Background
The microfluidic chip is a main platform for realizing microfluidic technology, and can integrate basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a small chip. The whole analysis process is automatically completed through the micro-channel, so as to realize various functions of a conventional chemistry or biology laboratory. The microfluidic chip has the advantages of light volume, small amount of used sample and reagent, high reaction speed, capability of parallel processing in a large amount, capability of being used and discarded, and the like, has great potential in the fields of biology, chemistry, medicine and the like, and has been developed into a brand-new research field with crossing subjects of biology, chemistry, medicine, fluid, electronics, materials, machinery and the like in recent years.
The existing microfluidic chip is mostly detected by adopting a fluorescent microsphere labeling method, a fluorescent analyzer is required to detect a detection result, different types of fluorescent microspheres are required to be excited by different excitation lights, an adaptive detection instrument is required to detect the fluorescent microspheres, so that the detection cost is increased, meanwhile, the sensitivity of the fluorescent microsphere labeling method is not high enough, and errors can be generated for certain detection requiring higher sensitivity, so that the detection result is inaccurate.
The chemiluminescent immunoassay combines a chemiluminescent system with an immune reaction, is a novel label immunoassay technology for detecting trace antigens or antibodies, has the advantages of good selectivity, high sensitivity, strong specificity, no radioactive hazard, high analysis speed and the like, is widely applied to the fields of environment, clinic, food, drug detection and the like, and becomes a research hotspot and development trend of an immunoassay method.
The common chemiluminescent reaction comprises chemiluminescent enzyme immunoassay and direct chemiluminescent immunoassay, wherein the chemiluminescent enzyme immunoassay is to label an antigen or an antibody by utilizing the catalysis of a label enzyme (horseradish peroxidase or alkaline phosphatase), perform antigen-antibody reaction to form a solid-phase antibody-antigen to be detected-enzyme-labeled antibody complex, and add a substrate (luminescent agent) after washing, enzyme catalyze and decompose the substrate to emit light, so as to calculate the concentration of a measured object, wherein the luminescent substrate catalyzed by the horseradish peroxidase is luminol and derivatives thereof, and the luminescent substrate catalyzed by the alkaline phosphatase is AMPPD; the direct chemiluminescence immune analysis is to directly label an antibody (antigen) by using a chemiluminescent agent (such as acridinium ester), and after immunoreaction with the corresponding antigen (antibody) in a sample to be detected, forming an acridinium ester-labeled antibody complex of a solid-phase coated antibody to be detected, wherein only an oxidant (H2O2) and a correction solution (NaOH) are added to make luminescence, so that quantitative or qualitative analysis can be carried out through the determination of the luminous intensity of a combined state.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art, and provides a chemiluminescent microfluidic chip which is used for solving the technical problems in the background art.
In order to achieve the above purpose, the technical scheme of the invention is that the chemiluminescent microfluidic chip comprises a substrate and a cover plate, wherein the substrate and the cover plate are surrounded to form a microchannel, the microchannel comprises an auxiliary sample adding region, a reaction region and a control region, the auxiliary sample adding region, the reaction region and the control region are sequentially communicated along the length direction of the microchannel, the control region is provided with a control valve, the control valve can be contacted with or separated from the microchannel, the control valve controls the flow of liquid in the reaction region to the control region, the reaction region comprises a marking region, a detection region, a reference region and a sample adding region, the marking region, the detection region, the reference region and the sample adding region are sequentially arranged in the length direction of the microchannel, the reaction region is also provided with a blank region, the blank region and the reference region are respectively provided with a microelectrode, the microelectrode is embedded in the surface of the substrate at a corresponding position, the surface of the substrate is coated with a luminescent agent, and the antibody can be combined with a target analyte, and the antibody can be detected, and the antibody can be combined with the antibody in the target analyte.
Further, the chemiluminescent agent is one of an enzyme-catalyzed luminescent enzyme and an acridinium ester.
