CN110496658B - Combined diagnosis paper-based micro-fluidic chip and preparation method thereof - Google Patents
Combined diagnosis paper-based micro-fluidic chip and preparation method thereof Download PDFInfo
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- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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Abstract
The invention belongs to the technical field of biomedical detection, and relates to a micro-fluidic chip for detecting a human whole blood sample and a preparation method thereof. The chip mainly comprises various functional regions of a paper base (7), a sample injection part (2) and a sample separation part (6), and is characterized in that the chip (1) is disc-shaped, and the paper base (7), a chip substrate (9) for placing the paper base (7) and a sealing film (8) on the paper base (7) are arranged on the chip; a circular hole is arranged at the center of the chip (1), a sample injection part (2) is placed in the circular hole, and more than 2 sample processing areas (3) are uniformly and symmetrically arranged around the sample injection part (2) along the diameter extension line; each sample processing area (3) is connected with 1 sample dividing part (6) at the outlet along the diameter extension line; each sample distribution component is connected with a detection area (5); more than 2 screw holes (4) are arranged on the chip (1) along the extension direction of the diameter for fixing.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and relates to a micro-fluidic chip for detecting a human whole blood sample and a preparation method thereof.
Background
Immunochromatography (immuno-chromatography) is a rapid diagnostic technique that has emerged abroad in recent years. The principle is an immunity detection technology based on chromatography technology and antigen-antibody specificity immunoreaction, which takes strip fiber layer material fixed with detection line (T line) and quality control line (C line) as fixed phase, the object to be detected is mobile phase, the object to be detected moves on the chromatography strip by capillary action, the object to be detected starts specificity immunoreaction at T line, and the free object reacts at C line. In recent years, the rapid development of fluorescence technology pushes the fluorescence immunochromatographic technology to break through forward continuously, and a novel detection method is pushed to develop rapidly aiming at the defects of common methods.
At present, in practical diagnosis application, a single index is generally adopted for diagnosis, and the diagnosis result is often not accurate enough. For example, C-reactive protein (CRP) and Procalcitonin (PCT) are a common indicator of clinical diagnosis of inflammatory responses during the course of the activity of inflammatory diseases such as autoimmune diseases, such as lupus erythematosus, reactive arthritis, and inflammatory bowel disease. If the PCT content is measured only, no obvious difference from the content of normal people can be found; however, when CRP is measured, the CRP content is significantly higher than that of normal persons. Therefore, a combination of these two criteria is required to determine the presence of an inflammatory response in a patient. However, a clinical means capable of simultaneously detecting multiple indices such as CRP and PCT is still lacking.
Common methods for detecting CRP and PCT include turbidimetry, latex agglutination, enzyme-linked immunosorbent assay (ELISA), colloidal gold and the like. The turbidimetric and ELISA methods have high accuracy, but the operation is complicated, and the detection time is long. The colloidal gold method has short detection time and simple operation, but has low sensitivity and can not realize quantitative detection.
In view of the defects of the prior detection technology, the invention provides a quantum dot microfluidic chip and a preparation method thereof. Quantum Dots (QDs) are semiconductor nanoparticles that have a very significant advantage in their photochemical properties compared to organic fluorescent dyes commonly used in other immunofluorescence analysis methods. When the QDs nano-particles are excited by an external light source, the emitted fluorescence spectrum is narrow, the scattering is less, the photobleaching effect is less, the photochemical property is stable, and the QDs nano-particles are not easily degraded by biological metabolism and chemical factors. Furthermore, quantum dots can also be covalently coupled to protein molecules via coupling agents. The chip of the invention can realize the qualitative and quantitative detection of CRP and PCT in human whole blood samples, plays an important role in the prediction and differential diagnosis of inflammatory infection, and can be widely applied to ICU wards, hematology departments, oncology departments, pediatrics departments, premature infants, neonatal care rooms, surgeries, internal medicine departments, organ transplantation departments, emergency departments, treatment laboratories and the like.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a combined diagnostic paper-based microfluidic chip.
A joint diagnosis paper-based microfluidic chip mainly comprises various functional regions of a paper base (7), a sample injection part (2) and a sample separation part (6), wherein the chip (1) is disc-shaped, and is provided with three layers, namely a paper base (7), a chip substrate (9) for placing the paper base (7) and a sealing film (8) on the paper base (7); a circular hole is arranged at the center of the chip (1), a sample injection part (2) is placed in the circular hole, and more than 2 sample processing areas (3) are uniformly and symmetrically arranged around the sample injection part (2) along the diameter extension line; each sample processing area (3) is connected with 1 sample dividing part (6) at the outlet along the diameter extension line; each sample distribution component is connected with a detection area (5); more than 2 screw holes (4) are arranged on the chip (1) along the extension direction of the diameter for fixing.
