CN113295859B

CN113295859B - Paper-based microfluidic chip detection system comprising gyroscopic sample microcentrifuge and application thereof

Info

Publication number: CN113295859B
Application number: CN202110556205.8A
Authority: CN
Inventors: 李博峰; 金腾川; 巩凡吾; 魏华兴
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2023-08-22
Anticipated expiration: 2041-05-21
Also published as: CN113295859A

Abstract

The invention provides a paper-based microfluidic chip detection system with a gyroscopic micro-sample centrifugation device, which can simply implement sample processing and sample analysis. The invention provides application of the antibody in antibody detection, especially in detection of trace samples requiring centrifugation. The system comprises a gyro centrifugal device and a paper-based microfluidic chip device, and can simply and conveniently detect novel coronavirus antibodies in samples without relying on complicated equipment by using a gyro as the centrifugal device to simply separate trace samples such as serum and performing enzyme-linked immunoassay on the paper-based microfluidic chip device with a movable valve.

Description

Paper-based microfluidic chip detection system comprising gyroscopic sample microcentrifuge and application thereof

Technical Field

The invention relates to the field of molecular biology detection, in particular to a detection system comprising a gyro micro-sample centrifugal device and a paper-based microfluidic chip, and particularly relates to application of the detection system in detecting antibodies in serum through gyro micro-sample centrifugation.

Background

Novel coronavirus pneumonia (Corona Virus Disease 2019, covd-19), abbreviated as novel coronavirus pneumonia, is an acute respiratory infectious disease caused by infection of mucosa cells on the surfaces of eyes, mouth, nose, throats and other parts by novel coronavirus (SARS-CoV-2), and has been developed into a global pandemic infectious disease. The new coronaries mainly show fever, dry cough and hypodynamia, and few symptoms of nasal obstruction, watery nasal discharge and diarrhea are accompanied. Severe patients often experience dyspnea, and severe patients can develop acute respiratory distress syndrome, septic shock, and multiple organ failure, even death. The risk of developing severe symptoms is greater in the elderly and those with chronic underlying diseases.

The new coronavirus has extremely strong infectivity, which is equivalent to that of the smallpox virus, and the main transmission ways are droplet transmission, aerosol transmission and contact transmission. Therefore, before the preparation and popularization of the novel coronavirus specific vaccine, strict prevention and control are the most effective measures for coping with the novel coronavirus epidemic situation, and mainly comprise the following steps: the transmission way is blocked (mask protection, keeping distance, reducing personnel flow and sanitary maintenance), symptoms are monitored (fever, cough and dyspnea), virus diagnosis is effectively isolated, and medical treatment is carried out in time.

After the invasion of human body by the new coronavirus, the human immune system recognizes the viral surface proteins and produces various specific antibodies, so the fact that the viral antibodies are detected from the blood can be used as evidence of infection with the virus. The main detection methods for virus diagnosis at present are antibody detection and nucleic acid detection. The main method for detecting antibodies generally requires centrifugation by using high-speed centrifugation equipment to obtain a serum sample, then an antigen-antibody enzyme-linked immunosorbent assay (ELISA) is used to generate a chromogenic reaction, and then an enzyme-labeled instrument is used to detect the serum sample to determine whether viral proteins or antibodies exist in the serum sample.

However, the above method requires expensive equipment (including high-speed centrifuges, q-PCR detectors, multifunctional enzyme-labeling instruments, etc.). However, hospitals and detection stations meeting the conditions in the later regions are more rare, and a large number of samples collected in a scattered mode are required to be transported and detected intensively at low temperature, so that the detection efficiency of the method is low, the instantaneity is poor, the total time is long, the result feedback is not timely, and the requirements of rapidly detecting a large number of suspected samples and even integrally screening people cannot be met.

Currently, third world countries after poverty and poverty-rich areas have become areas of viral spreading and heavy disaster (India' S covd-19 emergency.The Lancet,Vol 397,May 8, 2021, 1683, doi:10.1016/S0140-6736 (21) 01052-7), and existing detection means are particularly difficult to implement in these areas, greatly impeding new crown epidemic prevention and control efforts.

Therefore, in order to meet the demand of large-scale screening in the lagging area, the problem to be solved is to find a detection system which is more economical and convenient, can simply perform sample processing and sample analysis without complex equipment. Therefore, the time for overall use is shortened, the timeliness of result feedback is improved, and the time, manpower and material resources for transporting and concentrating the samples to a detection center or a hospital with higher equipment conditions are saved.

Disclosure of Invention

The detection system of the invention comprises a gyro micro-sample centrifugation and a paper-based microfluidic chip, and serum can be separated simply by using a gyro centrifugation device (such as a pull-cord gyro in particular) so as to be applied to the subsequent detection of the paper-based microfluidic chip.

In particular, in order to obtain a trace serum sample for detection, a trace blood centrifugal device is formed by a gyroscope and a capillary tube, and the gyroscope is driven to continuously rotate at a high speed by manually pulling a pull rope to generate centrifugal force, so that serum in the capillary tube is separated from blood, the serum separation process does not depend on complex and expensive equipment such as a centrifugal machine, and can be separated even without electricity, and the method is particularly suitable for later-economical areas, and has the advantages that the whole process from sample processing to sample analysis can be carried out cheaply and simply.

