cell;

Release the reagents in all the reagent pools, centrifuge the chip at low speed to the quantitative distribution unit for quantitative distribution, discharge the excess reagents into the waste liquid pool, and then introduce the quantitatively distributed reagents into the reaction through the microchannel drainage unit by high-speed centrifugation In the pool, due to the different lengths and resistances of the microchannels, the reagents will enter the reaction pools for reaction successively, and finally the detection signals in each reaction pool will be obtained in turn.

The detection method of the automated chemiluminescence immunoassay chip of the present invention comprises the following steps:

S1: Pre-embedding the target antibody (primary antibody) labeled with signal molecules in the chamber 17 on the first microchannel drainage unit 15, and pre-embedding the target antibody (secondary antibody) labeled with magnetic particles in the reaction cell 16;

S2: Add the sample to be detected at the sample injection port 11. Under the action of centrifugal force, the sample to be detected precipitates impurities in the sample separation tank 12; then, the sample solution enters the first quantitative distribution unit 13 for distribution, and enters through the quantitative area 14. In the first microchannel drainage unit 15, the target antibody (primary antibody) labeled with the signal molecule is reconstituted, the target protein in the sample solution and the target antibody labeled with the signal molecule form a first complex, and the first complex enters the reaction pool. 16, and react with the magnetic particle-labeled target antibody (secondary antibody) to form a second complex;

S3: Start the magnet adsorption to fix the second complex in the reaction cell 16, then open the valve 31, start centrifugation, and discharge the target antibody (primary antibody) labeled with the signal molecule that does not form the second complex into the waste liquid pool , the valve 31 is then closed.

S4: remove the magnet adsorption, release the reagents in the first reagent cell 21 and the second reagent cell 22 into the reaction cell 16, make the luminescent molecules captured in the second complex react to emit light, detect the light signal, and calculate The content of the target protein in the sample to be detected is obtained.

Example 1

The embodiment provided by the present invention is that the chip is applied to acridinium ester chemiluminescence immunoassay, and the immunoassay principle is that the immunosandwich principle is used for the detection of C-reactive protein in whole blood. The specific technical scheme is as follows:

1. Antibody Modification

The acridine ester is combined with the anti-CRP antibody through the EDC and NHS systems to prepare a signal-labeled antibody complex. Similarly, the magnetic particles are combined with the anti-CRP monoclonal antibody through the EDC and NHS systems to prepare the capture antibody complex;

2. Chip processing

The chip processing method adopts CNC numerical control processing. Each layer of the chip is processed separately, and the sample processing layer and the reagent processing layer are processed on both sides to form a microchannel drainage unit on the lower surface and a quantitative distribution unit on the upper surface. , the chip processing material is PMMA material;

It can be understood that the processing technology of the chip can be a chip processing technology such as laser engraving technology, CNC numerical control technology, soft lithography technology, etc., but is not limited to this;

It can be understood that the processing material of the chip can be PMMA, PC, PDMS and other plexiglass or polymers, but is not limited thereto.

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 is assembled by double-sided adhesive bonding for sealing and combination. Before the combination, the acridine ester-labeled CRP antibody was pre-embedded in the chamber 17 of the first microchannel drainage unit 15 (as shown in FIG. 7 ), and the magnetic particle-labeled CRP monoclonal antibody was pre-embedded in the reaction cell 16; Before the upper cover 54 seals the photo, an excitation solution (NaOH) reagent pack is pre-packaged in the first reagent pool 21, and a pre-excitation solution (H ₂ O ₂ ) is pre-packaged in the first reagent pool 22, and then the chip is sealed to form a complete Check the chip.