Further, a groove is formed in the lower surface of the cover plate along the length direction of the cover plate, and the groove and the upper surface of the base plate are enclosed to form the micro-channel.
Still further, the blank region is disposed between the marking region and the detection region.
Furthermore, the upper surface of the cover plate is coated with a light-shielding material, the positions of the upper surface of the cover plate corresponding to the blank area, the detection area and the reference area are provided with light-transmitting areas which are not coated with the light-shielding material, and the light-transmitting areas are round.
Furthermore, the auxiliary sample adding area is provided with auxiliary sample adding holes which are communicated with the upper surface and the lower surface of the cover plate, and the sample adding area is provided with sample adding holes which are communicated with the upper surface and the lower surface of the cover plate.
Still further, the control valve is a movable absorbent material that is capable of contacting or moving away from the microchannel.
The detection method of the chemiluminescence microfluidic chip, which is used for detection by adopting the chemiluminescence microfluidic chip and adopts a double-antibody sandwich mode, comprises at least the following steps:
1) Separating the control valve from the micro-channel, injecting a certain volume of sample to be tested into the sample loading area, allowing the sample to flow into the micro-channel and flow towards the detection area under the action of capillary force, and staying for 3-5 min to enable the reaction to be fully carried out, if the sample contains target antigen, capturing the target antigen by antibody coated by the detection area to form an antibody-target antigen complex, and after the reaction is finished, enabling the control valve to contact the micro-channel, and allowing unreacted redundant sample to flow towards the control area;
2) Separating the control valve from the micro-channel, adding buffer solution through the auxiliary sample adding area, enabling the buffer solution to flow towards the direction of the marking area and dissolve the antibody coated by the marking area, then driving the dissolved antibody in the marking area to continuously flow, forming an antibody-target antigen-marked antibody compound in the detection area, enabling unbound marked antibody to continuously flow along with the liquid, capturing the liquid in the reference area by the corresponding substance, enabling the control valve to contact the micro-channel after the reaction is finished, and continuously washing the micro-channel by redundant liquid and entering the control area;
3) Adding a corresponding luminescent substrate or oxidant into the auxiliary sample adding area according to the type of the marker in the marker area, regulating the temperatures of the blank area, the detection area and the reference area by using microelectrodes according to the reaction requirement, so that the luminescent substrate or oxidant flows into the blank area, the detection area and the reference area in sequence, and then the luminescent substrate or oxidant and the marker are combined to emit light;
4) Continuously collecting signal values of the blank area, the detection area and the reference area through the detector until the reaction is finished;
5) Acquiring a calibration function;
6) And obtaining the concentration of the target analyte in the sample to be tested through the calibration function.
The chemiluminescence micro-fluidic chip obtained through the technical scheme has the beneficial effects that:
1. The chemiluminescence is applied to the microfluidic chip, so that the sensitivity of the reaction is increased, and meanwhile, the visible light generated in the reaction process can be directly detected, so that the detection cost is reduced.
2. The empty area is arranged in the detection area, so that the interference of nonspecific luminescence in the chip can be eliminated, and the detection result is more accurate.
3. Microelectrodes are arranged below the blank area, the detection area and the reference area, and the required temperature can be regulated according to the chemiluminescent reaction conditions, so that the chemiluminescent reaction is more sufficient.