Paper-based microfluidic chips are devices that drive fluids using capillary principles, whose manipulation of the fluid is determined primarily by its design structure, and can be operated without any external pump. The paper-based microfluidic chip has the advantages of convenient and free control of fluids without timing, convenient and complex multi-step operation by untrained users, and good sensitivity and repeatability. The paper-based chip is light in weight and small in size, is easy to transport to a region with low economy, and is particularly suitable for a region without advanced detection conditions because the paper-based chip does not need to use complex equipment such as PCR and the like, and therefore has lower requirements for operators.

The invention realizes better fluid manipulation by arranging the movable valve in the paper-based micro-fluidic chip detection system. Specifically, the paper-based microfluidic chip detection system of the invention can connect the detection area with a specific shape and distribution of the hydrophobic area and the hydrophilic area with the washing flow passage part in a lamination manner through rotation, and can realize opening and closing of the flow passage through rotation of the movable valve, thereby realizing control of the flow rate and the flow quantity of the fluid, ensuring that the detected marker is not easy to run off and can fully react with the antigen aiming at the marker to achieve the color development effect.

THE ADVANTAGES OF THE PRESENT INVENTION

The standard curve of the system of the invention is linear (R ² More than 0.95), can detect three different novel coronavirus antibodies (IgA, igM and IgG) in human serum at the same time, has good sensibility and improves the accuracy.

In particular, the method for centrifugally separating the trace sample serum by the gyroscope realizes no power consumption in the whole process, avoids the problem of long total time length of collection, transportation and detection in areas without advanced detection conditions, improves the instantaneity of result feedback, and saves time, manpower and material resources spent on transporting and concentrating samples to a detection center or a hospital with higher equipment conditions. This effect is of great significance when the system is used for detecting rapidly transmitted diseases caused by pathogens in areas where economy is relatively poor, in particular if a new coronavirus is infected.

The present invention includes the following.

1. A detection system comprising a paper-based microfluidic chip device,

the paper-based microfluidic chip device comprises a pivot, a detection part and a movable valve which can at least horizontally rotate around the pivot to be overlapped with the detection part,

the detection part and the movable valve respectively comprise paper-based micro-fluidic chips,

the detection part is provided with a hydrophobic area (1) and a hydrophilic area (1), the hydrophilic area (1) is divided into more than one group (preferably 1-10 groups) of sample dripping areas and washing areas by the hydrophobic area (1),

the movable valve is provided with a runner part, a hydrophobic area (2) and a hydrophilic area (2) are arranged in the runner part, the hydrophilic area (2) is divided into runner areas with the same number as the sample dripping areas (1) through the hydrophobic area (2),

when the movable valve rotates to be overlapped with the detection part, the hydrophobic area (2) and the hydrophilic area (2) on the flow passage part are respectively overlapped and contacted with the hydrophobic area (1) and the hydrophilic area (1) on the detection part correspondingly, the flow passage between the sample dripping area and the washing area is communicated, the movable valve is in an open state at the moment, when the movable valve continues to rotate, the flow passage is disconnected, the movable valve is in a closed state at the moment,

the sample drop zone is a well (preferably a circular well, more preferably a circular well having a diameter in the range of 4 to 6 mm) for coating the antigen against the marker, and the wash zone comprises a wash drop well (preferably a circular well, more preferably a circular well having a diameter in the range of 4 to 6 mm).

2. The detection system of item 1, further comprising a gyroscopic centrifugation device for centrifuging a microsample,

the gyro centrifugal device comprises a gyro main body and a bracket with a gyro driving part,

the gyro body has a fixing member for fixing the sampling capillary on the upper surface and a gear D on the lower surface,

the gyro driving part comprises gears A, B and C, wherein the diameter of the gear A is larger than that of the gear B and is clamped with the gear B, the diameter of the gear B is smaller than that of the gear C and is fixed on the gear C and rotates coaxially with the gear C,

in a state where the gyro body is attached to the holder, the gear C engages with the gear D on the gyro body, and thereby, the gyro body is rotated by the gear a when the gear a is rotated by an external force.

3. The detection system according to item 1 or 2, wherein a pull rope is connected to the gear A,

when the pull rope is continuously pulled (preferably by manual pulling), the pull rope drives the gear A to rotate, so that the gyro main body is driven to rotate.

4. The detection system according to item 3, wherein the fixing member symmetrically fixes a plurality of (preferably 1 to 10, more preferably 2 to 8) sampling capillaries on the top surface of the gyro body through the center of the circle.

5. The detection system according to item 3, wherein the fixing member is selected from the group consisting of a snap, a bayonet, a hot melt adhesive (preferably a hot melt adhesive), the inner diameter of the sampling capillary is 0.5-2.0 mm, and the length thereof is 30-60 mm, and is made of a material selected from the group consisting of glass, plastic, polystyrene, and other organic polymer materials, preferably made of glass.

6. The detection system according to any one of claims 1 to 5, wherein the pivot is selected from the group consisting of a snap-able plastic ring, a metal shaft or a blind rivet, more preferably a snap-able plastic ring,

the hydrophobic region is preferably formed by hydrophobic treatment with a hydrophobic substance (e.g. polystyrene, paraffin, preferably paraffin), the washing region optionally comprises a flow guiding region connected to the washing drop well, preferably the washing region is symmetrically distributed on both sides of the sample drop region, optionally a color chip for judging the detection result is included in the paper-based microfluidic chip device.

