CN108226536B

CN108226536B - Micro-fluidic immunodetection chip for cardiac troponin I

Info

Publication number: CN108226536B
Application number: CN201810138655.3A
Authority: CN
Inventors: 沈林; 张华�; 戈军; 肖长河; 余琼林; 陈远超
Original assignee: Hunan Luyang Biotechnology Co ltd; Hunan Yonghe Sunshine Biotechnology Co ltd
Current assignee: Hunan Luyang Biotechnology Co ltd; Hunan Yonghe Sunshine Biotechnology Co ltd
Priority date: 2018-02-10
Filing date: 2018-02-10
Publication date: 2020-08-04
Anticipated expiration: 2038-02-10
Also published as: CN108226536A

Abstract

The invention discloses a microfluidic immunodetection chip and a microfluidic immunodetection method for cardiac troponin I. In the detection chip, the sample storage pool, the reaction detection pool and the waste liquid pool are sequentially arranged on the main microchannel path, one end of the main microchannel path is provided with a sample inlet, the other end of the main microchannel path is connected with a first pump, one end of the branch microchannel path is communicated with the storage pool through a second pump, the other end of the branch microchannel path is connected on the main microchannel path between the reaction detection pool and the waste liquid pool, the branch microchannel path is sequentially provided with a reagent B filling pool and a high-value calibration pool, the low-value calibration pool is connected with the high-value calibration pool in parallel, and the branch microchannel between the reagent B filling pool and the high-value calibration pool is further communicated with the main microchannel between the sample storage pool and the reaction detection pool through a microchannel connection branch path. The invention can greatly shorten the sample processing time, reduce the cost, improve the detection precision and realize the functions of mobile detection, home self-service detection and the like.

Description

Micro-fluidic immunodetection chip for cardiac troponin I

Technical Field

The invention relates to the technical field of biological detection, in particular to a micro-fluidic immunodetection chip for cardiac troponin I.

Background

Cardiac troponin (cTn) is a regulatory protein of cardiac muscle cell contraction, which is composed of subunits of three different genes: cardiac troponin t (ctnt), cardiac troponin i (ctni) and cardiac troponin c (ctnc). The detection of cTnI has the highest sensitivity among the three, so the detection requirement precision is also the highest, and the conventional means has difficulty in great accuracy and precision in the detection of the item. However, cTnI has very important clinical significance, mainly expressed in: (1) the cTnI is not affected by the damage of skeletal muscle, appears earlier after the onset of disease of a patient, has long duration, has the advantages of unique myocardial specificity and longer diagnosis window period, and is a better determination marker for diagnosing myocardial damage at present. (2) The release of cTnI is linearly related to the myocardial infarction area, the correlation is best after 9h after AMI, and the concentration of cTnI after 9h after AMI can estimate the myocardial infarction area. The area of myocardial infarction has close relation with the weakening of ventricular function and the occurrence of ventricular arrhythmia, and the estimation of the area of myocardial infarction is greatly helpful for the evaluation and prognosis of subsequent diseases. (3) The cTnI has higher sensitivity and specificity for detecting the micro myocardial damage, can be used for monitoring the perioperative period of the heart, and has been proved by researches to have the highest prediction value of the cTnI on the postoperative micro myocardial damage. (4) The cTnI has unique myocardial specificity and can identify whether patients with skeletal muscle injury or chronic renal failure are accompanied by myocardial injury.

At present, the detection of the cardiac troponin I is mainly carried out by a highly sensitive enzyme immunoassay (E L ISA) for quantitative detection or a rapid solid-phase immunochromatography for qualitative detection.

