CN109718877B - Centrifugal disc type micro-fluidic chip and using method thereof - Google Patents
Centrifugal disc type micro-fluidic chip and using method thereof Download PDFInfo
- Publication number
- CN109718877B CN109718877B CN201910012131.4A CN201910012131A CN109718877B CN 109718877 B CN109718877 B CN 109718877B CN 201910012131 A CN201910012131 A CN 201910012131A CN 109718877 B CN109718877 B CN 109718877B
- Authority
- CN
- China
- Prior art keywords
- reaction
- chamber
- magnetic beads
- washing
- magnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
The invention provides a centrifugal disc type microfluidic chip which comprises a chip body and a magnetic bead transfer module, wherein a plurality of centrifugal units are arranged on the chip body, the centrifugal units comprise different fluid chambers, reaction reagents, magnetic beads and organic reagents are contained in the fluid chambers, the magnetic beads can move among the different fluid chambers, the organic reagents are used for reducing the viscosity and the interfacial tension of the reaction reagents, the magnetic beads and the reaction reagents can be conveniently separated and then move to other fluid chambers, and the magnetic bead transfer module is used for providing magnetic driving force to transfer the magnetic beads to the different fluid chambers. According to the invention, the viscosity and the interfacial tension of the reaction reagent are reduced by adding the organic reagent, the problem of difficulty in transferring magnetic beads in the prior art, particularly the problem of difficulty in transferring micro magnetic beads is solved, and the magnetic bead transfer is completed without increasing the quantity of the magnetic beads or increasing the magnetic force.
Description
Technical Field
The invention relates to the field of microfluidic chips, in particular to a centrifugal disc type microfluidic chip based on immunochemiluminescence reaction and a using method thereof.
Background
Immunoassay technology based on magnetic beads is increasingly widely used on centrifugal disc type microfluidic chips. In a centrifugal disk microfluidic chip, antibody-coated magnetic beads are generally used to capture and enrich target antigens to be detected in a sample solution. When the target antigen to be detected is sufficiently bound to the surface of the magnetic bead, the magnetic bead needs to be separated from the sample solution by using a magnetic bead separation or magnetic bead transfer technology so as to perform subsequent operation or analysis and test steps. The magnetic bead separation refers to that the magnetic beads in flowing fluid are gathered at a certain position in a channel/chamber of the microfluidic chip, and the fluid flows out of the position where the magnetic beads are gathered in the flowing fluid; magnetic bead transfer refers to transferring magnetic beads from a fluid that is stationary in a microfluidic chip channel/chamber. In general, magnetic bead separation or magnetic bead transfer may be achieved depending on the difference in properties between the magnetic beads and the sample solution. For example, the magnetic beads and the sample solution may be separated by filtration according to the size of the size. However, the filtration method generally requires the use of a filter membrane or a filter, and integrates the filter membrane on a centrifugal disk type microfluidic chip or directly processes a filtration structure on the chip by using a relatively harsh high-precision micro-nano processing technology, which not only has high requirements for the processing technology, but also increases the cost. This difficulty is particularly acute when filtering magnetic beads of nanometer or submicron size. Magnetic bead separation can also be achieved according to the density difference between the magnetic beads and the sample solution. Generally, the magnetic beads have a density greater than the density of the sample solution. Although centrifugal disk microfluidic chips are used in centrifugal devices, which have centrifugal force fields, they usually only allow magnetic beads to settle at the bottom of the chamber in the centrifugal disk microfluidic chip, and the magnetic beads and the sample solution still exist in the same chamber. Magnetic separation is a preferred method over filtration and centrifugation. Magnetic separation is achieved by enriching magnetic beads with a magnet (typically a permanent magnet) and then transferring the magnetic beads out of the sample solution by using the fact that the attraction force between the magnet and the magnetic beads is greater than the viscous resistance and interfacial tension of the sample solution. Unfortunately, in the centrifugal disk type microfluidic chip, especially when micro (super) paramagnetic nano magnetic beads are used, even if the strongest commercialized ndfeb grade ndfeb permanent magnet of N52 is used, the magnetic force applied to the magnetic beads is weak, which is not enough to overcome the viscous resistance and interfacial tension of the sample solution, thereby failing to realize the magnetic bead transfer. Increasing the amount of beads will facilitate bead transfer, but will substantially increase the cost of the assay; the assembly of multiple N52 grade ndfeb permanent magnets results in an increased magnetic force on the beads, but in addition to the added cost savings, when integrated into an analytical detection instrument, creates relatively large magnetic field disturbances on the other components of the instrument.
Therefore, it is necessary to develop a centrifugal disk microfluidic chip based on immunochemiluminescence reaction to solve the problem of difficulty in magnetic bead transfer in the prior art.
Disclosure of Invention
The centrifugal disc type microfluidic chip based on immunochemiluminescence reaction solves the problem of difficulty in magnetic bead transfer in the prior art, particularly the problem of difficulty in trace magnetic bead transfer, and achieves magnetic bead transfer without increasing the quantity of magnetic beads or increasing magnetic force.
Therefore, an object of the present invention is to provide a centrifugal disk microfluidic chip based on immunochemiluminescence reaction, including a chip body and a magnetic bead transfer module, wherein a plurality of centrifugal units are disposed on the chip body, the centrifugal units include different fluid chambers, the fluid chambers include reaction reagents, magnetic beads and organic reagents, the magnetic beads are movable between the different fluid chambers, the organic reagents are used to reduce viscosity and interfacial tension of the reaction reagents, so that the magnetic beads are separated from the reaction reagents and then moved to other fluid chambers, and the magnetic bead transfer module is used to provide a magnetic driving force to transfer the magnetic beads to the different fluid chambers.
As a preferable technical scheme, the concentration of the organic reagent is 15-50% by volume fraction.
As a preferred technical scheme, the organic reagent is isopropanol or n-propanol.