7. The detection system according to item 6, wherein the marker is one or more of human antibodies IgA, igM and IgG that specifically bind to the RBD protein of the novel coronavirus, and the paper-based microfluidic chip device is used for performing ELISA method.

8. The detection system of any one of claims 1 to 7, wherein the paper-based microfluidic chip device is prepared by:

in the assembled paper-based microfluidic chip device, a chitosan solution (preferably 5 mu L of 0.25 mg/mL) is dropwise added to a detection part in a closed state of a movable valve, the solution is dried at room temperature, glutaraldehyde solution (preferably 5 mu L of 2.5%) is dropwise added, the solution is reacted for 2 hours at room temperature, and then the solution is rotated to an open state of the movable valve, the detection part is washed by a washing solution (preferably deionized water), and the solution is dried at room temperature;

in the "closed" state of the movable valve, 5 to 10. Mu.L of an antigen (preferably human novel coronavirus RBD antigen) 1 to 4. Mu.g/mL (preferably 5. Mu.L, 2. Mu.g/mL) is added dropwise to the detection part, and the reaction is carried out at room temperature for 20 to 60 minutes.

9. Use of the detection system according to any one of claims 1 to 8 for detecting antibodies in a sample, preferably whole blood, which antibodies are one or more (preferably 3) antibodies IgA, igM and IgG that specifically bind to the RBD protein of a novel coronavirus.

10. A method for detecting an antibody, which is carried out using the detection system according to any one of items 1 to 8, comprising:

10 to 30. Mu.L of a sample (preferably whole blood) is centrifuged by a gyro centrifugation device at an angular velocity of 1000 to 500rad/s (preferably 700 rad/s) for 1 to 20 minutes to obtain serum,

ELISA was performed on the paper-based microfluidic chip device to detect antibodies in serum,

the antibody is one or more (preferably 3) of IgA, igM and IgG antibodies which specifically bind to the RBD protein of the novel coronavirus.

11. The method for producing a detection system according to any one of items 1 to 8, comprising:

the pattern with the distribution of the hydrophobic area and the hydrophilic area is designed by using drawing software, filter paper is used as a base material, a wax spraying printer is used for micro-spraying, after printing, the printing is finished, the printing is heated and baked for 15 to 40 seconds at the temperature of 120 to 180 ℃ to enable wax to permeate into paper, so that a paper-based micro-fluid chip with the distribution of the hydrophobic area and the hydrophilic area is obtained, all parts of the paper-based micro-fluid chip are cut, and holes are optionally punched, so that the paper-based micro-fluid chip device is assembled.

12. The detection method according to item 10, comprising:

the whole blood (preferably 10-30 mu L) is sucked by a sampling capillary tube by utilizing the self-priming phenomenon, one end of the sampling capillary tube is sealed, the sampling capillary tube is horizontally fixed on a centrifugal device by utilizing a fixing component, the unsealed end points to the centrifugal circle center along the centrifugal radius, the sealed end deviates from the centrifugal circle center along the centrifugal radius, a pull rope drives a gyro centrifugal device to carry out centrifugation, the upper serum sample is sucked from the unsealed end,

dripping chitosan solution into the sample dripping area, drying at room temperature, dripping glutaraldehyde solution for activation,

coating, sealing, incubating an antibody standard or serum dilution sample, incubating a secondary antibody, incubating a tertiary antibody and performing a substrate chromogenic reaction on the novel coronavirus RBD antigen.

Comparing the color development of the sample dripping area with a reference color development obtained by using the concentration gradient of a standard substance or a reference color card prepared in advance, and judging that the subject is infected with the novel coronavirus when the color development depth is equal to or deeper than the standard substance (concentration), otherwise, judging that the subject is not infected.

In one embodiment of the invention, a spinning top based on a gear structure driven by a pull rope is used as a spinning top centrifugal device, and the spinning top can be a spinning top shown in fig. 2, specifically can have a gear structure shown in fig. 3, and details of the gear structure and the pull rope coefficient are shown in table 1. The number of gears, the number of teeth, the length of the string, etc. of the gear structure may be appropriately changed, or a reed or the like may be further provided to automatically reset the string after the string reaches the end.

TABLE 1

Gear reference number	Radius of gear (millimeter)	Tooth number of gear	Weight of gear (g)
				A	13	28	2.988
B	4	8	0.106
				C	7	13	0.391
D	6	12	26.671

Pull rope	Rope length (millimeter)	Rope weight (g)
					219	0.2957

The structure of the driving gyro body is not limited, and a structure in which a string is replaced with a spring or the like for storing energy may be employed. The pull rope gyroscope is preferably manually pulled by manpower from the standpoint of light weight and low manufacturing cost.

In one embodiment of the invention, a capillary tube with a certain length can be used for directly collecting the blood of the fingertip of the object to be tested, the capillary tube is sealed by blowing out hot melt adhesive through a common hot melt adhesive gun after blood collection, and the capillary tube can be adhered and fixed on a gyroscope through the hot melt adhesive, and at the moment, the fixing component is the hot melt adhesive.

The top surface of the top may be symmetrically provided with a bayonet or a buckle, etc. for clamping the capillary tube, so long as the capillary tube or its extension line passes through the center of the circle after being fixed.