The Chip is mainly composed of a micro-channel, a micro-pump, a micro-valve, a micro-reaction chamber and the like, and has various functional characteristics of high integration, automation and a biochemical laboratory, the biochemical operation on the micro-fluidic Chip has the advantages of convenient and rapid sample processing, low cost and the like, and the specific surface area of the sample is far larger than that of a macroscopic sample under the microscopic scale, so that a plurality of biochemical reactions with long time consumption under the macroscopic scale can be more fully completed in a short time, and the detection result is more accurate than that of the traditional detection means, therefore, the enzyme reaction, the immunoreaction, the PCR reaction, the enzyme-linked immunosorbent assay (E L ISA) and the like which are carried out by taking drug screening or biochemical detection analysis as the purposes can be realized on the Chip, the sample processing time can be greatly shortened, the reagent and the micro-fluidic detection cost can be greatly reduced, and the detection function of a mobile home self-service detection instrument and the like can be greatly improved.

Disclosure of Invention

The invention aims to provide a micro-fluidic immunodetection chip and a micro-fluidic immunodetection method for cardiac troponin I, so as to overcome the problems in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a micro-fluidic immunodetection chip for cardiac troponin I comprises an upper cover of the chip and the chip with a structure, wherein the chip with the structure is provided with a sample inlet, a sample storage tank, a micro-channel, a reagent B filling tank, a high-value calibration tank, a low-value calibration tank, a reaction detection tank, a waste liquid tank, a water storage tank, a second pump and a first pump, the micro-channel comprises a main micro-channel circuit and a micro-channel branch circuit, the sample storage tank, the reaction detection tank and the waste liquid tank are sequentially arranged on the main micro-channel circuit, one end of the main micro-channel circuit is provided with the sample inlet, the other end of the main micro-channel circuit is connected with the first pump, one end of the micro-channel branch circuit is communicated with the water storage tank through the second pump, the other end of the micro-channel branch circuit is connected on the main micro-channel circuit between the reaction detection tank and the waste liquid tank, the reagent B filling tank and the high-value calibration tank are sequentially, the low-value calibration pool is connected with the high-value calibration pool in parallel, and a micro-channel branch between the reagent B filling pool and the high-value calibration pool is also communicated with a micro-channel main circuit between the sample storage pool and the reaction detection pool through a micro-channel connecting branch; and a blood filtering membrane is arranged in the sample storage pool.

Further, the one end of microchannel branch road is equipped with second pump interface, be equipped with first medical rubber buffer in the second pump interface, the output of second pump passes through syringe needle and second pump interface intercommunication.

Further, the other end of microchannel main road is equipped with first pump interface, be equipped with the medical rubber buffer of second in the first pump interface, the output of first pump passes through syringe needle and first pump interface intercommunication.

The invention also provides a detection method of the microfluidic immunodetection chip for the cardiac troponin I, which comprises the following steps,

s1, injecting a quantitative whole blood or serum sample into the detection chip through the sample inlet;

s2, starting a first pump to vacuumize, enabling two ends of fluid in a main path of a micro channel of the detection chip to generate pressure difference, enabling the sample to flow into a sample storage pool under the action of surface tension, and enabling blood serum to enter a reaction detection pool after the sample is filtered by a blood filtering membrane in the sample storage pool;

s3, combining the sample flowing into the reaction detection pool with the pre-filled reagent A for reaction, pumping the redundant sample into the waste liquid pool by the first pump after the reaction is sufficient, and then closing the first pump;

s4, starting a second pump to pump water in the water storage pool into a reagent B filling pool, conveying the water into the reaction detection pool, the high-value calibration pool and the low-value calibration pool, and closing the second pump to enable the reagent to fully react;

s5, starting a first pump, sucking away redundant reagents in the reaction detection pool, the high-value calibration pool and the low-value calibration pool, and allowing the reagents to flow into a waste liquid pool;

and S6, detecting, processing and displaying the reaction detection pool, the high-value calibration pool and the low-value calibration pool by external instrument equipment.

Compared with the prior art, the invention has the advantages that: the invention can greatly shorten the sample processing time, greatly reduce the reagent cost, improve the detection accuracy, realize the functions of mobile detection, household self-service detection and the like, and bring foreseeable great economic benefits.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic structural diagram of a microfluidic immunodetection chip for cardiac troponin I according to the present invention.