As a preferred technical solution, the different fluid chambers include a sample reaction chamber, a washing chamber, and a chemiluminescence reaction chamber, the sample reaction chamber and the washing chamber, and the washing chamber and the chemiluminescence reaction chamber are communicated by a fluid channel, the reaction reagent includes a reaction mixture, a washing solution, and a luminescence substrate solution, the reaction mixture and an organic reagent enter the sample reaction chamber, the washing solution and an organic solvent enter the washing chamber, and the luminescence substrate solution enters the chemiluminescence reaction chamber.
As a preferred technical solution, the different fluid chambers further include a sample inlet chamber, a washing buffer inlet chamber, and a luminescent substrate inlet chamber, and the sample inlet chamber and the sample reaction chamber, the washing buffer inlet chamber and the washing chamber, the luminescent substrate inlet chamber, and the chemiluminescent reaction chamber are respectively connected through a fluid channel.
As a preferable technical solution, the magnetic bead transfer module includes a permanent magnet, and the permanent magnet is a neodymium iron boron magnet formed by splicing N50 and N48.
As a preferred technical solution, the centrifugal disk microfluidic chip further comprises an optical detection module and a temperature control module, wherein the optical detection module is used for detecting and collecting an optical signal after a chemiluminescence reaction; the temperature control module is used for controlling the temperature in the chemiluminescence reaction process.
As a preferred technical scheme, the reaction reagent, the magnetic beads and the organic reagent are added into the chip in a direct adding mode when in use.
As a preferred technical scheme, the chip body is prepared by adopting a transparent base material.
In a preferred embodiment, the transparent substrate is selected from any one of polymer materials such as polystyrene, polymethyl methacrylate, and cyclic olefin copolymer.
One objective of the present invention is to provide a method for using a centrifugal disk microfluidic chip based on immunochemiluminescence reaction, comprising the following steps:
s1: the reaction mixed liquid and the magnetic beads enter a sample reaction chamber to react;
s2: the organic reagent enters the sample reaction chamber to be mixed with the reaction mixed liquid, the washing liquid and the organic reagent enter the washing chamber to be washing mixed liquid, and the luminous substrate liquid enters the chemiluminescence reaction chamber;
s3: under the drive of the magnetic bead transfer module, the reacted magnetic beads are transferred from the sample reaction chamber to the washing chamber and are fully washed;
s4: under the drive of the magnetic bead transfer module, transferring the washed magnetic beads from the washing chamber to the chemiluminescence reaction chamber to generate an optical signal;
s5: the optical detection module detects and collects the optical signals to obtain a detection result.
Compared with the prior art, the invention has the advantages and beneficial effects that:
according to the centrifugal disc type micro-fluidic chip based on immunochemiluminescence reaction, the organic solvent is added into the reaction reagent, so that the interfacial tension between the reaction reagent and the magnetic beads is reduced, and the magnetic bead transfer is completed without increasing the quantity of the magnetic beads and increasing the magnetic force.
The centrifugal disc type micro-fluidic chip based on immunochemiluminescence reaction selects proper organic reagent types and proper organic reagent concentrations, does not generate protein variation phenomenon while reducing the viscosity and interfacial tension of reaction reagents, and does not reduce the luminous intensity of the whole chemiluminescence system.
The operation method of the centrifugal disc type microfluidic chip based on the immunochemiluminescence reaction is simple and convenient, and rapid and accurate detection can be realized without adding additional parts and complicated operation.
Drawings
FIG. 1 is a schematic view of a chip body according to one embodiment;
FIG. 2 is a schematic view of a bottom plate layer of the chip body according to one embodiment;
FIG. 3 is a schematic view of a channel layer of a chip body according to an embodiment;
FIG. 4 is a schematic view of a top plate layer of the chip body according to one embodiment;
FIG. 5 is a schematic view of a chip body in the third embodiment;
FIG. 6 is a schematic view of a top plate layer of the chip body in the third embodiment;
FIG. 7 is a linear relationship between the concentration of different antigens and the luminescence intensity in the fifth example;
FIG. 8 is a linear relationship between the concentration of different antigens and the luminescence intensity in the sixth example.
Wherein, the chip body 1; the bottom plate layer 2, a first central hole 21 and a first positioning hole 22; channel layer 3, second central hole 31, second positioning hole 32, sample inlet chamber 33, washing buffer inlet chamber 34, luminescent substrate inlet chamber 35, sample reaction chamber 36, washing chamber 37, chemiluminescence reaction chamber 38, and exhaust channel 39; top plate layer 4, third central hole 41, third positioning hole 42, sample inlet 43, wash buffer inlet 44, luminogenic substrate inlet 45, vent hole 46, sampling hole 47.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings and the embodiment. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be further noted that, for the convenience of description, only some but not all of the elements associated with the present invention are shown in the drawings.
Example one
The embodiment provides a centrifugal disc formula micro-fluidic chip based on immunochemiluminescence reaction, shifts the module including chip body and magnetic bead, set up a plurality of centrifugal units on the chip body, centrifugal unit contains different fluid cavity, contain reactant, magnetic bead and organic reagent in the fluid cavity, the magnetic bead can remove between different fluid cavity, organic reagent is used for reducing reactant's viscosity and interfacial tension, and the magnetic bead of being convenient for removes to other fluid cavity after separating with reactant, the magnetic bead shifts the module and is used for providing magnetism drive power so that the magnetic bead shifts to in the different fluid cavity.
Specifically, each centrifugal unit can complete one detection index, and therefore joint detection of multiple indexes can be completed according to the setting and combination of the centrifugal units. In some examples of the present invention, the number of the centrifugal units is 1 to 100, in some examples of the present invention, the number of the centrifugal units is 1 to 80, in some examples of the present invention, the number of the centrifugal units is 1 to 60, in some examples of the present invention, the number of the centrifugal units is 1 to 50, in some examples of the present invention, the number of the centrifugal units is 1 to 30, in some examples of the present invention, the number of the centrifugal units is 1 to 20, and in some examples of the present invention, the number of the centrifugal units is 1 to 16. The number of the centrifugal units can be set according to specific needs, and is not limited by the invention. In this embodiment, the number of the centrifugal units is 4.