Since a plurality of sampling capillaries can be simultaneously fixed on the same gyroscope, the system of the invention can simultaneously centrifuge, for example, 1 to 10 samples, for example, 8 blood (whole blood) samples, and has good practicability. Accordingly, the size of the gyroscope can be determined according to the number and the length of the sampling capillary, for example, the diameter is 20-100 mm.

Hereinafter, the "paper-based microfluidic chip" is sometimes simply referred to as "paper chip".

In one embodiment, the paper chip is designed to assemble two movable valves with a snap-in plastic ring (e.g., a commercially available page holder) to the detection section, enabling flexible flipping and centering movement. The pivot is not particularly required, and a metal shaft, a hollow rivet or the like can be used as long as the paper chip can flexibly rotate without hindrance.

In the opened state of the movable valve, the movable valve and the detection portion may be temporarily fixed in the water-repellent region using a jig or the like in order to ensure that the movable valve and the detection portion do not move relative to each other and are in good contact with each other.

The area of the single sample dripping area should be not less than 7-12 mm ² 。

Although an example in which the washing zone is symmetrically distributed on both sides of the sample addition zone is given in the later-described embodiment, the washing zone may be distributed on only one side of the sample addition zone. In the case of distribution on both sides, the sample addition region preferably has washing regions on both sides thereof, since the sample addition region receives more washing liquid at the same time during washing, facilitating the liquid to permeate through the back surface of the paper chip and be removed.

The washing area can form a flow channel with the sample dripping area in the opening state of the movable valve, and can be arranged at an upper or lower position on the plane of the paper chip, and is not necessarily parallel to the sample dripping area.

The paper-based microfluidic chip detection system of the present invention can also be used for detection of substances other than the novel coronavirus antibodies by changing the kind of antigen immobilized on the paper chip. The detection substance is not particularly limited as long as it can be measured by an immunoassay such as ELISA method, and is preferably used for detecting antibodies against the etiology of other infectious diseases, for example.

Because the invention can be provided with a plurality of sample cells on the same paper-based micro-fluidic chip device, not only can the simultaneous detection of samples of different subjects be completed, but also the simultaneous detection of a plurality of novel coronavirus antibodies of the samples of the same or different subjects can be carried out, thereby improving the accuracy of diagnosing the infection of the novel coronavirus.

In one embodiment of the present invention, the paper-based microfluidic chip is a material that allows for coating of antigens or antibodies, antigen-antibody reaction, affinity reaction and/or color reaction, including polydimethylsiloxane, polymethyl methacrylate, polystyrene, filter paper, etc., preferably filter paper.

In one embodiment of the present invention, three antibodies specifically recognizing the RBD protein on the surface of a novel coronavirus in human serum were detected, namely, anti-RBD IgA, anti-RBD IgM and anti-RBD IgG (IgA, igM, igG).

In one embodiment, when the threshold value is set for the concentration of each antibody recognizing RBD protein on the surface of a novel coronavirus, and IgA is 0.6. Mu.g/mL, igM is 0.5. Mu.g/mL, and IgG is 1.25. Mu.g/mL, the threshold value or more is defined as positive for viral infection, and the threshold value or less is negative for viral infection, and when at least one of the results of IgA, igM, igG antibodies from the same sample is positive, the sample is judged as positive for viral infection.

Drawings

Fig. 1 is a photograph of one embodiment of a paper-based microfluidic chip device for use in the present invention, patterned using the il ustrate software, printed and fabricated using a wax printing process. The black areas are hydrophobic waxes and the white areas are hydrophilic papers (filter papers). A is in an unassembled state, B is in a valve-open (on) state, and C is in a valve-closed (off) state.

Fig. 2 is a photograph showing an embodiment of the centrifugal device of the present invention, (a) showing a top body for centrifuging a blood sample, and a hand-held holder having a string and gear structure, and (B) showing a state in which a capillary tube containing the blood sample is fixed to the top surface of the top. (C) is a state in which a plurality of capillaries are simultaneously fixed.

The gear structure of the gyro of fig. 3 is schematically shown, a is a photograph of a bracket with a housing removed, the relationship between gears is shown, and B, C, D are photographs of various parts.

FIG. 4 is a standard chart prepared from the standard gray scale values obtained in examples 1,2 and 3. A, B, C are standard curves of IgA, igM, and IgG of anti-RBD in sequence.

FIG. 5 is the concentration of the corresponding antibodies in the raw serum (μg/mL) calculated from the gray scale value of each serum dilution sample using the standard curve formula of examples 1,2, 3. A, B, C are the concentrations of anti-RBD IgA, igM, igG in the raw serum.

Detailed Description

One embodiment of the invention comprises a gyroscopic micro-sample centrifugation-paper-based microfluidic chip detection system using a pull-cord gyroscope with a gear device as a gyroscopic centrifugation device.

In one embodiment, the pull-cord top has, for example, the following gear configuration and pull-cord factor (table 2), the gear arrangement being shown in fig. 3.