The sample injection part (2) is a hollow cylinder with a bottom surface, more than 2 layers of circular step-shaped platforms are arranged in the cylinder from bottom to top from big to small, a concave sedimentation flow equalizing pool (2-4) is processed on each platform, and a circle of the bottom of the hollow cylinder is a residual liquid pool (2-5); the sedimentation flow equalizing tanks (2-4) are uniformly divided into more than 2 by partition walls (2-3) passing through the circle center of the sample injection part (2) according to the same number with the sample processing areas (3) of the chip (1); sample equalizing ports (2-2) with the same opening height on the same platform are arranged in each sedimentation flow equalizing tank (2-4) in a staggered mode; 1 sample inlet pipe (2-1) is arranged at the circle center of the topmost partition wall (2-3) of the sample inlet component (2), and the bottom opening of the sample inlet pipe (2-1) is uniformly divided by the partition wall (2-3) of the sedimentation flow equalizing tank (2-4); a paper-based channel sample inlet (2-8) which is in sealing fit with the control needle (2-6) is also arranged in the middle position of the bottom of each sedimentation flow equalizing pool (2-4) on the lowest layer; the sample injection part (2) is provided with a shallow groove for installing the sample injection channel (3-1) of the paper-based sample processing area (3) on the lower bottom surface of the sample injection port (2-8) of each paper-based channel; the control needles (2-6) are all arranged below one control needle frame (2-7) and can be inserted into or pulled out of the paper-based channel sample injection ports (2-8) together; the control needle frame (2-7) can be arranged on a matched detection instrument to control the downward and upward movement.
The sample processing area (3) is uniformly distributed around the circumference of the sample injection part (2) along the diameter extension line direction and comprises three functional areas of functional channels which are arranged side by side; firstly, one end of a sample injection channel (3-1) is communicated with a sample injection port (2-8) of each paper-based channel at the bottommost layer of the sample injection part (2); the other end head of the sample feeding channel (3-1) is obliquely inserted into the side wall opening at one end of the outer side of the circumference of the parallel centrifugal channels (3-2) and is communicated with the centrifugal channels (3-2); the other end of the inner side of the circumference of the centrifugal channel (3-2) is connected with one end of the parallel quantum dot reaction channel (3-3) through a U-shaped pipeline head; the other end of the quantum dot reaction channel (3-3) is connected with the detection sample main channel (10) through a corner and a head; the main detection sample channel (10) is communicated with a sample dividing part (6) on the circumferential diameter extension line of the sample feeding part (2) outside the sample processing area (3).
A reverse stop membrane (3-6) is arranged on the side wall where the sample feeding channel (3-1) is connected with the centrifugal channel (3-2), and the reverse stop membrane (3-6) is provided with a plurality of one-way valves on one side of the centrifugal channel (3-2) and can allow a liquid mixture to flow through under normal pressure, but the one-way valves are closed under pressurization to prevent the liquid mixture from flowing backwards; one end of the centrifugal channel (3-2) connected with the sample introduction channel (3-1) is close to the outer side of the center of a circle of the chip, a reverse stop membrane (3-6) is also arranged at the opening of the side wall of the centrifugal channel (3-2) close to the outer side of the center of a circle of the chip, two sides of the reverse stop membrane (3-6) are respectively processed with non-communicated one-way valves with different opening and closing forces, the one-way valves at the end head facing the outer side of the center of a circle of the chip under normal pressure can not pass through closed sample liquid, but the sample liquid can open the one-way valves to flow through under the centrifugal pressure of external force, and the centrifuged sample liquid can pass through the other side of the reverse stop membrane (3-; the reverse stop membrane (3-6) is provided with a centrifugal channel (3-2) towards the outer side of the center of the chip, the total length of the centrifugal channel is 1/4-1/3, the centrifugal capture channel (3-7) is long, and the end of the centrifugal capture channel (3-7) is provided with an exhaust hole (3-8) on the sealing membrane (8).
A coarse filter membrane (3-5) is arranged in the middle of the centrifugal channel (3-2) and can intercept particulate matters on the surface of the paper base; one end of the centrifugal channel (3-2) close to the inner side of the circumference of the chip is provided with a color-changing induction valve (3-4); the color-changing induction valve (3-4) enables the photosensitive valve to touch the thin sheet to block the centrifugal channel (3-2) through color-changing paper which is arranged in the end of the centrifugal channel (3-2) and is subjected to color change when meeting liquid; the color-changing paper is automatically faded after 3-5min, and the photosensitive valve touches and cuts off the thin slice for cutting off the centrifugal channel (3-2).