4. Sample detection

Specifically, 100-200 μl of the whole blood sample is added to the sample addition port 11, and the chip is sealed and placed in the matching detection instrument;

Specifically, in the instrument, the chip is firstly centrifuged at a low speed, and the cells and impurities in the whole blood sample are precipitated at the bottom of the sample separation tank 12, and the plasma, blood cells and impurities in the complete blood are separated, and the centrifugation speed is increased, and the whole blood supernatant is separated. Due to the continuous centrifugal force, it will enter the first quantitative distribution unit 13 for distribution, and the final volume of the sample will be accurately quantified by the size of the quantitative area 14. The excess waste liquid will enter the waste liquid pool 32, and then the centrifugation ends;

Specifically, high-speed centrifugation of the chip is started, and a quantitative sample solution is injected into the first microchannel drainage unit 13. The sample solution will redissolve the acridinium ester-labeled CRP antibody and react with the CRP antigen in the sample to form the acridine ester-labeled CRP antibody- The CRP complex can control the fluid flow rate through the meandering microchannels, and at the same time make the reaction more sufficient. Finally, under the action of continuous high-speed centrifugation, the meandering microchannels that extend vertically or parallel to the center of the circle can be combined with the help of science and technology. The influence of the Ulrich force and the Euler force on the deflection of the fluid improves the mixing effect, and finally all the liquid will be discharged into the reaction tank 16, and the valve 31 is in the closed state at this time;

Specifically, the high-speed centrifugation stop process will cause the solution in the reaction tank 16 to vibrate, and complete the reaction between the magnetic particle-labeled CRP monoclonal antibody and the acridinium ester-labeled CRP antibody-CRP complex, making the reaction faster, shortening the reaction time, and reacting The acridinium ester-labeled CRP antibody-CRP-magnetic particle-labeled CRP monoclonal antibody complex is formed. At this time, the magnet adsorption is started, so that the magnetic particles are fixed in the reaction cell 16, the valve 31 is opened, the chip centrifugation is started, and all the liquid is discharged into the waste liquid In the pool 32, the centrifugation was stopped; then the valve 31 was closed, the adsorption of the magnetic particles by the magnet was removed, and the pre-excitation liquid and the excitation liquid in the first reagent pool 21 and the second reagent pool 22 were released at the same time, and the low-speed centrifugation was started to accurately quantify the reagents. , followed by high-speed centrifugation, the pre-excitation liquid preferentially and quickly enters the reaction tank 16 to resuspend the magnetic particles, and then the excitation liquid passes through the meandering third microchannel drainage unit 27, as shown in Figures 5 and 6, by controlling the length of the microchannel and the resistance, so that the excitation liquid quickly enters the reaction tank 16 at the same time or in sequence, so that the captured acridine ester completes the rapid reaction and emits light, and the instrument detects the light signal to complete the accurate quantitative detection of the CRP content in the sample.

Understandably, as shown in Figure 1, each chip can complete the simultaneous detection of three different samples, and each detection unit in the chip can complete the analysis and detection of 7 target detection objects, and at the same time, it can sacrifice the last detection unit. Set up a positive control assay to complete the quality control of a single chip.

Example 2

Another embodiment provided by the present invention is to apply the chip to the luminol chemiluminescence immunoassay based on horseradish peroxidase (HRP), and the immunoassay principle is the principle of immunosandwich for procalcitonin (PCT) ) detection, the specific technical scheme is as follows:

1. Antibody Modification

Combine HRP with anti-PCT antibody through EDC and NHS systems to prepare signal-labeled antibody complexes, and similarly, combine magnetic particles with anti-PCT monoclonal antibodies through EDC and NHS systems to prepare capture antibody complexes;

2. The chip processing method is similar to the specific embodiment 1;

3. Chip assembly

As shown in Figure 10, the chip has a total of 3 layers, including an upper cover 54, a lower cover 55, and an intermediate layer 53. The chip is assembled by double-sided tape for sealing and assembly. Before assembly, the HRP-labeled PCT antibody is pre-packaged Buried at the beginning of the first microchannel drainage unit 15 (that is, the end connected to the quantitative unit 14), the magnetic particle-labeled PCT monoclonal antibody is pre-embedded in the reaction cell 16; The luminol reagent pack is pre-packaged in the pool 21, and the pre-hydrogen peroxide (H ₂ O ₂ ) is pre-packaged in the second reagent pool 22 , and then chip sealing is performed to form a complete detection chip.