Specifically, the concentration of the organic reagent is 15-50% by volume fraction. The effect of reducing the solution viscous resistance and the solution interfacial tension is not obvious and the effect of mediating the magnetic bead transfer cannot be achieved due to the fact that the organic solvent is too low; too high an organic solvent may increase the risk of denaturing the enzyme and/or antibody (antigen) used in the immunochemical luminescent reaction system, and it is therefore necessary to select the appropriate type of organic solvent while ensuring a reasonable concentration of the organic solvent used.
Specifically, the organic reagent is isopropanol and n-propanol. In the invention, the organic reagent is added into the reaction reagent, and the organic reagent can reduce the interfacial tension of the reaction reagent solution, namely, the resistance generated by the reaction reagent during the magnetic bead transfer can be reduced to increase the possibility of smooth magnetic bead transfer.
In the present invention, the reagent is a liquid reagent that needs to participate in the immunochemiluminescence reaction process, and includes reagent solutions such as antigens and antibodies that directly participate in the reaction, and reagent solutions such as washing reagents that are not directly involved in the reaction and are used only for washing. In the immunochemiluminescence detection, according to the specific reaction or the difference of the object to be detected, a suitable reagent can be selected to participate in the reaction to obtain the detection result, for example, a suitable enzyme-labeled antibody, a suitable biotinylated antibody and the like are selected, and it can be seen that the reagent based on the immunochemiluminescence reaction contains a protein reagent.
The purpose of adding the organic reagent in the invention is not to participate in the immunochemiluminescence reaction process, but only to change the viscosity and interfacial tension of the reaction reagent to ensure the smooth transfer of magnetic beads, while in the conventional cognition, the organic solvent is easy to cause protein modification, so that the addition of the organic reagent in an immunochemiluminescence reaction system is reduced or avoided.
In some examples of the invention, the principle of magnetic bead-based sandwich immunochemiluminescence reaction is as follows: the biotinylated antibody coated paramagnetic beads recognize and bind to a specific antigen in the sample solution after mixing with the sample solution. Separating the magnetic beads coated with the biotinylated antibody and combined with the antigen from the sample solution, and adding an enzyme-labeled antibody solution into the sample solution to ensure that the enzyme-labeled antibody is combined with the antigen, thereby forming a double-antibody sandwich structure. And then separating the magnetic beads coated with the biotinylated antibody and combined with the antigen and the enzyme-labeled antibody from the enzyme-labeled antibody solution, washing, mixing with a luminescent substrate solution, and generating a chemiluminescent reaction and an optical signal. The generated optical signal can be detected by an optical detection device, and the strength of the optical signal is positively correlated with the concentration of the antigen in the sample solution. The concentration of the substance to be detected in the sample solution can be obtained through the detected optical signal.
It will be appreciated that the magnetic bead-based sandwich immunochemiluminescence reaction can be performed in a single step or in two steps. In some examples of the present invention, in order to simplify the structure of the centrifugal disk type microfluidic chip and facilitate the processing, the magnetic bead-based sandwich immunochemiluminescence reaction is performed based on a one-step method.
Specifically, different fluid chambers include sample reaction chamber, washing chamber, chemiluminescence reaction chamber, communicate by fluid channel between sample reaction chamber and the washing chamber, wash chamber and the chemiluminescence reaction chamber, reaction reagent includes reaction mixed liquid, washing liquid and luminous substrate liquid, reaction mixed liquid and organic reagent get into sample reaction chamber, washing liquid and organic solvent get into the washing chamber, luminous substrate liquid gets into chemiluminescence reaction chamber.
More specifically, the reaction mixture includes a sample solution and an enzyme-labeled antibody solution. Therefore, the reaction mixed liquid is mixed with the magnetic beads in the sample reaction chamber firstly, the process of combining the antigen and the enzyme-labeled antibody is completed, a double-antibody sandwich structure is formed, the interfacial tension of the reaction mixed liquid is reduced through the organic reagent, the magnetic beads are smoothly transferred to the washing chamber, the interfacial tension of the washing liquid is reduced through the organic reagent after washing, the magnetic beads are smoothly transferred to the chemiluminescence reaction chamber, the magnetic beads are combined with the luminescence substrate liquid to generate optical signals, and corresponding detection results are obtained through detecting the optical signals.
More specifically, the different fluid chambers further comprise a sample inlet chamber, a washing buffer inlet chamber and a luminescent substrate inlet chamber, wherein the sample inlet chamber and the sample reaction chamber, the washing buffer inlet chamber and the washing chamber, the luminescent substrate inlet chamber and the chemiluminescent reaction chamber are respectively connected through a fluid channel.
Specifically, the chip body further comprises a whole blood separation unit and a distribution unit, wherein the whole blood separation unit is used for separating blood cells, and the distribution unit is used for distributing the reaction reagent to the centrifugal units.
Specifically, the chip body is further provided with a center hole and a positioning hole.
Specifically, the chip body is prepared from a transparent base material, and the transparent base material is selected from any one of polymer materials such as polystyrene, polymethyl methacrylate and cyclic olefin copolymer.
Specifically, the chip body comprises three layers of substrates, namely a bottom plate layer, a channel layer and a top plate layer. In this embodiment, the three-layered substrate is made of a transparent polymethyl methacrylate (PMMA) base material.
More specifically, a sample inlet, a wash buffer inlet, a luminogenic substrate inlet and a vent are provided on the top plate layer.
More specifically, a sample inlet chamber, a washing buffer inlet chamber, a luminescent substrate inlet chamber, a sample reaction chamber, a washing chamber and a chemiluminescence reaction chamber are arranged on the channel layer.
More specifically, the bottom plate layer, the channel layer and the top plate layer are all provided with a central hole and a positioning hole so as to ensure the positioning and the centrifugal action of each substrate of the chip.