And a plurality of nano fiber holes (3-9) are processed in the quantum dot reaction channel (3-3).
The sample distribution component (6) is a hollow cylinder with a bottom surface, an opening is formed in the side wall of the bottom surface of the hollow cylinder, facing the circle center of the chip (1), and is connected with the main detection sample channel (10), and the bottom surface of the hollow cylinder of the sample distribution component (6) does not exceed the height of the main detection sample channel (10); an arc-shaped liquid storage tank (6-1) is formed by a retaining wall (6-2) in the hollow cylinder facing the main detection sample channel (10); the arc convex surface of the retaining wall (6-2) of the liquid storage tank (6-1) faces the outer side of the circle center of the chip (1), and the arc convex surface of the retaining wall (6-2) and the rest part of the hollow cylinder of the sample distribution component (6) form a liquid distribution tank (6-3); an opening is processed on the outer wall of the bottom of the hollow cylinder of the liquid separation pool (6-3) and is connected with more than 2 paper bases (7) for sample separation detection channels (11); the detection channels (11) with the number of more than 2 are uniformly distributed on the side surface of the hollow cylinder of the liquid separating pool (6-3) of the sample separating component (6), and the distance from the opening of the hollow cylinder of the sample separating component (6) to the arc-shaped retaining wall (6-2) is consistent in each detection channel (11), so that the liquid separation is guaranteed to be uniform and consistent.
1-2 detection reaction chambers (5-1) and 1 quality detection reaction chamber (5-2) are sequentially processed on each detection channel (11) from one end of the sample separation component (6) to the outside, and the other end of each detection channel (11) to the outside is connected to 1 waste liquid chamber (5-3) to form an independent detection area (5); each waste liquid chamber (5-3) is provided with a vacuum tube (5-4); after the centrifugation is finished, the vacuum tube (5-4) is connected with a vacuum pump to accelerate the flow of the centrifugal sample liquid, accelerate the detection and shorten the detection time; the detection channels (11) are all marked with different detection channels.
Furthermore, the sample injection component (2) and the sample separation component (6) are put into a reserved circular hole of the paper base (7) of the chip (1) after being processed in advance, a sealing film (8) is not arranged on the upper surface of the sample injection component (2), and the sealing film (8) is arranged on the upper surface of the sample separation component (6); the chip (1) is fixed on a matched fluorescence detector through a screw hole (4); detection channel marks are arranged on the detection channels (11), the detection reaction chamber (5-1) and the quality detection reaction chamber (5-2) of the chip (1) are made of nitrocellulose membranes, and the quantum dot reaction channel (3-3) is made of glass fiber membranes; the paper base of the centrifugal catching channels (3-7) is a rough surface of the pits, and the surface is subjected to hydrophobic treatment to prevent the centrifuged supernatant from being adsorbed.
Furthermore, the paper base (7) of the sample injection channel (3-1) and the paper base (7) of the centrifugal channel (3-2) only play a role in drainage, and liquid channels are reserved above the paper base (7) of the sample injection channel (3-1) and the paper base (7) of the centrifugal channel (3-2).
The invention also aims to provide a preparation method of the chip.
According to the preparation method of the paper-based microfluidic chip for joint diagnosis, the paper base (7) with a certain thickness and the same diameter as the chip is adopted, the quantum dot reaction channel (3-3), the detection reaction chamber (5-1) and the quality inspection reaction chamber (5-2) in the functional area of the paper base (7) are replaced by material excavation, the thickness of the sample injection channel (3-1) and the centrifugal channel (3-2) is reduced, and the sample injection channel and the centrifugal channel are pasted on the chip substrate (9); then installing a sample injection part (2) and a sample separation part (6) which are hermetically matched at corresponding positions; the upper surface of the paper base (7) except the sample injection part (2) is sealed and covered by a sealing film (8).
Further, the sealing film (8) is colorless and transparent, the light transmittance is more than 90%, and the thickness is not more than 1mm (not more than 1 mm).
Further, the preparation method also comprises the following steps:
1) manufacturing a chip paper base: manufacturing a microfluidic chip channel on the silanized monolayer paper-based baseplate by using an ultraviolet lithography method to obtain a paper base (7);
2) manufacturing a detection reaction chamber (5-1) and a quality control reaction chamber (5-2): depositing the antibody on a paper-based chip to respectively obtain a detection reaction chamber (5-1) and a quality control reaction chamber (5-2);
3) manufacturing a quantum dot reaction channel (3-3): etching a nanofiber hole (3-9) on the paper base (7) by using a laser etching method, and depositing the quantum dot marked antibody in the nanofiber hole (3-9) by using a spraying machine to obtain a quantum dot reaction channel (3-3);
4) packaging the chip: the transparent film is adhered to the surface of the paper-based chip to enable the thickness to meet the requirement, and then the transparent film is stood in vacuum and then baked and cured to realize air pump sealing.