4. Sample detection

Specifically, 100-200 μl of the whole blood sample is added to the sample addition port 11, and the chip is sealed and placed in the matching detection instrument;

Specifically, in the instrument, the chip is firstly centrifuged at a low speed, and the cells and impurities in the whole blood sample are precipitated at the bottom of the sample separation tank 12, and the plasma, blood cells and impurities in the complete blood are separated, and the centrifugation speed is increased, and the whole blood supernatant is separated. Due to the continuous centrifugal force, it will enter the first quantitative distribution unit 13 for quantitative distribution, the final volume of the sample will be accurately quantified by the size of the quantitative area 14, and the excess waste liquid will enter the waste liquid pool 32, and then the centrifugation ends;

Specifically, high-speed centrifugation of the chip is started, and a quantitative sample solution is injected into the first microchannel drainage unit 15, and the sample solution will reconstitute the HRP-labeled PCT antibody and react with the PCT antigen in the sample to form the HRP-labeled PCT antibody-PCT complex, Through the meandering microchannels, the fluid flow rate can be controlled, and the reaction can be made more sufficient. Finally, under the action of continuous high-speed centrifugation, the meandering microchannels extending vertically or parallel to the center of the circle can be combined with the help of Coriolis force and European The effect of the pulling force on the deflection of the fluid improves the mixing effect, and finally all the liquid will be discharged into the reaction tank 16, and the valve 31 is closed at this time;

Specifically, the high-speed centrifugation stop process will cause the solution in the reaction tank 16 to vibrate, and complete the reaction between the magnetic particle-labeled PCT monoclonal antibody and the HRP-labeled PCT antibody-PCT complex, making the reaction faster, shortening the reaction time, and forming HRP. Label the PCT antibody-PCT-magnetic particle-labeled PCT monoclonal antibody complex, start the magnet adsorption at this time, make the magnetic particles immobilized in the reaction cell 16, open the valve 31, start the chip centrifugation, discharge all the liquid into the waste liquid pool, stop Centrifuge; then close the valve 31, remove the adsorption of the magnet to the magnetic particles, release the luminol in the first reagent pool 21 at the same time, start the low-speed centrifugation to accurately quantify the reagent, and then high-speed centrifuge, so that the reagent enters the reaction pool 16, and then The hydrogen peroxide solution in the second reagent cell is rapidly injected into the reaction cell 16 by pressing or injecting simultaneously or sequentially, and the rapid reaction with the captured HRP enzyme is completed. Precise quantitative detection.

Understandably, as shown in Figure 1 and Figure 9, each chip can complete the simultaneous detection of three different samples, and each detection unit in the chip can complete the analysis and detection of 7 kinds of target detection substances, and can also sacrifice the last one. Detection unit, set up positive control detection, complete the quality control of a single chip.

The verification of the effect of the drainage channel provided by the present invention overcoming the Coriolis force and Euler force is shown in Figure 8: drainage channels with different extension lengths are designed, and the final result is verified that from left to right, the deflection of the liquid becomes larger and the channel extends to a certain When the length is longer, the deflection of the liquid is basically not affected by the Coriolis force and the Euler force, which further ensures the reliability and repeatability of the analysis and detection structure.

Those skilled in the art can easily understand that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, etc., All should be included within the protection scope of the present invention.

Claims (9)