More specifically, the bottom plate layer, the channel layer and the top plate layer may be provided with a plurality of positioning holes to further ensure positioning between the substrates.
In this embodiment, as shown in fig. 1 to 4, the chip body 1 includes a bottom plate layer 2, a channel layer 3, and a top plate layer 4, and the bottom plate layer 2 is provided with a first central hole 21 and a first positioning hole 22; the channel layer 3 is provided with a second central hole 31, a second positioning hole 32, a sample inlet chamber 33, a washing buffer inlet chamber 34, a luminescent substrate inlet chamber 35, a sample reaction chamber 36, a washing chamber 37 and a chemiluminescence reaction chamber 38; the top plate layer 4 is provided with a third central hole 41, a third positioning hole 42, a sample inlet 43, a washing buffer inlet 44, a luminescent substrate inlet 45, and a vent hole 46. The chambers on the channel layer 3 are directly communicated together through fluid channels respectively; specifically, sample reaction chamber 36 and wash chamber 37, wash chamber 37 and chemiluminescent reaction chamber 38 are in communication. The channel layer 3 is also provided with an exhaust channel 39 for exhausting air. The radial distance between the centers of the sample reaction chamber 36, the wash chamber 37, the chemiluminescent reaction chamber 38 to the center of the second central bore 31 decreases in sequence, but the linear distance from the sample inlet chamber 33 to the sample reaction chamber 36, the wash buffer inlet chamber 34 to the wash chamber 37, and the luminogenic substrate inlet chamber 35 to the chemiluminescent reaction chamber 38 are equal.
The holes, fluid channels and/or fluid chambers in the bottom plate layer 2, the channel layer 3 and the top plate layer 4 may be obtained using CNC engraving of a PMMA substrate. After the bottom plate layer 2, the channel layer 3 and the top plate layer 4 are aligned through the positioning holes (the first center hole 21, the second center hole 31 and the third center hole 41 are aligned with each other; and the first positioning hole 22, the second positioning hole 32 and the third positioning hole 42 are aligned with each other), the substrates of the layers are bonded together by using a hot-press packaging method.
Specifically, the magnetic bead transfer module includes a permanent magnet, the permanent magnet is neodymium iron boron magnet that is formed by N50 and N48 concatenation.
Specifically, the centrifugal disk type microfluidic chip further comprises an optical detection module and a temperature control module (not shown), wherein the optical detection module is used for detecting and collecting optical signals after chemiluminescence reaction; the temperature control module is used for controlling the temperature in the chemiluminescence reaction process.
The application method of the centrifugal disc type microfluidic chip based on the immunochemiluminescence reaction comprises the following steps:
s1: the reaction mixture and the magnetic beads enter the sample reaction chamber 36 for reaction;
s2: the organic reagent enters the sample reaction chamber 36 to be mixed with the reaction mixed liquid, the washing liquid and the organic reagent enter the washing chamber 37 to be washing mixed liquid, and the luminous substrate liquid enters the chemiluminescence reaction chamber 38;
s3: under the driving of the magnetic bead transfer module, the reacted magnetic beads are transferred from the sample reaction chamber 36 to the washing chamber 37 and are fully washed;
s4: under the driving of the magnetic bead transfer module, the washed magnetic beads are transferred from the washing chamber 37 to the chemiluminescence reaction chamber 38, and an optical signal is generated;
s5: the optical detection module detects and collects the optical signals to obtain a detection result.
More specifically, in the use process, the magnetic beads coated with the biotinylated antibody are mixed with the sample solution and the enzyme-labeled antibody (i.e., reaction mixture) and immediately added to the sample inlet chamber 33 of the chip body 1. The reaction mixture flows into the sample reaction chamber 36 driven by centrifugal force. Subsequently, the centrifugal device is alternately rotated clockwise and counterclockwise at a specific rotation speed so that the antigen in the mixed solution reacts with the antibody for a certain period of time at a suitable temperature. The centrifuge stops rotating, an appropriate amount of organic solvent is added to the sample inlet chamber 33, and the centrifuge is then operated again to allow the organic solvent to mix uniformly with the reaction mixture. The centrifuge stops rotating and the magnetic bead transfer module is activated so that the biotinylated antibody-coated magnetic beads, which have bound the antigen and the enzyme-labeled antibody, are transferred to the washing chamber 37 under the drive of the magnetic force applied by the permanent magnet. The washing chamber 37 contains a proper amount of a mixed solution of a washing buffer and an organic solvent (referred to as a washing mixed solution), and the mixed solution flows into the washing chamber 37 along a fluid path from the washing buffer inlet chamber 34 by a centrifugal force. After the centrifuge apparatus is rotated alternately clockwise and counterclockwise at a specific rotation speed for a period of time, the magnetic beads are washed sufficiently, and then the magnetic bead transfer module is activated to transfer the magnetic beads to the chemiluminescent reaction chamber 38 containing the luminescent substrate solution flowing from the luminescent substrate inlet chamber 35 into the chemiluminescent reaction chamber 38. At this time, the luminescent substrate undergoes a chemical reaction catalyzed by the enzyme to generate an optical signal. By collecting the optical signal at a specific time point, the antigen concentration in the sample solution can be analyzed.
In the present invention, the amount of paramagnetic beads used is very small, and the magnetism exhibited by the magnetization of the permanent magnet is weak. Therefore, transferring magnetic beads from one fluid chamber to another is difficult to achieve with a permanent magnet such as rubidium, iron, and boron directly. The invention mediates the magnetic bead transfer by using an organic solvent to reduce the interfacial tension of the solution, because the organic solvent, especially the organic solvent which is miscible with water (such as isopropanol or n-propanol), can reduce the interfacial tension of the solution. Therefore, when the magnetic beads are transferred from the reaction mixture or the washing mixture containing the organic solvent by using the permanent magnet, the magnetic force of the permanent magnet on the magnetic beads overcomes the viscous resistance and the interfacial tension of the reaction mixture or the washing mixture, so that the magnetic beads can be smoothly transferred.