Preferably, the uncured PDMS is coated on the surface of the paper-based chip in the step 4), and is centrifugally laid on the paper base by a glue spreader until the thickness meets the requirement, and the air pump sealing is realized by vacuum standing for bubble removal treatment, baking and curing.
Further, the quantum dots in the step 3) are CdSe/ZnS quantum dots, or metal nanocluster quantum dots, or quantum dots formed by combining the CdSe/ZnS quantum dots and the metal nanoclusters.
Furthermore, the detection reaction chamber (5-1) and the quality detection reaction chamber (5-2) are made of nitrocellulose membranes, and the quantum dot reaction channel (3-3) is made of glass fiber membranes.
The invention has the beneficial effects that:
1) the invention provides a paper-based microfluidic chip for joint diagnosis, which is additionally provided with a whole blood centrifugal unit during design, can quickly and automatically separate whole blood, greatly saves detection time and simplifies detection steps.
2) The paper-based micro-fluidic chip provided by the invention can realize automatic sample introduction, whole blood separation and multi-channel detection of samples, and can meet the detection precision.
3) The invention adopts the paper-based micro-fluidic chip, has low cost, simple and easy manufacture and convenient use, and has great practicability in poor areas and occasions with limited conditions.
Drawings
Fig. 1 is a schematic top view of a chip according to the present invention.
Fig. 2 is a schematic top view of a sample injection component according to the present invention.
Fig. 3 is a schematic front sectional view of a sample injection component according to the present invention.
Fig. 4 is a schematic top view of a sample injection component with a control needle holder according to the present invention.
FIG. 5 is a schematic cross-sectional view of a sample injection part with a control needle holder according to the present invention.
FIG. 6 is an enlarged front view of a control pin according to the present invention.
FIG. 7 is a schematic top view of a sample processing and detection zone according to the present invention.
Fig. 8 is a schematic top view of a sample-separating member according to the present invention.
Fig. 9 is a schematic front sectional view of a sample separating member according to the present invention.
Fig. 10 is a schematic top view of a chip according to the present invention.
Detailed Description
The following describes in detail preferred embodiments of the present invention. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
1. And (3) detecting the C-reactive protein by adopting a quantum dot immunochromatography technology. The quantum dots were labeled on CRP murine monoclonal antibody and sprayed on the quantum dot reaction channel (3-3). Meanwhile, a CRP mouse monoclonal antibody is used as a detection line, a goat anti-mouse IgG secondary antibody is used as a quality control line to be coated on a nitrocellulose membrane, and the test strip is assembled with a water absorption pad and an optimized sample pad.
2. Synthetic quantum dot-labeled CRP antibodies
And (3) activation: adding the water-soluble quantum dots with the surfaces coated with the carboxyl into a BS solution, adding a mixed solution of NHS and BS and a solution of EDC and MES, uniformly mixing by vortex, and performing ultrasonic activation. Volume was increased, centrifuged using a low temperature ultracentrifuge, and the supernatant was discarded.
Coupling: and adding the BS solution into the activated quantum dots, redissolving under the conditions of vortex and ultrasound, and adding the CRP antibody. Vortex, mix evenly, put into rotary incubator to react. Respectively adding a terminator and a sealant, uniformly mixing, placing on a rotary incubator at room temperature for reaction and volume expansion, performing low-temperature centrifugal separation, removing supernatant, and repeating the operation twice. Adding a preservation solution and resuspending. The sample is stored in a refrigerator for later use.
Example 2
Synthesis of Quantum dot-labeled PCT antibody
The quantum dot water-solubility method for combining oil-soluble CdSe/ZnS quantum dots, or metal nano-cluster quantum dots, or CdSe/ZnS quantum dots and metal nano-clusters is characterized in that acetone is used for precipitating and re-dispersing in trichloromethane, a proper amount of thioglycolic acid is added, and the mixture is fully mixed and then stands for reaction for 2 hours. Centrifuging, removing supernatant, adding buffer solution to completely dissolve the supernatant, adding acetone to purify, repeating the steps for 2-3 times, and finally dispersing the precipitate in phosphate buffer solution for storage and standby.
Cross-reactivity can be substantially eliminated by using anti-calcineurin monoclonal antibodies and anti-calcitonin polyclonal antibodies that bind to two different sites of PCT, respectively. Connecting the monoclonal antibody to the surface of the quantum dot by a covalent cross-linking method to obtain the quantum dot labeled monoclonal antibody, which comprises the following specific steps: adding quantum dots, EDC, 15 mu g NHS solution and anti-calcium monoclonal antibody solution into phosphate buffer solution, mixing uniformly, reacting at room temperature, and adding glycine for sealing. Separating and purifying by using a chromatographic column or a chromatographic column to obtain the quantum dot labeled anti-calcium monoclonal antibody.