FIG. 2 is an internal schematic view of a microfluidic immunodetection chip for cardiac troponin I according to the present invention.

FIG. 3 is a perspective view of a microfluidic immunodetection chip for cardiac troponin I according to the present invention.

Fig. 4 is a perspective view of the internal structure of the microfluidic immunodetection chip for cardiac troponin I of the present invention.

Description of reference numerals: 1. sample inlet, 2, sample reservoir, 3, microchannel, 4, detect the chip, 5, second pump interface, 6, reagent B fills the pond, 7, high value calibration pond, 8, low value calibration pond, 9, reaction detection pond, 10, waste liquid pond, 11, first pump interface, 12, storage water pond, 13, the second pump, 14, first medical rubber buffer, 15, the medical rubber buffer of second, 16, the syringe needle, 17, first pump, 21, chip upper cover, 22, the chip of structure of band.

Detailed Description

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, and the scope of the present invention will be more clearly and clearly defined.

Referring to fig. 1 to 4, the present invention provides a microfluidic immunoassay chip 4 of cardiac troponin I, comprising an upper chip cover 21 and a chip 22 with a structure, wherein the chip 22 with a structure is provided with a sample inlet 1, a sample reservoir 2, a microchannel 3, a reagent B filling reservoir 6, a high value calibration reservoir 7, a low value calibration reservoir 8, a reaction detection reservoir 9, a waste liquid reservoir 10, a water reservoir 12, a second pump 13 and a first pump 17.

The micro-channel 3 comprises a micro-channel main path and a micro-channel branch path, the sample storage pool 2, the reaction detection pool 9 and the waste liquid pool 10 are arranged on the micro-channel main path in sequence, one end of the main microchannel path is provided with a sample inlet 1, the other end of the main microchannel path is connected with a first pump 17, one end of the micro-channel branch is communicated with a water storage pool 12 through a second pump 13, the other end of the micro-channel branch is connected on a micro-channel main path between the reaction detection pool 9 and the waste liquid pool 10, a reagent B filling pool 6 and a high-value calibration pool 7 are sequentially arranged from one end to the other end of the micro-channel branch, the low-value calibration pool 8 is connected with the high-value calibration pool 7 in parallel, and a micro-channel branch between the reagent B filling pool 6 and the high-value calibration pool 7 is also communicated with a micro-channel main path between the sample storage pool 2 and the reaction detection pool 9 through a micro-channel connecting branch; and a blood filtering membrane is arranged in the sample storage pool 2.

One end of the micro-channel branch is provided with a second pump interface 5, a first medical rubber plug 14 is arranged in the second pump interface 5, and the output end of the second pump 13 is communicated with the second pump interface 5 through a needle 16.

The other end of microchannel main road is equipped with first pump interface 11, be equipped with second medical rubber buffer 15 in the first pump interface 11, the output of first pump 17 passes through syringe needle 16 and first pump interface 11 intercommunication.

The detection chip 4 of the present invention is pre-embedded with a reagent A (capture antibody) in a reaction detection cell 9, and pre-embedded with a reagent B (labeled antibody coupled with fluorescein) in a reagent B filling cell 6.

The basic principle of the invention is as follows: the fluid has the effect of surface tension in the microchannel, the detection chip 4 is externally connected with a pump to ensure that the fluid has pressure difference in the microchannel through vacuum pumping, the fluid can flow in the microchannel under the action of the pressure difference, and the flowing distance is related to the surface roughness of the microchannel, the viscosity coefficient of the fluid, the pressure difference and the like. The surface of the micro-channel can be modified into a hydrophilic surface by a surface modification technology, so that the resistance of fluid in the channel can be reduced, and the phenomenon that the liquid is remained in the flow channel and does not flow away is avoided. When the pump is not driven, the fluid stays in place and no longer flows.