Although organic solvents can reduce the interfacial tension of aqueous solutions, organic solvents also risk denaturing the antibody or antigen. Therefore, it is extremely critical to select the appropriate organic solvent and the amount of organic solvent used. This may in practice need to be selected according to the particular bioreagent system. In the present invention, the organic solvent does not adversely affect the whole sandwich immunochemiluminescence reaction reagent system within the concentration range used for the sandwich immunochemiluminescence reaction reagent system based on magnetic beads used in the present invention.
Example two
In order to show that the addition of an organic reagent is beneficial to the transfer of magnetic beads, the following experiment was performed using a centrifugal disk microfluidic chip based on immunochemiluminescence reaction as shown in example one.
After 70. mu.L of a magnetic bead dispersion (0.2857mg/mL) of magnetic beads to which biotinylated antibodies are bound, 70. mu.L of a commercial alkaline phosphatase-labeled antibody solution (6. mu.g/mL) diluted with an enzyme-labeled diluent, and 10. mu.L of a carcinoembryonic antigen (CEA) solution (1000ng/mL) are mixed in a centrifuge tube (reaction mixture for short), the mixture is immediately added into the sample inlet chamber 33 from the sample inlet 43 of the chip body 1. The chip body 1 is fixed in a rotating shaft of centrifugal equipment through a central hole on the chip body 1, and corresponding working parameters of the centrifugal equipment are set. The centrifuge is activated and the reaction mixture is driven by centrifugal force to flow into sample reaction chamber 36. Then, the centrifugal device is rotated alternately clockwise and counterclockwise at a specific rotation speed, so that the reaction mixture is reacted for 10min under the condition of oscillation. The centrifuge stops rotating and 150. mu.L of an aqueous solution containing 50% (v: v) isopropanol is added to the sample inlet chamber 33 from the sample inlet 43. To the wash buffer inlet chamber 34 was added 300. mu.L of a wash mixture of wash buffer (10mM 7.2PBS containing 0.05% TW-20) and isopropanol 4:1(v: v) from the wash buffer inlet 44 and to the luminogenic substrate inlet chamber 35 was added 230. mu.L of a commercially available luminogenic substrate solution whose major component was 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt (AMPPD) from the luminogenic substrate inlet 45. Then the centrifugal equipment is operated again, so that the isopropanol enters the sample reaction chamber 36 and can be uniformly mixed with the reaction mixed liquid; at the same time, the washing mixture and the luminescent substrate solution enter the washing chamber 37 and the chemiluminescent reaction chamber 38, respectively. And stopping the rotation of the centrifugal equipment, and immediately starting the magnetic bead transfer module on the centrifugal equipment to transfer the magnetic beads. It was finally observed that the magnetic beads coated with the biotinylated antibody, to which the antigen and the alkaline phosphatase-labeled antibody were bonded, were smoothly transferred to the washing chamber 37 by the magnetic force applied by the permanent magnet. And then, operating a centrifugal device to enable the centrifugal disc type microfluidic chip to rotate clockwise and anticlockwise for 3min alternately, and washing the magnetic beads. The rotation of the centrifuge was stopped and the magnetic beads were then transferred in the same manner as described above. It is also observed that magnetic beads can be transferred from the washing chamber 37 to the chemiluminescent reaction chamber 38 containing the luminescent substrate solution.
Comparative example 1
In this comparative example, the apparatus, operation and reagents were the same as those in example two except that in this comparative example, isopropanol was not added to the reaction mixture and the washing mixture, but the same volume of washing buffer was added. However, during the experiment, the magnetic beads cannot be transferred from the sample reaction chamber 36, and thus the transfer from the washing chamber 37 to the chemiluminescence reaction chamber 38 cannot be continued.
Experiments of example two and comparative example one show that the sandwich immunochemiluminometric reagent system with isopropanol is more beneficial to magnetic bead transfer under the condition of using the same amount of magnetic beads and the same permanent magnet. This is because the organic solvent isopropanol is miscible with the aqueous solution to form a homogeneous solution after being added to the aqueous solution and shaken, thereby reducing the viscous resistance and interfacial tension of the aqueous solution.
EXAMPLE III
In order to verify whether isopropanol adversely affects the magnetic bead-based sandwich immunochemiluminescence reaction system, in this embodiment, after the magnetic bead-based sandwich immunochemiluminescence reaction performed in the centrifugal disk microfluidic is completed, the reaction solution is taken out from the chemiluminescent reaction chamber 38, and then signal detection is performed on a microplate reader. Fig. 5 shows a chip body 1 used in this embodiment, which is different from the first embodiment in that the top plate layer 4 is slightly different, and the rest is the same.
The top plate layer 4 of the chip body 1 of the present embodiment is shown in fig. 6. The top plate layer 4 differs from the first embodiment in that it further comprises a sampling hole 47. The sampling hole 47 is positioned at the upper right side of the chemiluminescence reaction chamber 38 and is sealed by a film containing a pressure-sensitive adhesive in the sandwich immunochemiluminescence reaction process, and the sampling hole 47 is used for taking out a reaction solution from the chemiluminescence reaction chamber 38 after the reaction of the sandwich immunochemiluminescence reaction based on magnetic beads in centrifugal disk type microfluidic is finished, and then carrying out signal detection on a microplate reader.