TABLE 2

Gear number	Radius of gear (millimeter)	Tooth number of gear	Weight of gear (g)
				A	13	28	2.988
B	4	8	0.106
				C	7	13	0.391
D	6	12	26.671

Pull rope	Rope length (millimeter)	Rope weight (g)
					219	0.2957

The invention includes a method for detecting antibodies (e.g., novel coronavirus antibodies) in serum using a paper-based microfluidic chip detection system, comprising:

collecting: collecting fingertip blood of an object to be detected by using a disposable blood sampling tool, sucking a proper amount of blood by using a capillary tube with the length of 10-60 mm, sealing one end of the capillary tube, and fixing the capillary tube on the upper surface of the gyroscope. The unsealed end of the capillary points to the center of the gyroscope along the radius of the gyroscope, and the sealed end deviates from the center of the gyroscope along the radius of the gyroscope;

and (3) centrifuging: placing the gyroscope into a handheld bracket, rotating a pull rope for 3-20 minutes, and then taking down a capillary tube to obtain an upper serum sample (at least 1 microliter is enough for detection after dilution by 40 times);

the preparation step of the paper chip comprises the following steps: and (3) designing a pattern capable of realizing distribution of a hydrophobic region and a hydrophilic region which meet the characteristics of the item 1 by using drawing software (for example, illuster software), printing the pattern on a common filter paper substrate by adopting a paraffin printing process, and heating and baking after printing, so as to obtain the paper chip with the water conveying functional channel. Cutting each part of the paper chip and then completing assembly;

and (3) an activation step: and activating the detection part of the paper chip prepared by the steps. Specifically, in the movable valve closed state, to the paper chip detection part sample drop zone drop chitosan solution, room temperature drying, drop glutaraldehyde solution, at room temperature reaction for 1-5 hours, rotating the movable valve to open state to connect the flow path, washing liquid (such as deionized water, physiological saline, PBS, containing or not containing Tween substance PBST, preferably containing 0.05% Tween 20 PBST) drop to the washing area, from the sample drop zone paper chip back side (for example by suction) to remove the seepage washing liquid, repeated several times, room temperature drying;

ELISA steps: performing enzyme-linked immunosorbent assay (ELISA) using the above paper chip, detecting the concentration of antibodies (e.g., one or more selected from the group consisting of viral antibodies IgA, igM, and IgG) in each test subject serum;

the analysis step: analyzing the gray value of a paper chip detection part (comprising a sample dripping area), making a standard curve by combining the concentration of the standard substance and a formula thereof, and calculating the concentration of the antibody of the corresponding standard substance in the serum sample; or by comparing the color development of the sample drop zone with a reference color chart or prepared in advance.

Alternatively, the force used to pull the spinning top during the centrifugation step is 1-20 newtons, and the spinning time is 3-20 minutes, preferably 10 minutes, when pulled in a continuous pulling manner.

Optionally, in the activation step, the method specifically comprises the steps of dripping 2-30 mu L of 0.25mg/mL chitosan solution, airing at room temperature, dripping 2-30 mu L of 2.5% glutaraldehyde solution, reacting for 1-4 hours, preferably 2 hours at room temperature, switching on a washing channel, washing a paper chip detection part with deionized water for 3-5 times, and drying at room temperature.

Optionally, the ELISA step specifically includes the following steps:

(1) Diluting the concentration of RBD antigen to 2-10 mu g/mL by using PBS buffer solution with pH of 7.4, dripping 2-20 mu L of RBD antigen into a sample dripping area, reacting for 10-60 minutes at room temperature, rotating a movable valve to be opened, dripping 10-30 mu L of PBST solution into a washing area, and washing the paper chip detection part for 3-5 times;

(2) 2-20 mu L of the liquid containing 0.5% -movable valve is dripped into the sample dripping area to be opened, after reacting for 10-60 minutes at room temperature, 10-30 mu L of PBST solution is used for washing the paper chip detection part for 3-5 times; optionally, after drying, storing in a refrigerator at a refrigeration temperature (e.g., 0-4 ℃) for transportation or storage until use;

(3) Diluting a serum sample with PBS solution in a ratio of 1:40-1:100 to obtain a serum diluted sample;

(4) The standard of the antibody (for example, one or more of virus RBD antibodies IgA, igM, igG) is diluted according to the concentration gradient of 100ng/mL,50ng/mL,25ng/mL,12.5ng/mL,6.25ng/mL and 0ng/mL respectively to be used as the standard;

(5) 2-20 mu L of serum is dripped into the sample dripping area to dilute the sample or the standard substance, after the reaction is carried out for 5-40 minutes at room temperature, the movable valve is rotated to be in an opened state, and the PBST solution, for example, 10-30 mu L of washing paper chip detection part is used for 3-5 times;

(6) Preparing 500-2000 ng/mL secondary antibody (for example, specifically recognizing IgA or IgM or IgG), dripping 2-20 mu L secondary antibody into a sample dripping area, reacting for 5-40 minutes at room temperature, rotating a movable valve to an open state, and washing 3-5 times by 10 mu L PBST;

(7) When the secondary antibody is not connected with HRP (for example, the detected virus antibody is IgG), 2-20 mu L of tertiary antibody connected with HRP is dripped into the dripping area of the sample, and after 10-60 minutes of reaction, 10-30 mu L of PBST is used for washing 3-5 times;

(8) To each detection portion was added dropwise 10 to 40. Mu.L of the reaction substrate TMB solution, and after 10 minutes, the depth of blue color developed in the sample addition area was observed.

Examples

Hereinafter, examples are given for the purpose of illustrating the present invention, but the present invention is not limited to the following examples.

Washing operation: the washing operations described in the examples were all performed with the movable valve open, the washing liquid was sucked from the back of the sample drop zone of the chip, and the movable valve was returned to the closed state after the washing was completed.