4. The light-shielding material is coated on the upper surface of the cover plate, and the light-transmitting area is reserved, so that the light value emitted by a single area can be detected, the scattering of light at other positions is blocked, and the accuracy of the detection result is improved.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
As shown in fig. 1 and fig. 3-4, a chemiluminescent microfluidic chip comprises a substrate 1 and a cover plate 2, wherein the substrate 1 and the cover plate 2 are surrounded to form a microchannel 3, the microchannel 3 comprises an auxiliary sample adding region 31, a reaction region 32 and a control region 33, the auxiliary sample adding region 31, the reaction region 32 and the control region 33 are sequentially communicated along the length direction L of the microchannel 3, the control region 33 is provided with a control valve, the control valve can be contacted with or far away from the microchannel 3, the control valve controls the flow of a liquid in the reaction region 32 to the control region 33, the reaction region 32 comprises a labeling region 32a, a detection region 32b, a reference region 32c and a sample adding region 32d, the labeling region 32a, the detection region 32b, the reference region 32c and the sample adding region 32d are sequentially arranged in the reaction region 32 along the length direction L of the microchannel 3, the reaction region 32 is also provided with a blank region 32e, the blank region 32e is arranged between the labeling region 32a and the detection region 32b and the blank region 32d, the blank region can be combined with an antibody in the surface of the substrate 32b, the antibody can be bound with a detection region 4, and the antibody can be bound to a target region 32b, and the antibody can be bound to the surface of the antibody in the detection region 32b, and the antibody can be bound to the target region 32b and the antibody can be detected by the antibody.
The chemiluminescent agent is one of an enzymatic light-emitting enzyme, an acridinium ester, including but not limited to horseradish peroxidase HRP (or alkaline phosphatase AP).
Specifically, a groove is formed in the lower surface of the cover plate 2 along the length direction of the cover plate, and the groove and the upper surface of the substrate 1 enclose to form the micro-channel 3.
Specifically, the blank area 32e is disposed between the mark area 32a and the detection area 32 b.
As shown in fig. 2, the upper surface of the cover plate 2 is coated with a light-shielding material, the positions of the upper surface of the cover plate 2 corresponding to the blank area 32e, the detection area 32b and the reference area 32c are provided with a light-transmitting area 21 which is not coated with the light-shielding material, and the light-transmitting area 21 is circular.
Specifically, the auxiliary sample loading area 31 is provided with an auxiliary sample loading hole 22 for connecting the upper and lower surfaces of the cover plate 2, and the sample loading area 32d is provided with a sample loading hole 23 for connecting the upper and lower surfaces of the cover plate 2.
Specifically, the control valve is a movable water absorbent material that is capable of contacting or moving away from the micro-channel 3, and when the water absorbent material contacts the micro-channel 3, the liquid in the micro-channel 3 flows into the control area 33, and when the water absorbent material moves away from the micro-channel 3, the liquid in the micro-channel 3 does not flow into the control area 33.
The detection method of the chemiluminescence microfluidic chip, which is used for detection by adopting the chemiluminescence microfluidic chip and adopts a double-antibody sandwich mode, comprises at least the following steps:
1) Separating the control valve from the micro-channel, injecting a certain volume of sample to be tested into the sample loading area, allowing the sample to flow into the micro-channel and flow towards the detection area under the action of capillary force, and staying for 3-5 min to enable the reaction to be fully carried out, if the sample contains target antigen, capturing the target antigen by antibody coated by the detection area to form an antibody-target antigen complex, and after the reaction is finished, enabling the control valve to contact the micro-channel, and allowing unreacted redundant sample to flow towards the control area; (this step requires adjustment of the amount of sample to be added according to parameters such as the type and viscosity of the sample to be measured so as to ensure that the sample does not flow to the marking zone.)
2) Separating the control valve from the micro-channel, adding buffer solution through the auxiliary sample adding area, enabling the buffer solution to flow towards the direction of the marking area and dissolve the antibody coated by the marking area, then driving the dissolved antibody in the marking area to continuously flow, forming an antibody-target antigen-marked antibody compound in the detection area, enabling unbound marked antibody to continuously flow along with the liquid, capturing the liquid in the reference area by the corresponding substance, enabling the control valve to contact the micro-channel after the reaction is finished, and continuously washing the micro-channel by redundant liquid and entering the control area;
3) Adding a corresponding luminescent substrate (or oxidant) into the auxiliary sample adding area according to the type of the marker in the marker area, regulating the temperatures of the blank area, the detection area and the reference area by using microelectrodes according to the reaction requirement, and enabling the luminescent substrate (or oxidant) to flow into the blank area, the detection area and the reference area in sequence, so that the luminescent substrate (or oxidant) and the marker are combined and then emit light; (enzyme-free chemiluminescent reaction adds corresponding luminescent substrate, and direct chemiluminescent reaction adds corresponding oxidant and correction fluid.)