Before sandwich immunochemiluminescence reaction based on magnetic beads is carried out in the chip body 1, paramagnetic magnetic beads need to be carried out in a centrifugal tubeBlocking treatment and treatment with a biotinylated antibody. Paramagnetic beads were first incubated for 2h at 37 ℃ in blocking solution (10mM 7.2PBS buffer containing 1% BSA, 0.05% TW-20) and then magnetically attracted using a permanent magnet to remove the supernatant. Next, the beads were washed with a diluted solution of biotinylated antibody (10mM 7.2PBS buffer containing 0.1% BSA, 0.05% TW20, 0.05% PC300), magnetically attracted, and the supernatant removed, and repeated 3 times. The commercial biotinylated antibody is diluted by biotinylated antibody diluent, then mixed with the closed magnetic beads uniformly, and then incubated at 37 ℃ for 10 min. After completion of the reaction, the supernatant was removed by magnetic attraction using a permanent magnet, and then an enzyme-labeled diluent (20mM 7.2PBS buffer containing 5mM MgCl)2、0.1mM ZnCl20.1% BSA, 0.05% TW-20, 0.05% PC300) on the magnetic beads to which the biotinylated antibody was bound, 1 time. Finally, an enzyme-labeled diluent is used to prepare a magnetic bead dispersion of magnetic beads combined with biotinylated antibodies, so that the concentration of the magnetic bead dispersion is 0.5714 mg/mL. The magnetic beads combined with the biotinylated antibody can be stored for at least 1 month under proper conditions, and the quantity of the magnetic beads used in the centrifugal disc type microfluidic chip is small, so that more magnetic beads can be processed at one time for storage and standby when the magnetic beads are subjected to sealing treatment and combined with the biotinylated antibody for convenience of operation.
After 70. mu.L of the magnetic bead dispersion of the magnetic beads to which the biotinylated antibody is bound, 70. mu.L of a commercial alkaline phosphatase-labeled antibody solution (6. mu.g/mL) diluted with an enzyme-labeled diluent, and 10. mu.L of a carcinoembryonic antigen (CEA) solution (1000ng/mL) are mixed in a centrifuge tube (reaction mixture for short), the mixture is immediately added into the sample inlet chamber 33 from the sample inlet 43 of the chip body 1. The chip body 1 is fixed in a rotating shaft of centrifugal equipment through a central hole on the chip body 1, and corresponding working parameters of the centrifugal equipment are set. The centrifuge is activated and the reaction mixture is driven by centrifugal force to flow into sample reaction chamber 36. Then, the centrifugal device is rotated alternately clockwise and counterclockwise at a specific rotation speed, so that the reaction mixture is reacted for 10min under the condition of oscillation. The centrifuge stops rotating and 150. mu.L of an aqueous solution containing 50% (v: v) isopropanol is added to the sample inlet chamber 33 from the sample inlet 43, 300. mu.L of a wash mixture of wash buffer (10mM 7.2PBS containing 0.05% TW-20) and isopropanol 4:1(v: v) is added to the wash buffer inlet chamber 34 from the wash buffer inlet 44, and 230. mu.L of a commercial AMPPD luminescence substrate solution is added to the luminescence substrate inlet chamber 35 from the luminescence substrate inlet 45. Then, the centrifugal equipment is operated again, so that the isopropanol enters the sample reaction chamber 36 and can be uniformly mixed with the reaction mixed solution; at the same time, the washing mixture and the luminescent substrate solution enter the washing chamber 37 and the chemiluminescent reaction chamber 38, respectively. The centrifuge stops rotating and the magnetic bead transfer module on the centrifuge is immediately activated so that the biotinylated antibody-coated magnetic beads, which have bound the antigen and the alkaline phosphatase-labeled antibody, are transferred to the washing chamber 37 under the drive of the magnetic force applied by the permanent magnet. And then, operating a centrifugal device to enable the centrifugal disc type microfluidic chip to rotate clockwise and anticlockwise for 3min alternately, and washing the magnetic beads. The rotation of the centrifuge is stopped and the beads are transferred to the chemiluminescent reaction chamber 38 containing the luminescent substrate solution in the same manner as described above. Next, the centrifuge apparatus was operated to rotate the centrifugal disk type microfluidic chip alternately clockwise and counterclockwise for 1min, then the sealing film above the sampling hole 47 was pierced with a micrometer needle, and then 200. mu.L of the reaction solution was taken out of the chemiluminescent reaction chamber 38 into a 96-well plate and the luminescence intensity at 10min was measured on a microplate reader (Molecular Devices, USA, model: SpectraMax. O R i 3X).
Comparative example No. two
In this comparative example, the apparatus, operation and reagents were the same as those in example three except that in this comparative example, isopropanol was not added to the reaction mixture and the washing mixture, but the same volume of washing buffer was added. Since it is necessary to ensure that the magnetic beads in the control experiment group can be smoothly transferred in order to verify whether isopropanol has an effect on the sandwich immunochemiluminescence reaction system based on the magnetic beads, the amount of the magnetic beads used in the present comparative example was 40. mu.g.
The results of the tests of example three and comparative example two show that the isopropanol test group in example three has a luminous intensity of 3110134, the control test group in comparative example has a luminous intensity of 2961999, and the difference of the chemiluminescence signals is less than 10%. This is an acceptable deviation. Therefore, it is shown that the addition of isopropanol to a magnetic bead-based sandwich immunochemiluminescence reaction system in a centrifugal disk microfluidic chip does not significantly adversely affect the sandwich immunochemiluminescence reaction system, such as inactivation or activity reduction of antigen and/or antibody, insufficient washing of magnetic beads.
Example four:
this example was conducted to examine whether n-propanol adversely affects the magnetic bead-based sandwich immunochemiluminescence reaction system used.
In this example, the apparatus, operation, and reagents were the same as in example three except that in this comparative example, the organic solvent added to the reaction mixture and the washing mixture was not isopropyl alcohol, but n-propyl alcohol in the same amount.
Comparative example No. three
In this comparative example, the apparatus, operation and reagents were the same as those in example four except that in this comparative example, the same volume of washing buffer was added to the reaction mixture and the washing mixture, instead of n-propanol. Since it is necessary to ensure that the magnetic beads in the control experiment group can be smoothly transferred in order to verify whether n-propanol has an effect on the sandwich immunochemiluminescence reaction system based on the magnetic beads, the amount of the magnetic beads used in the present comparative example was 40. mu.g.