Example 1 detection of novel coronavirus antibody IgA

The subjects tested were groups of 35 persons from healthy volunteers and patients diagnosed with a new coronavirus infection.

Step one: taking blood from the finger of the test subject with a disposable lancet, and sucking 20. Mu.L of blood with a capillary having a length of 50 mm;

step two: sealing one end of the capillary tube by using a sol gun (model RJQHAC50-GC113 of Ganchun practice Co., ltd., shanghai) and using a matched hot melt adhesive rod (Shanghai Ganchun practice Co., ltd.), and fixing the capillary tube on the upper surface of the gyro by using the hot melt adhesive, wherein the unsealed end points to the center of the gyro along the radius of the gyro and the sealed end faces away from the center of the gyro along the radius of the gyro, as shown in FIG. 2B;

step three: putting the gyroscope into a pull rope device shown in fig. 2A, pulling the rope to rotate for 10 minutes with a force of 10N, taking down the capillary, and sucking out the capillary from the opening end by using a Hamiltonian needle to obtain an upper serum sample;

step four: the pattern of fig. 1A was designed using the il-luster software, using ashless analytical filter paper as a substrate, and printing was performed using a paraffin printer (XEROX Phaser 8560 DN), and a heating bake at 150 ℃ for 30 seconds allowed the wax to penetrate the paper, thereby obtaining a paper chip master having a distribution of hydrophobic and hydrophilic regions as the pattern of fig. 1A. Punching after cutting the original sheet, and fixing the detection part and the movable valve together by using a page buckle to form a state of B, C in FIG. 1;

step five: dropwise adding 5 mu L of 0.25mg/mL chitosan solution into a sample dropwise adding area of the paper chip, airing at room temperature, dropwise adding 5 mu L of 2.5% glutaraldehyde solution, reacting for 2 hours at room temperature, rotating to an open state of a movable valve, dropwise adding 20 mu L of deionized water into a washing area, removing liquid from the back of the sample dropwise adding area of the paper chip by using multi-layer filter paper, repeatedly washing for three times, and drying at room temperature;

step six: the ELISA detection method specifically comprises

(1) Human novel coronavirus RBD antigen (see, e.g., weihong Zeng, et al, 2021) was diluted with PBS buffer pH7.4 to a concentration of 2. Mu.g/mL and 5. Mu.L was added dropwise to the sample drop zone, and after 30 minutes of reaction at room temperature, the sample drop zone was washed 3 times with 10. Mu.L of PBST solution in the manner described above;

(2) To the sample drop zone drop 5 u L containing 0.5%BSA PBS solution, room temperature reaction for 20 minutes, with 10 u L PBST solution washing 3 times;

(3) Diluting each serum sample of the two groups of detection objects with PBS solution in a ratio of 1:100 to obtain a serum diluted sample;

(4) The virus antibody IgA standard (for example, the preparation method is shown in Weihong Zeng, et al, 2021) is diluted according to concentration gradients of 100ng/mL,50ng/mL,25ng/mL,12.5ng/mL,6.25ng/mL and 0ng/mL respectively, so as to obtain a series of gradient standard products;

(5) Dropwise adding 5 mu L of serum dilution sample or IgA standard substance into a sample dropwise adding area, reacting for 10 minutes at room temperature, and washing 3 times with 10 mu L of PBST solution;

(6) Specifically recognized IgA secondary antibody (HRP-goat anti-human IgA (HRP Conjugated AffiniPure Goat Anti-human IgA) manufacturer Boster biological, product No. BA 1066) was prepared according to instructions to a final concentration of 500ng/mL, and 5. Mu.L of secondary antibody was added dropwise to the sample addition area, reacted at room temperature for 10 minutes, and washed 3 times with 10. Mu.L of PBST as above;

(7) To the sample drop zone was added dropwise 20. Mu.L of a reaction substrate TMB solution (Biyun Tian, cat. No. P0209) and reacted for 1 minute.

Step seven: and photographing a sample dripping area of the detection part, analyzing gray values corresponding to the samples, making a standard curve by combining with the concentration of the IgA standard substance and a formula thereof, and calculating the concentration of the virus antibody IgA in the original serum sample.

The standard curve and its formula and corresponding color development are shown in FIG. 4A, and the IgA antibody concentration in the original serum sample calculated is shown in FIG. 5A.

As shown in FIG. 4A, the paper-based microfluidic chip detection system of the present invention has good linearity of the standard curve (R ² > 0.975), demonstrating that the system used in the present invention has good response.

As shown in FIG. 5, when the threshold value of the concentration of the IgA antibody against the novel coronavirus was set to 0.6. Mu.g/mL based on the upper limit of the serum IgA concentration of the detected healthy human, and the threshold value or more was set to be positive for viral infection and the threshold value or less was negative for viral infection, the detection rate of IgA in the novel coronavirus positive group was 97.1% (34/35), and no healthy group had false positive. It is known that the method for ELISA detection of the novel coronavirus RBD protein specific antibody by using the paper-based microfluidic chip detection system is practical and effective.

The inventor performs three repeatability tests under the same conditions, and as a result, the error between repeated measurements of the paper-based microfluidic chip detection system is smaller, and the error of a healthy subject is less than 0.05 mug/mL; the error of the new crown infected person is less than 1.0 mug/mL, and the repeatability is good.