The method comprises the following concrete steps:

step one, preparing a chip: the microfluidic chip for detection can be manufactured by injection molding, photoetching, corrosion, mold transfer, stamping and the like. The chip can be made of glass, PDMS, PC plastic, PMMA and other materials, and the chip material can be various or one of the materials. After the chip is processed, the surface structure of the chip is chemically modified according to requirements, and the chemical modification can be hydrophilic modification or hydrophobic modification.

Step two, reagent pre-filling: the reagent is pre-embedded into a reaction detection pool, a reagent filling pool and a high-low value calibration pool in the modes of physical adsorption, chemical crosslinking, sol-gel embedding, microbead fixing, membrane fixing, crystallization treatment, freezing and the like.

Step three, chip bonding and packaging: the bonding of the chip selects different bonding modes according to different chip preparation materials. Including direct bond packaging, ultrasonic bond packaging, laser bond packaging, solution assisted bond packaging, thermal bond packaging, introduction of interlayer bond packaging (glue, film, etc.), and the like.

The fourth step, pour into quantitative whole blood or serum sample into toward the chip with quantitative capillary or other equipment, two external first pumps and second pump insert the chip in, and medical rubber buffer has been inlayed to the inserted hole, guarantees that the phenomenon of gas leakage and liquid refluence can not appear, and concrete detection flow is as follows:

s1, injecting a quantitative whole blood or serum sample into the detection chip 4 through the sample inlet 1;

s2, starting the first pump 17 to vacuumize, so that pressure difference is generated at two ends of fluid in the main path of the micro-channel of the detection chip 4, the sample flows into the sample storage pool 2 under the action of surface tension, and the serum enters the reaction detection pool 9 after being filtered by a blood filtering membrane in the sample storage pool 2;

s3, the sample flowing into the reaction detection pool 9 is combined with the pre-filled reagent A for reaction, after the reaction is fully performed, the first pump 17 pumps the redundant sample away to flow into the waste liquid pool 10, and then the first pump 17 is closed;

s4, the second pump 13 is started to pump the water in the water storage pool 12 into the reagent B filling pool 6, and the water is conveyed into the reaction detection pool 9, the high value calibration pool 7 and the low value calibration pool 8, and the second pump 13 is closed to enable the reagent to fully react;

s5, starting the first pump 17, sucking away the redundant reagents in the reaction detection pool 9, the high-value calibration pool 7 and the low-value calibration pool 8, and flowing into the waste liquid pool 10;

The principle of the reaction detection is a double-antibody sandwich method principle, a reagent A (capture antibody) and a reagent B (coupled fluorescein labeled antibody) are pre-embedded in a detection chip 4 in advance, filtered serum flows into a reaction detection pool 9 and then fully contacts and reacts, the antigen in the serum is captured, the combination of the antigen and the antibody is completed, after the reaction is completed, the redundant serum is pumped away, then a second pump 13 is started, water is pumped into the chip dissolving reagent B and conveyed into the reaction detection pool 9, a high-value calibration pool 7 and a low-value calibration pool 8, the antibody with the fluorescent label in the reaction detection pool 9 is combined with the antigen which is combined with the capture antibody in the reaction pool again, and a double-antibody antigen-sandwiched structure is formed; the high value calibration cell 7 and the low value calibration cell 8 are the antigen bound to the fluorescein labeled antibody. The bound antigens are all provided with fluorescent labels, and only have absorption effect on a certain wavelength or a certain wavelength range. Through the optical detection mode, the antigen in the serum can be rapidly detected, and the detection result can be accurately judged and corrected according to the calibration of the high and low values.

The detection chip provided by the invention is provided with a self-contained calibration system, so that the detection result is accurate and correct.