Example 3
A joint diagnostic paper-based microfluidic chip: in one embodiment, the chip mainly comprises various functional areas of a paper base (7), a sample injection part (2) and a sample separation part (6), and is characterized in that the chip (1) is in a disc shape, and is provided with three layers, namely a paper base (7), a chip substrate (9) for placing the paper base (7), and a sealing film (8) on the paper base (7); a circular hole is arranged at the center of the chip (1), a sample injection part (2) is placed in the circular hole, and more than 2 sample processing areas (3) are uniformly and symmetrically arranged around the sample injection part (2) along the diameter extension line; each sample processing area (3) is connected with 1 sample dividing part (6) at the outlet along the diameter extension line; each sample distribution component is connected with a detection area (5); more than 2 screw holes (4) are arranged on the chip (1) along the extension direction of the diameter for fixing.
In one embodiment, the sample injection part (2) is a hollow cylinder with a bottom surface, more than 2 layers of circular step-shaped platforms are arranged in the cylinder from bottom to top from large to small, each platform is processed with a concave sedimentation flow equalizing pool (2-4), and a circle of the bottom of the hollow cylinder is a residual liquid pool (2-5); the sedimentation flow equalizing tanks (2-4) are uniformly divided into more than 2 by partition walls (2-3) passing through the circle center of the sample injection part (2) according to the same number with the sample processing areas (3) of the chip (1); sample equalizing ports (2-2) with the same opening height on the same platform are arranged in each sedimentation flow equalizing tank (2-4) in a staggered mode; 1 sample inlet pipe (2-1) is arranged at the circle center of the topmost partition wall (2-3) of the sample inlet component (2), and the bottom opening of the sample inlet pipe (2-1) is uniformly divided by the partition wall (2-3) of the sedimentation flow equalizing tank (2-4); a paper-based channel sample inlet (2-8) which is in sealing fit with the control needle (2-6) is also arranged in the middle position of the bottom of each sedimentation flow equalizing pool (2-4) on the lowest layer; the sample injection part (2) is provided with a shallow groove for installing the sample injection channel (3-1) of the paper-based sample processing area (3) on the lower bottom surface of the sample injection port (2-8) of each paper-based channel; the control needles (2-6) are all arranged below one control needle frame (2-7) and can be inserted into or pulled out of the paper-based channel sample injection ports (2-8) together; the control needle frame (2-7) can be arranged on a matched detection instrument to control the downward and upward movement.
In one embodiment, the sample processing area (3) is uniformly distributed along the diameter extension line direction around the circumference of the sample feeding part (2) and comprises three functional areas of functional channels which are arranged side by side; firstly, one end of a sample injection channel (3-1) is communicated with a sample injection port (2-8) of each paper-based channel at the bottommost layer of the sample injection part (2); the other end head of the sample feeding channel (3-1) is obliquely inserted into the side wall opening at one end of the outer side of the circumference of the parallel centrifugal channels (3-2) and is communicated with the centrifugal channels (3-2); the other end of the inner side of the circumference of the centrifugal channel (3-2) is connected with one end of the parallel quantum dot reaction channel (3-3) through a U-shaped pipeline head; the other end of the quantum dot reaction channel (3-3) is connected with the detection sample main channel (10) through a corner and a head; the main detection sample channel (10) is communicated with a sample dividing part (6) on the circumferential diameter extension line of the sample feeding part (2) outside the sample processing area (3).
In one embodiment, a reverse stop membrane (3-6) is arranged on the side wall of the sample feeding channel (3-1) connected with the centrifugal channel (3-2), and the reverse stop membrane (3-6) is provided with a plurality of one-way valves on one side of the centrifugal channel (3-2) and can allow the liquid mixture to flow through under normal pressure, but the one-way valves close under the pressure to prevent the liquid mixture from flowing backwards; one end of the centrifugal channel (3-2) connected with the sample introduction channel (3-1) is close to the outer side of the center of a circle of the chip, a reverse stop membrane (3-6) is also arranged at the opening of the side wall of the centrifugal channel (3-2) close to the outer side of the center of a circle of the chip, two sides of the reverse stop membrane (3-6) are respectively processed with non-communicated one-way valves with different opening and closing forces, the one-way valves at the end head facing the outer side of the center of a circle of the chip under normal pressure can not pass through closed sample liquid, but the sample liquid can open the one-way valves to flow through under the centrifugal pressure of external force, and the centrifuged sample liquid can pass through the other side of the reverse stop membrane (3-; the reverse stop membrane (3-6) is provided with a centrifugal channel (3-2) towards the outer side of the center of the chip, the total length of the centrifugal channel is 1/4-1/3, the centrifugal capture channel (3-7) is long, and the end of the centrifugal capture channel (3-7) is provided with an exhaust hole (3-8) on the sealing membrane (8).