Example 2 detection of novel coronavirus antibody IgM

In this example, the groups were classified into a healthy person group (number of people: 35) and an infection positive group (number of people: 35), and the sources of the two groups of subjects were the same as in example 1.

The same procedure as in example 1 was performed except that a virus antibody IgM standard (for example, weihong Zeng, et al, 2021) was used as the standard, and the secondary antibody was (HRP-goat anti-Human IgM secondary antibody (HRP Conjugated AffiniPure Goat Anti-Human IgM. Mu. Chain) manufacturer Boster Biological, product No. BA 1077). The standard curve and its formula and corresponding color development are shown in FIG. 4B, and the IgM antibody concentration in the original serum sample calculated is shown in FIG. 5B.

As shown in FIG. 4A, the paper-based microfluidic chip detection system of the present invention has good linearity of the standard curve (R ² > 0.956), demonstrating that the system used in the present invention has good response.

As shown in FIG. 5B, when the threshold value of the concentration of the novel coronavirus IgM antibody was set to 0.5. Mu.g/mL based on the upper limit of the serum IgM concentration of the healthy person detected, and when the threshold value or more was positive for viral infection and the threshold value or less was negative for viral infection, the detection rate of the novel coronavirus IgM positive group was 91.4% (32/35). It is known that the method for ELISA detection of the novel coronavirus RBD protein specific antibody by using the paper-based microfluidic chip detection system is practical and effective.

EXAMPLE 3 detection of novel coronavirus antibody IgG

Among them, as a standard, a virus antibody IgG standard (for example, see Weihong Zeng, et al 2021) was used, and the secondary antibody was (Goat Anti-Human IgG secondary antibody (gold Anti-Human IgG-Fc Secondary Antibody) manufacturer Sino Biological accession number SSA 015), with the addition of the following steps: after the secondary antibody reaction and washing were completed, 5. Mu.L of an HRP-linked tertiary antibody (HRP-labeled rabbit anti-Goat IgG (HRP-conjugated Rabbit anti-coat IgG) manufacturer's organism, cat. D110117) was added dropwise to the sample drop zone, reacted for 20 minutes, and then washed 3 times with 10. Mu.L of PBST.

Otherwise, the same operations as in example 1 were performed. The standard curve and its formula and corresponding color development are shown in FIG. 4C, and the IgM antibody concentration in the original serum sample calculated is shown in FIG. 5C.

As shown in FIG. 4C, the paper-based microfluidic chip detection system of the present invention has good linearity of the standard curve (R ² > 0.966), demonstrating that the system used in the present invention has good response.

As shown in FIG. 5C, when the threshold value of the concentration of the IgG antibody against the novel coronavirus was set to 1.25. Mu.g/mL based on the upper limit of the serum IgG concentration of the healthy human detected, and when the threshold value or more was positive for viral infection and the threshold value or less was negative for viral infection, the detection rate of IgM in the positive group for novel coronavirus was 85.7% (30/35), and no false positive occurred in the healthy group.

It is important to note that since the system of the present invention allows for multiple channels, simultaneous determination of multiple analytes in a sample of the same subject can be achieved, and by combining the results of the multiple analytes, the detection accuracy of the detection method can be improved.

The inventors comprehensively analyze the results of the three antibodies in examples 1,2 and 3, and found that if at least one antibody in the IgA, igM, igG three antibodies of the same sample is positive, the sample is judged to be positive for virus infection, and the following conditions can be obtained: the paper-based microfluidic chip detection system provided by the invention has the advantages that the detection rate of the antibody in the serum of a new crown infection sample is 99.9%, and the missed detection of an infected person can be successfully stopped. The ELISA detection method by using the paper-based microfluidic chip detection system is practical and effective, and is particularly suitable for detecting the novel coronavirus RBD protein specific antibody.

In addition, as can be seen from the chromogenic photographs in FIGS. 4A, B, and C, the color between the wells of each concentration gradient standard exhibits a blue color with gradually increasing depth, and the difference is significant. Therefore, from the viewpoint of practicality, a color chart in which a color corresponding to a specific concentration of a specific analyte is printed in a color is considered to be attached to a system package or a specification, and thus, work for repeating establishment and calculation of a standard curve is saved, and a result can be intuitively obtained by an untrained person.

In addition, when the color chart is not used, the photos of the detection result can be collected and transmitted to a computer with common software for analysis in a data form, and the transmission can be completed instantaneously. Compared with the direct transportation of serum samples, the instantaneity of the result feedback is greatly improved, the requirements on the detection environment and the knowledge level of operators can be reduced to be extremely low, and huge cost saving of manpower and material resources is realized.

Industrial applicability

The invention realizes the effects of simply and conveniently implementing sample treatment and sample analysis flow by utilizing the paper-based microfluidic chip detection system comprising the centrifugal device, is particularly suitable for the situation that serum is required to be obtained from blood centrifugation, and is particularly suitable for the identification of various infectious pathogens (especially novel coronavirus) antibody infection, the rapid screening of crowds and the like.

The system and the method have the characteristics of easily available detection tools, low cost, portability, no need of expensive large-scale instruments, rapid and convenient detection process, difficult space restriction and the like, and are particularly suitable for being used in areas with undeveloped public health conditions so as to rapidly screen a large number of suspected samples. In addition, the paper-based microfluidic chip detection system has good accuracy, and can detect various analytes in body fluid samples such as whole blood at the same time, so that the comprehensive judgment of multiple indexes is realized, and the detection accuracy is further improved.