4) Continuously collecting signal values of the blank area, the detection area and the reference area through the detector until the reaction is finished;
5) Acquiring a calibration function: detecting a series of concentration standard substance solutions containing target analytes through the steps, and respectively obtaining a signal value A of the standard substance solution in a blank area, a signal value T of a detection area and a signal value R of a reference area to obtain detection signal values: (T-A)/(R-A), and obtaining a calibration function by taking the detection signal value as an ordinate and the concentration of the standard solution as an abscissa;
6) And obtaining the concentration of the target analyte in the sample to be tested through the calibration function.
Examples
1. Chip preparation
1) Manufacture of chip cover plate
The micro-fluidic chip is manufactured by adopting polymethyl methacrylate (PMMA) material, designing a micro-channel structure of a chip cover plate by adopting CAD software, and then processing and manufacturing the chip cover plate by using a laser etching machine. The preparation of the chip upper cover is completed by fixing round films with diameters of 2mm on the upper surface of the cover plate corresponding to the blank area, the detection area and the reference area, then performing ink-jet treatment on the upper surface of the cover plate, and finally removing the round films of the blank area, the detection area and the reference area.
2) Chip surface modification treatment
The lower surface of the chip cover plate and the upper surface of the substrate are modified in a conventional manner, such as a plasma treatment manner, so that the originally hydrophobic surface is modified into a hydrophilic surface.
3) Fabrication of biochips
Firstly, respectively spotting streptavidin in a detection area and a reference area of the substrate, after incubating for 1h, washing off the streptavidin which does not participate in the reaction, as shown in figure 5, then, respectively spotting biotinylated anti-human IgE antibody and biotinylated purified human IgE in the detection area and the reference area, continuously incubating for 1h, and after washing the substrate again, spotting HRP-anti-human IgE antibody solution in a labeling area, and drying at room temperature.
4) Assembly of chemiluminescent microfluidic chip
And (3) tightly attaching the biochip to a chip cover plate with a channel structure, and bonding the biochip to the chip cover plate by using acetone until the chip is prepared.
2. Detection program
1) As shown in fig. 6, the control valve of the control region is moved rightward to be far away from the micro-channel, then 5 μl of the serum sample to be measured is injected into the sample loading hole, the sample flows leftward in the micro-channel, after reaction 5 min, the control valve is moved leftward to be embedded in the micro-channel, and then the liquid in the micro-channel flows toward the control region.
2) As shown in FIG. 7, the control valve of the control zone was moved to the right away from the microchannel, then 35. Mu.l of buffer was injected into the auxiliary sample well, the buffer flowed to the right in the microchannel, after reaction 5min, the control valve was moved to the left to embed it in the microchannel, and the excess buffer was continuously washed the microchannel and entered the control zone.
3) As shown in FIG. 8, 10. Mu.l of HRP-containing luminescent substrate solution (luminol) was injected into the auxiliary sample well under dark conditions and the microelectrodes under the blank, detection and reference areas were activated for reaction at 37℃for 3min.
4) And capturing and imaging luminous signals of the blank area, the detection area and the reference area by using a CCD camera in the detector, then analyzing gray values of the areas, obtaining a blank area signal value A, a detection area signal value T and a reference area signal value R, finally, representing the detected signal values by (T-A)/(R-A), and quantifying the concentration of tIgE in the sample by using a prefabricated calibration curve.
3. Detection result
The above technical solution only represents the preferred technical solution of the present invention, and some changes that may be made by those skilled in the art to some parts of the technical solution represent the principles of the present invention, and the technical solution falls within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first", "a second", etc. may explicitly or implicitly include one or more such feature. In the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "configured" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art in a specific case.