In one embodiment, the middle part of the centrifugal channel (3-2) is provided with a coarse filter membrane (3-5) which can trap particulate matters on the surface of the paper substrate; one end of the centrifugal channel (3-2) close to the inner side of the circumference of the chip is provided with a color-changing induction valve (3-4); the color-changing induction valve (3-4) enables the photosensitive valve to touch the thin sheet to block the centrifugal channel (3-2) through color-changing paper which is arranged in the end of the centrifugal channel (3-2) and is subjected to color change when meeting liquid; the color-changing paper is automatically faded after 3-5min, and the photosensitive valve touches and cuts off the thin slice for cutting off the centrifugal channel (3-2).
In one embodiment, the quantum dot reaction channel (3-3) is internally processed with a plurality of nano fiber holes (3-9).
In one embodiment, the sample separation part (6) is a hollow cylinder with a bottom surface, an opening is arranged on the side wall of the bottom surface of the hollow cylinder facing the circle center of the chip (1) and is connected with the main detection sample channel (10), and the bottom surface of the hollow cylinder of the sample separation part (6) does not exceed the height of the main detection sample channel (10); an arc-shaped liquid storage tank (6-1) is formed by a retaining wall (6-2) in the hollow cylinder facing the main detection sample channel (10); the arc convex surface of the retaining wall (6-2) of the liquid storage tank (6-1) faces the outer side of the circle center of the chip (1), and the arc convex surface of the retaining wall (6-2) and the rest part of the hollow cylinder of the sample distribution component (6) form a liquid distribution tank (6-3); an opening is processed on the outer wall of the bottom of the hollow cylinder of the liquid separation pool (6-3) and is connected with more than 2 paper bases (7) for sample separation detection channels (11); the detection channels (11) with the number of more than 2 are uniformly distributed on the side surface of the hollow cylinder of the liquid separating pool (6-3) of the sample separating component (6), and the distance from the opening of the hollow cylinder of the sample separating component (6) to the arc-shaped retaining wall (6-2) is consistent in each detection channel (11), so that the liquid separation is guaranteed to be uniform and consistent.
In one embodiment, 1-2 detection reaction chambers (5-1) and 1 quality detection reaction chamber (5-2) are processed on each detection channel (11) from one end of the sample separation part (6) to the outside in sequence, and the other end of each detection channel (11) facing to the outside is connected to 1 waste liquid chamber (5-3) to form an independent detection area (5); each waste liquid chamber (5-3) is provided with a vacuum tube (5-4); after the centrifugation is finished, the vacuum tube (5-4) is connected with a vacuum pump to accelerate the flow of the centrifugal sample liquid, accelerate the detection and shorten the detection time; the detection channels (11) are all marked with different detection channels.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (8)
1. A joint diagnosis paper-based microfluidic chip mainly comprises various functional regions of a paper base (7), a sample injection part (2) and a sample separation part (6), and is characterized in that the chip (1) is disc-shaped, and is provided with three layers, namely a paper base (7), a chip substrate (9) for placing the paper base (7), and a sealing film (8) on the paper base (7); a circular hole is arranged at the center of the chip (1), a sample injection part (2) is placed in the circular hole, and more than 2 sample processing areas (3) are uniformly and symmetrically arranged around the sample injection part (2) along the diameter extension line; each sample processing area (3) is connected with 1 sample dividing part (6) at the outlet along the diameter extension line; each sample distribution component is connected with a detection area (5); more than 2 screw holes (4) are arranged on the chip (1) along the extension line direction of the diameter for fixing;
the sample injection part (2) is a hollow cylinder with a bottom surface, more than 2 layers of circular step-shaped platforms are arranged in the cylinder from bottom to top from big to small, a concave sedimentation flow equalizing pool (2-4) is processed on each platform, and a circle of the bottom of the hollow cylinder is a residual liquid pool (2-5); the sedimentation flow equalizing tanks (2-4) are uniformly divided into more than 2 by partition walls (2-3) passing through the circle center of the sample injection part (2) according to the same number with the sample processing areas (3) of the chip (1); sample equalizing ports (2-2) with the same opening height on the same platform are arranged in each sedimentation flow equalizing tank (2-4) in a staggered mode; 1 sample inlet pipe (2-1) is arranged at the circle center of the topmost partition wall (2-3) of the sample inlet component (2), and the bottom opening of the sample inlet pipe (2-1) is uniformly divided by the partition wall (2-3) of the sedimentation flow equalizing tank (2-4); a paper-based channel sample inlet (2-8) which is in sealing fit with the control needle (2-6) is also arranged in the middle position of the bottom of each sedimentation flow equalizing pool (2-4) on the lowest layer; the sample injection part (2) is provided with a shallow groove for installing the sample injection channel (3-1) of the paper-based sample processing area (3) on the lower bottom surface of the sample injection port (2-8) of each paper-based channel; the control needles (2-6) are all arranged below one control needle frame (2-7) and can be inserted into or pulled out of the paper-based channel sample injection ports (2-8) together; the control needle frame (2-7) can be arranged on a matched detection instrument to control the downward and upward movement;