The test results of example four and comparative example three show that the n-propanol test group in example four has a luminous intensity of 2914671, the control test group in comparative example three has a luminous intensity of 2961999, and the difference of the chemiluminescence signals is less than 10%. This is an acceptable deviation. Therefore, it is shown that the addition of n-propanol to a magnetic bead-based sandwich immunochemiluminescence reaction system in a centrifugal disk microfluidic chip does not significantly adversely affect the sandwich immunochemiluminescence reaction system, such as inactivation or activity reduction of antigen and/or antibody, insufficient washing of magnetic beads.
Example five:
in order to examine the linear variation trend of the luminescence intensity of a sandwich immunochemiluminescence reaction system based on magnetic beads when the system detects different CEA concentrations after isopropanol is added into the system, CEA solutions with different concentrations are respectively detected in the embodiment. In this example, the apparatus, operation and reagents were the same as those in example three except that the CEA solution was used at concentrations of 7.8125ng/mL, 31.25ng/mL, 125ng/mL, 250ng/mL and 500ng/mL, respectively. Finally, the linear relationship between the different antigen concentrations and the luminescence intensity is shown in FIG. 7. Within the range of the concentration gradient, the linear correlation coefficient R2Was 0.985. This demonstrates that not only does the addition of isopropanol to the above described magnetic bead-based sandwich immunochemiluminescence reaction system not significantly adversely affect the reaction system, but also the relatively broad range of antigen concentrations as low as 7.8125ng/mL and as high as 500ng/mL still maintains a good linear dependence.
Example six:
in order to examine the linear variation trend of the luminescence intensity of a sandwich immunochemiluminescence reaction system based on magnetic beads when the system detects different CEA concentrations after adding n-propanol, CEA solutions with different concentrations are respectively detected in the embodiment. In this example, the apparatus, operation and reagents were the same as those in example three except that the CEA solution was used at concentrations of 7.8125ng/mL, 31.25ng/mL, 125ng/mL, 250ng/mL and 500ng/mL, respectively. Finally, the linear relationship between the different antigen concentrations and the luminescence intensity is shown in FIG. 8. Indicating a linear correlation coefficient R within the concentration gradient range2Is 0.998. This demonstrates that not only does the addition of isopropanol to the above described magnetic bead-based sandwich immunochemiluminescence reaction system not significantly adversely affect the reaction system, but also the relatively broad range of antigen concentrations as low as 7.8125ng/mL and as high as 500ng/mL still maintains a good linear dependence.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A centrifugal disc type micro-fluidic chip is characterized by comprising a chip body and a magnetic bead transfer module, wherein a plurality of centrifugal units are arranged on the chip body, the centrifugal units comprise different fluid chambers, reaction reagents, magnetic beads and organic reagents are contained in the fluid chambers, the magnetic beads can move among the different fluid chambers, the organic reagents are used for reducing the viscosity and the interfacial tension of the reaction reagents, the magnetic beads can be conveniently separated from the reaction reagents and then move to other fluid chambers, and the magnetic bead transfer module is used for providing a magnetic driving force to transfer the magnetic beads to the different fluid chambers; the reaction reagent is a liquid reagent which needs to participate in the immunochemical reaction process; the concentration of the organic reagent is 15-50% by volume fraction, and the organic reagent is isopropanol or n-propanol.
2. The microfluidic chip according to claim 1, wherein the different fluid chambers comprise a sample reaction chamber, a washing chamber, and a chemiluminescence reaction chamber, the sample reaction chamber and the washing chamber, the washing chamber and the chemiluminescence reaction chamber are communicated by a fluid channel, the reaction reagent comprises a reaction mixture, a washing solution, and a luminescence substrate solution, the reaction mixture and the organic reagent enter the sample reaction chamber, the washing solution and the organic solvent enter the washing chamber, and the luminescence substrate solution enters the chemiluminescence reaction chamber.
3. The microfluidic chip of claim 2, wherein said different fluid chambers further comprise a sample inlet chamber, a wash buffer inlet chamber, and a luminophore inlet chamber, said sample inlet chamber and said sample reaction chamber, said wash buffer inlet chamber and said wash chamber, said luminophore inlet chamber and said chemiluminescent reaction chamber are connected by fluid channels, respectively.
4. The centrifugal disk microfluidic chip of claim 1, wherein said magnetic bead transfer module comprises a permanent magnet, said permanent magnet is a neodymium iron boron magnet spliced by N50 and N48.
5. The centrifugal disc microfluidic chip according to claim 1, further comprising an optical detection module and a temperature control module, wherein the optical detection module is used for detecting and collecting optical signals after chemiluminescence reaction; the temperature control module is used for controlling the temperature in the chemiluminescence reaction process.
6. The centrifugal disk microfluidic chip according to claim 1, wherein the chip body is made of a transparent substrate, and the transparent substrate is selected from any one of polystyrene, polymethyl methacrylate, and cyclic olefin copolymer.
7. The centrifugal disk microfluidic chip of claim 1, wherein said chip body comprises three layers of substrates, respectively a bottom plate layer, a channel layer and a top plate layer.