1. An automated chemiluminescence immunoassay chip, comprising at least one detection unit (200), each of the detection units comprising a sample processing layer (51) and a reagent processing layer (52); The sample processing layer (51) includes a sample injection port (11), a sample separation pool (12), a first quantitative distribution unit (13), a quantitative area (14), a first microchannel drainage unit (15) and a reaction a pool (16); the sample adding port (11), the sample separation pool (12), the first quantitative distribution unit (13) and the quantitative area (14) are connected in sequence, and the first microchannel drainage unit (15) The first microchannel drainage unit (15) is provided with a chamber (17), and the chamber (17) is used for embedding substances required for the reaction; The reagent treatment layer (52) includes a first reagent pool (21) and a second reagent pool (22), and both the first reagent pool (21) and the second reagent pool (22) are connected to the reaction pool (16) ; The first reagent pool (21) and the second reagent pool (22) are used for pre-packaged reagents required for the reaction. 2. The automated chemiluminescence immunoassay chip according to claim 1, wherein the first reagent pool (21) and the second reagent pool (22) pass through the second quantitative distribution unit (23) and the first reagent pool respectively. The quantitative chamber (24) and the second quantitative chamber (25) are connected, and the first quantitative chamber (24) and the second quantitative chamber (25) are respectively connected with the second microchannel drainage unit (26) and The third microchannel drainage unit (27) is connected, the second microchannel drainage unit (26) is connected to the third microchannel drainage unit (27) through the second quantitative chamber (25), and the third microchannel drainage The unit (27) is connected with the reaction pool (16); the first reagent pool (21) and the second reagent pool (22) are used for pre-packaged reagents required for the reaction. 3. The automated chemiluminescence immunoassay chip according to claim 1, wherein the first reagent pool (21) is connected to the first quantitative chamber (24) through the second quantitative distribution unit (23), The first quantitative chamber (24) is connected with the second microchannel drainage unit (26), and the second microchannel drainage unit (26) is connected with the reaction pool (16); the second reagent pool (22) ) is directly connected to the reaction pool (16); the first reagent pool (21) and the second reagent pool (22) are used for pre-packaged reagents required for the reaction. 4. The automated chemiluminescence immunoassay chip according to claim 1, wherein the detection unit (200) further comprises a valve (31), a waste liquid pool (32) and an air hole (33); the valve (200) 31) are respectively connected with the reaction pool (16) and the waste liquid pool (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 connected with the first quantitative distribution unit (13) and the second quantitative distribution unit (23); The liquid pool (32) is used for collecting the 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 to the waste liquid pool (32) Connection for balancing the air pressure inside the chip. 5. The automated chemiluminescence immunoassay chip according to claim 1, wherein the first microchannel drainage unit (15) has a serpentine microchannel combination, and the serpentine microchannel combination is used for centrifugation by means of a chip During the process, the effect of Coriolis force and Euler force on the deflection of the fluid increases the mixing effect of the liquid and controls the fluid flow rate. 6. the detection method of the automatic chemiluminescence immunoassay chip as described in any one of claim 1-5, is characterized in that, comprises the following steps: S1: pre-embedding the target antibody labeled with signal molecules in the chamber (17) on the first microchannel drainage unit (15), and pre-embedding the target antibody labeled with magnetic particles in the reaction pool (16); S2: Add the sample to be tested at the sample inlet (11), and under the action of centrifugal force, the sample to be tested precipitates impurities in the sample separation tank (12); then, the sample solution enters the first quantitative distribution unit (13) for distribution , enter the first microchannel drainage unit (15) through the quantitative area (14), and reconstitute the target antibody labeled with the signal molecule, the target protein in the sample solution and the target antibody labeled with the signal molecule form a first complex. A complex enters the reaction cell (16), and reacts with the magnetic particle-labeled target antibody to form a second complex; S3: start the magnet adsorption, so that the second complex is fixed in the reaction tank (16), then open the valve (31), start centrifugation, and discharge the target antibody labeled with the signal molecule that does not form the second complex into the waste liquid tank , then close the valve (31); S4: remove the magnet adsorption, release the reagents in the first reagent pool (21) and the second reagent pool (22) into the reaction pool (16), make the luminescent molecules captured in the second complex react to emit light, and react to the light The signal is detected, and the content of the target protein in the sample to be detected is calculated. 7. The detection method of claim 6, wherein the signal molecule is acridine ester, acridine or horseradish peroxidase. 8. The detection method according to claim 6 or 7, wherein the target protein is acute time response protein, procalcitonin or interleukin-6; the target antibody is acute time response protein antibody, calcitonin primary antibody or interleukin-6 antibody. 9 . The detection method according to claim 6 , wherein the reagents in the first reagent pool ( 21 ) and the second reagent pool ( 22 ) are pre-excitation liquid and excitation liquid, respectively. 10 .

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