Reference is made to:

1.Weihong Zeng,Huan Ma,Chengchao Ding,et al.Characterization of SARS-CoV-2-specific antibodies in COVID-19 patients reveals highly potent neutralizing IgA,Si gnal Transduction and Targeted Therapy(2021)6：35；https：//doi.org/10.1038/s41392-0 21-00478-7.Supplementary Materials for Characterization of SARS-CoV-2 Specific A ntibodies in COVID-19 Patients Reveals Highly Potent Neutralizing IgA。

Claims

1. a detection system comprising a paper-based microfluidic chip device and a gyroscopic centrifugation device for centrifuging a microsample,

the paper-based microfluidic chip device comprises a pivot, a detection part and a movable valve, wherein the movable valve can at least horizontally rotate around the pivot to be overlapped with the detection part,

the detection part is provided with a hydrophobic area (1) and a hydrophilic area (1), the hydrophilic area (1) is divided into more than one group of sample dripping areas and washing areas through the hydrophobic area (1),

when the movable valve rotates to be overlapped with the detection part, the hydrophobic area (2) and the hydrophilic area (2) on the flow passage part are respectively overlapped and contacted with the hydrophobic area (1) and the hydrophilic area (1) on the detection part correspondingly, and the flow passage between the sample dripping area and the washing area is communicated, and the movable valve is in an open state at the moment; when the movable valve continues to rotate, the flow path is disconnected, and the movable valve is in a closed state,

the sample dripping area is a hole for coating the antigen aiming at the marker, and the washing area comprises a washing dripping hole;

2. The detection system according to claim 1, wherein the hydrophilic region (1) is partitioned into 1 to 10 groups of sample addition and washing regions by the hydrophobic region (1).

3. The detection system of claim 1, wherein the sample drop zone is a circular aperture and the wash drop aperture is a circular aperture.

4. A detection system according to claim 3, wherein the diameter of the circular aperture is in the range 4-6 mm.

5. The detection system according to any one of claims 1 to 4, wherein a pull rope is connected to the gear A,

when the pull rope is continuously pulled, the pull rope drives the gear A to rotate, so that the gyro main body is driven to rotate.

6. The detection system of claim 5, wherein the continuous pulling is by manual pulling.

7. The detection system according to claim 5, wherein the fixing member symmetrically fixes the plurality of sampling capillaries on the top surface of the gyro body through the center of a circle.

8. The detection system of claim 7, wherein the number of sampling capillaries is 1-10.

9. The detection system of claim 7, wherein the number of sampling capillaries is 2-8.

10. The detection system according to claim 5, wherein the fixing member is selected from a group consisting of a snap, a bayonet, a hot melt adhesive, and the sampling capillary has an inner diameter of 0.5 to 2.0mm and a length of 30 to 60mm, and is made of a material selected from an organic polymer material of glass, plastic or polystyrene.

11. The detection system according to any one of claims 1-4, wherein the pivot is selected from the group consisting of a snap-in plastic ring, a metal shaft or a blind rivet,

the washing area comprises a diversion area connected with the washing liquid dripping hole, and the washing area is symmetrically distributed on two sides of the sample dripping area.

12. The detection system of claim 11, wherein the hydrophobic substance is polystyrene or paraffin.

13. The detection system according to any one of claims 1 to 4, wherein a color chart for judging a detection result is included in the paper-based microfluidic chip device.

14. The detection system of claim 11, wherein the marker is one or more of human antibodies IgA, igM and IgG that specifically bind to the RBD protein of the new coronavirus, and a paper-based microfluidic chip device is used to perform an ELISA method.

15. The detection system of any one of claims 1-14, wherein the paper-based microfluidic chip device is prepared by:

in the assembled paper-based micro-fluidic chip device, dropwise adding chitosan solution into a detection part in a closed state of a movable valve, airing at room temperature, dropwise adding glutaraldehyde solution, reacting for 2 hours at room temperature, rotating to an open state of the movable valve, cleaning the detection part by using a cleaning solution, and drying at room temperature;

in the state of the movable valve being closed, 5-10 mu L of antigen with 1-4 mu g/mL is dripped into the detection part, and the reaction is carried out for 20-60 minutes at room temperature.

16. The detection system of claim 15, wherein the wash solution is deionized water.

17. The detection system of claim 15, wherein the antigen is a human neocoronavirus RBD antigen.

18. Use of the detection system of any one of claims 1-17 for detecting antibodies in a sample, said sample being whole blood, said antibodies being one or more of antibodies IgA, igM and IgG that specifically bind to the RBD protein of a novel coronavirus.

19. The use of claim 18, wherein the antibodies are antibodies IgA, igM and IgG that specifically bind to the RBD protein of the new coronavirus.

20. A method of detecting an antibody, the method performed using the detection system of any one of claims 1-17, comprising:

using a gyro centrifugal device to centrifuge 10-30 mu L of sample at an angular velocity of 1000-500 rad/s for 1-20 min to obtain serum,

the antibody is one or more of IgA, igM and IgG which specifically bind to the RBD protein of the novel coronavirus.

21. The method of claim 20, wherein the sample is whole blood.

22. The method of claim 21, wherein the antibodies are antibodies IgA, igM, and IgG that specifically bind to the RBD protein of the new coronavirus.