the sample processing area (3) is uniformly distributed around the circumference of the sample injection part (2) along the diameter extension line direction and comprises three functional areas of functional channels which are arranged side by side; firstly, one end of a sample injection channel (3-1) is communicated with a sample injection port (2-8) of each paper-based channel at the bottommost layer of the sample injection part (2); the other end head of the sample feeding channel (3-1) is obliquely inserted into the side wall opening at one end of the outer side of the circumference of the parallel centrifugal channels (3-2) and is communicated with the centrifugal channels (3-2); the other end of the inner side of the circumference of the centrifugal channel (3-2) is connected with one end of the parallel quantum dot reaction channel (3-3) through a U-shaped pipeline head; the other end of the quantum dot reaction channel (3-3) is connected with the detection sample main channel (10) through a corner and a head; the main detection sample channel (10) is communicated with a sample dividing part (6) on the circumferential diameter extension line of the sample feeding part (2) at the outer side of the sample processing area (3);
a reverse stop membrane (3-6) is arranged on the side wall where the sample feeding channel (3-1) is connected with the centrifugal channel (3-2), and the reverse stop membrane (3-6) is provided with a plurality of one-way valves on one side of the centrifugal channel (3-2) and can allow a liquid mixture to flow through under normal pressure, but the one-way valves are closed under pressurization to prevent the liquid mixture from flowing backwards; one end of the centrifugal channel (3-2) connected with the sample introduction channel (3-1) is close to the outer side of the center of a circle of the chip, a reverse stop membrane (3-6) is also arranged at the opening of the side wall of the centrifugal channel (3-2) close to the outer side of the center of a circle of the chip, two sides of the reverse stop membrane (3-6) are respectively processed with non-communicated one-way valves with different opening and closing forces, the one-way valves at the end head facing the outer side of the center of a circle of the chip under normal pressure can not pass through closed sample liquid, but the sample liquid can open the one-way valves to flow through under the centrifugal pressure of external force, and the centrifuged sample liquid can pass through the other side of the reverse stop membrane (3-; the reverse stop membrane (3-6) is provided with a centrifugal channel (3-2) towards the outer side of the center of the chip, the total length of the centrifugal channel is 1/4-1/3, the centrifugal capture channel (3-7) is long, and the end of the centrifugal capture channel (3-7) is provided with an exhaust hole (3-8) on a sealing membrane (8);
a coarse filter membrane (3-5) is arranged in the middle of the centrifugal channel (3-2) and can intercept particulate matters on the surface of the paper base; one end of the centrifugal channel (3-2) close to the inner side of the circumference of the chip is provided with a color-changing induction valve (3-4); the color-changing induction valve (3-4) enables the photosensitive valve to touch the thin sheet to block the centrifugal channel (3-2) through color-changing paper which is arranged in the end of the centrifugal channel (3-2) and is subjected to color change when meeting liquid; after 3-5min, the color-changing paper is automatically faded, and the photosensitive valve touches and cuts off the thin slice for cutting off the centrifugal channel (3-2);
a plurality of nano fiber holes (3-9) are processed in the quantum dot reaction channel (3-3);
the sample distribution component (6) is a hollow cylinder with a bottom surface, an opening is formed in the side wall of the bottom surface of the hollow cylinder, facing the circle center of the chip (1), and is connected with the main detection sample channel (10), and the bottom surface of the hollow cylinder of the sample distribution component (6) does not exceed the height of the main detection sample channel (10); an arc-shaped liquid storage tank (6-1) is formed by a retaining wall (6-2) in the hollow cylinder facing the main detection sample channel (10); the arc convex surface of the retaining wall (6-2) of the liquid storage tank (6-1) faces the outer side of the circle center of the chip (1), and the arc convex surface of the retaining wall (6-2) and the rest part of the hollow cylinder of the sample distribution component (6) form a liquid distribution tank (6-3); an opening is processed on the outer wall of the bottom of the hollow cylinder of the liquid separation pool (6-3) and is connected with more than 2 paper bases (7) for sample separation detection channels (11); the more than 2 detection channels (11) are uniformly distributed on the side surface of the hollow cylinder of the liquid separation pool (6-3) of the sample separation part (6), and the distance from the opening of the hollow cylinder of the sample separation part (6) to the arc-shaped retaining wall (6-2) is consistent for each detection channel (11);
1-2 detection reaction chambers (5-1) and 1 quality detection reaction chamber (5-2) are sequentially processed on each detection channel (11) from one end of the sample separation component (6) to the outside, and the other end of each detection channel (11) to the outside is connected to 1 waste liquid chamber (5-3) to form an independent detection area (5); each waste liquid chamber (5-3) is provided with a vacuum tube (5-4); after the centrifugation is finished, the vacuum tube (5-4) is connected with a vacuum pump to accelerate the flow of the centrifugal sample liquid, accelerate the detection and shorten the detection time; the detection channels (11) are all marked with different detection channels.