8. A method for using a centrifugal disk microfluidic chip, wherein the centrifugal disk microfluidic chip of any one of claims 1 to 7 is used, comprising the following steps:
s1: the reaction mixed liquid and the magnetic beads enter a sample reaction chamber to react;
s2: the organic reagent enters the sample reaction chamber to be mixed with the reaction mixed liquid, the washing liquid and the organic reagent enter the washing chamber to be washing mixed liquid, and the luminous substrate liquid enters the chemiluminescence reaction chamber;
s3: under the drive of the magnetic bead transfer module, the reacted magnetic beads are transferred from the sample reaction chamber to the washing chamber and are fully washed;
s4: under the drive of the magnetic bead transfer module, transferring the washed magnetic beads from the washing chamber to the chemiluminescence reaction chamber to generate an optical signal;
s5: the optical detection module detects and collects the optical signals to obtain a detection result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910012131.4A CN109718877B (en) | 2019-01-07 | 2019-01-07 | Centrifugal disc type micro-fluidic chip and using method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910012131.4A CN109718877B (en) | 2019-01-07 | 2019-01-07 | Centrifugal disc type micro-fluidic chip and using method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109718877A CN109718877A (en) | 2019-05-07 |
| CN109718877B true CN109718877B (en) | 2021-05-11 |
Family
ID=66298166
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910012131.4A Active CN109718877B (en) | 2019-01-07 | 2019-01-07 | Centrifugal disc type micro-fluidic chip and using method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109718877B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021037099A1 (en) * | 2019-08-30 | 2021-03-04 | 东莞东阳光医疗智能器件研发有限公司 | Mobile device and detection device for magnetic particles of disk chip, and movement control method therefor |
| CN112986554B (en) * | 2019-12-17 | 2022-07-26 | 中国农业大学 | Centrifugal micro-fluidic based milk micromolecule detection method and special chip thereof |
| CN111879761A (en) * | 2020-06-22 | 2020-11-03 | 武汉新烽光电股份有限公司 | Micro-reagent-consumption water quality residual chlorine detection method based on micro-fluidic disc chip |
| CN113009136B (en) * | 2020-08-21 | 2024-04-05 | 东莞东阳光医疗智能器件研发有限公司 | Small multi-index detection sample analysis device |
| CN113049495A (en) * | 2021-03-24 | 2021-06-29 | 东莞东阳光医疗智能器件研发有限公司 | Chemiluminescence detection equipment |
| CN114609388B (en) * | 2022-05-11 | 2022-07-19 | 广州华澳生物科技有限公司 | Microfluidic immunodetection method and device |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1939629A3 (en) * | 2006-08-11 | 2011-03-09 | Samsung Electronics Co., Ltd. | Centrifugal Force Based Magnet Position Control Device and Disk-Shaped Micro Fluidic System |
| JP2009002806A (en) * | 2007-06-21 | 2009-01-08 | Hitachi Ltd | Chemiluminescence measuring device |
| CN102980996B (en) * | 2012-12-31 | 2014-09-10 | 广州市第一人民医院 | Chemiluminescence immunoassay system, as well as method and application thereof |
| CN104975003A (en) * | 2015-07-23 | 2015-10-14 | 上海美吉生物医药科技有限公司 | Nucleic acid recovery and purification kit and method |
| CN206838098U (en) * | 2017-04-07 | 2018-01-05 | 苏州含光微纳科技有限公司 | A kind of centrifugal immunological magnetic bead sorting micro-fluidic chip and device |
-
2019
- 2019-01-07 CN CN201910012131.4A patent/CN109718877B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109718877A (en) | 2019-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109718877B (en) | Centrifugal disc type micro-fluidic chip and using method thereof | |
| US10145843B2 (en) | Magnetic immunosensor and method of use | |
| US20220326232A1 (en) | Magnetic immunosensor with trench configuration and method of use | |
| US10126296B2 (en) | Immunoassay method and device with magnetically susceptible bead capture | |
| US10048258B2 (en) | Oscillating immunoassay method and device | |
| CN105259163B (en) | The direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection | |
| TWI545323B (en) | Centrifugal magnetic bead operating apparatus and operating method thereof | |
| Tekin et al. | Attomolar protein detection using a magnetic bead surface coverage assay | |
| KR101519379B1 (en) | Centrifugal Micro-fluidic Device and Method for immunoassay | |
| US9766230B1 (en) | System and method for detecting components of a mixture including a valving scheme for competition assays | |
| CN105233892A (en) | Magnetic particle chemiluminescence double-layer micro-fluidic chip used for whole-blood sample detection | |
| CN205449995U (en) | Magnetic particle chemiluminescence micro -fluidic chip | |
| Shen et al. | An enhanced centrifugation-assisted lateral flow immunoassay for the point-of-care detection of protein biomarkers | |
| CN109954524A (en) | A kind of micro-fluidic chip to be shone based on homogeneous chemistry | |
| CN205650212U (en) | A double -deck micro -fluidic chip of magnetic particle chemiluminescence for whole blood sample test | |
| CN113088446A (en) | Full-automatic nucleic acid rapid detection device and detection method | |
| EP4012410A1 (en) | Magnetic particle luminescence micro-fluidic chip for multi-marker detection, and detection device | |
| US20080309323A1 (en) | Method for biochemical analysis | |
| CA3019181C (en) | Dmf method and system for concentrating analyte from large volumes into smaller volumes using magnetic microparticles | |
| CN114814261A (en) | Automatic chemiluminescence immunoassay chip and detection method thereof | |
| WO2021068912A1 (en) | Magnetic particle luminescence micro-fluidic chip for multi-marker detection, and detection device | |
| JPWO2002073203A1 (en) | Whole blood measurement method | |
| Shih et al. | An enzyme-linked immunosorbent assay on a centrifugal platform using magnetic beads | |
| CN205449806U (en) | A magnetic particle chemiluminescence micro -fluidic chip for whole blood sample test | |
| CN205599183U (en) | Creatine kinase isoenzyme's magnetic particle chemiluminescence micro -fluidic chip in detection whole blood |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190801 Address after: 523871 Room 301, 3rd floor, 368 Zhenan Middle Road, Sha Community, Chang'an Town, Dongguan City, Guangdong Province Applicant after: Dongguan Dongyang Sunshine Medical Intelligent Device R&D Co.,Ltd. Address before: 523871 No. 368 Zhenan Middle Road, Chang'an Town, Dongguan City, Guangdong Province Applicant before: DONGGUAN HEC TECH R&D Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A centrifugal disk microfluidic chip and its use method Effective date of registration: 20220728 Granted publication date: 20210511 Pledgee: China Development Bank Shenzhen Branch Pledgor: Dongguan Dongyang Sunshine Medical Intelligent Device R&D Co.,Ltd. Registration number: Y2022980011450 |