The calibration principle of the calibration system (low value calibration principle) is: the high-value and low-value calibration pools are respectively filled with known quantitative antigens with different concentrations, the high-value calibration pool is filled with an antigen of cardiac troponin I with a higher concentration, the low-value calibration pool is filled with an antigen with a lower concentration, during calibration, the antigens in the two calibration pools are combined with a reagent B (a labeled antibody coupled with fluorescein), 2 different values, namely a low value and a high value can be detected during detection, each detection device is provided with a standard calibration curve corresponding to each detection value by each concentration, during the actual detection process, when the system is not calibrated, the detection result depends on the standard calibration curve in an instrument, the system can calculate the concentration of the cardiac troponin I in the blood of a detector according to the comparison between the detection data and the calibration curves, however, the actual detection is not simple, between the sample, all there is the difference between the chip, the simple detection result that appears easily to rely on standard calibration curve appears and deviates and inaccurate, under the condition that there is the height value calibration, 2 height values of survey can contrast with standard curve, form a conversion formula, every chip all has the difference, therefore the height value of every chip survey probably all is different, the conversion formula is also different, the result of the sample detection of every chip also can be according to the calibration conversion formula of this chip and make corresponding adjustment, thereby the testing result of this sample of more accurate determination.

The invention adopts a micro-fluidic chip, integrates the whole process of detecting the cardiac troponin I on the micro-fluidic chip, controls the entry of a sample and the reaction by using the driving of an external pump, and detects the reaction result by using a fluorescent label and an optical detection device. The invention has the following advantages:

1. the method comprises the steps of (1) greatly reducing the dosage of a sample and a reagent, completing a detection process only by whole blood or serum of 1-100 mu L, and greatly reducing the dosage of the corresponding reagent, (2) reducing the sample processing time, adding a blood filtering membrane in a chip, and quickly separating the serum, thereby overcoming the characteristic of long time consumption of the traditional centrifugal serum separation technology, improving the sample pretreatment efficiency, 3) realizing the clinical and mobile detection by miniaturizing and portability a detection instrument, or realizing the detection mode popularized in families, bringing great convenience to medical staff and patients, and 4) shortening the detection time and improving the detection precision, wherein the specific surface area of the reagent and the sample is far larger than that of the micro-scale, the combination of the reagent and the sample can be more sufficient and quick, the detection time is greatly reduced, and the detection precision is more accurate.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, various changes or modifications may be made by the patentees within the scope of the appended claims, and within the scope of the invention, as long as they do not exceed the scope of the invention described in the claims.

Claims

1. A micro-fluidic immunodetection chip of cardiac troponin I is characterized in that: the device comprises an upper cover of a chip and the chip with the structure, wherein the chip with the structure is provided with a sample inlet, a sample storage pool, a micro-channel, a reagent B filling pool, a high-value calibration pool, a low-value calibration pool, a reaction detection pool, a waste liquid pool, a water storage pool, a second pump and a first pump, the micro-channel comprises a micro-channel main path and a micro-channel branch path, the sample storage pool, the reaction detection pool and the waste liquid pool are sequentially arranged on the micro-channel main path, one end of the micro-channel main path is provided with the sample inlet, the other end of the micro-channel main path is connected with the first pump, one end of the micro-channel branch path is communicated with the water storage pool through the second pump, the other end of the micro-channel branch path is connected on the micro-channel main path between the reaction detection pool and the waste liquid pool, the reagent B filling pool and the high-value calibration pool are sequentially arranged from one end to the other end of the micro, the micro-channel branch between the reagent B filling pool and the high-value calibration pool is also communicated with a micro-channel main path between the sample storage pool and the reaction detection pool through a micro-channel connecting branch; a blood filtering membrane is arranged in the sample storage pool; a second pump interface is arranged at one end of the micro-channel branch, a first medical rubber plug is arranged in the second pump interface, and the output end of the second pump is communicated with the second pump interface through a needle head;

the other end of the main micro-channel path is provided with a first pump interface, a second medical rubber plug is arranged in the first pump interface, and the output end of the first pump is communicated with the first pump interface through a needle head;

and pre-burying a reagent B in a reagent B filling pool, wherein the reagent B is a labeled antibody coupled with fluorescein.