2. The combined diagnostic paper-based microfluidic chip according to claim 1, wherein the sample injection part (2) and the sample separation part (6) are placed in a reserved circular hole of the paper-based (7) of the chip (1) after being processed in advance, the upper surface of the sample injection part (2) is not provided with a sealing film (8), and the upper surface of the sample separation part (6) is provided with the sealing film (8); the chip (1) is fixed on a matched fluorescence detector through a screw hole (4); detection channel marks are arranged on the detection channels (11), the detection reaction chamber (5-1) and the quality detection reaction chamber (5-2) of the chip (1) are made of nitrocellulose membranes, and the quantum dot reaction channel (3-3) is made of glass fiber membranes; the paper base of the centrifugal catching channels (3-7) is a rough surface of the pits, and the surface is subjected to hydrophobic treatment.
3. The joint diagnosis paper-based microfluidic chip according to claim 1, wherein the paper-based (7) of the sample introduction channel (3-1) and the centrifugal channel (3-2) only play a role of drainage, and a liquid channel is left above the paper-based (7) of the sample introduction channel (3-1) and the centrifugal channel (3-2).
4. The preparation method of the paper-based micro-fluidic chip for joint diagnosis according to any one of claims 1 to 3, characterized in that a paper base (7) with a certain thickness and the same diameter as the chip is adopted, the quantum dot reaction channel (3-3) of the functional area of the paper base (7), the detection reaction chamber (5-1) and the quality inspection reaction chamber (5-2) are replaced by removing materials, the thickness of the sample injection channel (3-1) and the centrifugal channel (3-2) is reduced, and the sample injection channel and the centrifugal channel are pasted on the chip substrate (9); then installing a sample injection part (2) and a sample separation part (6) which are hermetically matched at corresponding positions; the upper surface of the paper base (7) except the sample injection part (2) is sealed and covered by a sealing film (8).
5. The production method according to claim 4, wherein the sealing film (8) is colorless and transparent, has a light transmittance of more than 90%, and has a thickness of not more than 1 mm.
6. The method of claim 4, further comprising the steps of:
1) manufacturing a chip paper base: manufacturing a microfluidic chip channel on the silanized monolayer paper-based baseplate by using an ultraviolet lithography method to obtain a paper base (7);
2) manufacturing a detection reaction chamber (5-1) and a quality control reaction chamber (5-2): depositing the antibody on a paper-based chip to respectively obtain a detection reaction chamber (5-1) and a quality control reaction chamber (5-2);
3) manufacturing a quantum dot reaction channel (3-3): etching a nanofiber hole (3-9) on the paper base (7) by using a laser etching method, and depositing the quantum dot marked antibody in the nanofiber hole (3-9) by using a spraying machine to obtain a quantum dot reaction channel (3-3);
4) packaging the chip: and tightly sticking the transparent film with the thickness meeting the requirement on the surface of the paper-based chip to realize the sealing of the air pump.
7. The preparation method according to claim 6, wherein the quantum dots in step 3) are CdSe/ZnS quantum dots, or metal nanocluster quantum dots, or quantum dots in which CdSe/ZnS quantum dots are combined with metal nanoclusters.
8. The preparation method according to claim 6, wherein the detection reaction chamber (5-1) and the quality detection reaction chamber (5-2) are made of nitrocellulose membranes, and the quantum dot reaction channel (3-3) is made of glass fiber membranes.
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| CN206334683U (en) * | 2016-07-28 | 2017-07-18 | 上海速创诊断产品有限公司 | A kind of CD plate-likes micro-fluidic chip |
| CN106770520B (en) * | 2016-12-22 | 2019-04-23 | 西安交通大学 | The paper substrate micro-fluidic chip of full blood hemoglobin detection and its production and application |
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