CN113295859A - Paper-based microfluid chip detection system containing gyroscope sample microcentrifuge device and application thereof - Google Patents
Paper-based microfluid chip detection system containing gyroscope sample microcentrifuge device and application thereof Download PDFInfo
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Abstract
The present invention provides a paper-based microfluidic chip detection system with a gyroscopic micro-sample centrifuge that enables simple sample processing and sample analysis. The invention provides the application of the polypeptide in antibody detection, in particular to the detection of trace samples needing centrifugation. The system comprises a gyro centrifugal device and a paper-based microfluidic chip device, and can simply and conveniently separate a trace amount of sample such as serum by using a gyro as the centrifugal device and carry out enzyme-linked immunoassay on the paper-based microfluidic chip device with a movable valve, thereby simply and conveniently realizing the detection of the novel coronavirus antibody in the sample without depending on complicated equipment.
Description
Technical Field
The invention relates to the field of molecular biological 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 gyro micro sample centrifugal device in detecting antibodies in serum.
Background
The novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is called novel coronavirus pneumonia for short, is an acute respiratory infectious Disease caused by infection of surface mucosal cells of eyes, mouth, nose, throat and the like by the novel coronavirus (SARS-CoV-2), and has been developed into a global pandemic infectious Disease. The new coronary pneumonia is mainly manifested by fever, dry cough, debilitation, and few symptoms accompanied by nasal obstruction, watery nasal discharge, and diarrhea. Severe patients often develop dyspnea, and severe patients may develop acute respiratory distress syndrome, septic shock, and multiple organ failure, or even death. It has been found clinically that the elderly and those with chronic underlying disease are more at risk of developing severe illness.
The new coronavirus has extremely strong infectivity, is equivalent to smallpox virus, and has main transmission paths of droplet transmission, aerosol transmission and contact transmission. Therefore, before the specific vaccine of the new coronavirus is prepared and popularized, strict prevention and control are the most effective measures for dealing with the new coronary epidemic situation, and the method mainly comprises the following steps: the system has the advantages of blocking of transmission paths (mask protection, distance keeping, personnel flow reduction, health maintenance), monitoring of symptoms (fever cough and dyspnea), effective isolation, virus diagnosis and timely hospitalization.
After invasion of the human body by the new coronavirus, the human immune system recognizes the virus surface protein and produces various specific antibodies, and thus, the fact that the virus antibodies are detected from 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 the antibody usually needs to use a high-speed centrifuge to obtain a serum sample, then an antigen-antibody enzyme-linked immunosorbent assay (ELISA) is used to generate a color reaction, and then an enzyme-linked immunosorbent assay (ELISA) is used to detect the presence of viral proteins or antibodies in the serum sample.
However, the above method requires the use of relatively expensive instruments (including high-speed centrifuges, q-PCR detectors, multi-purpose microplate readers, etc.). However, hospitals and detection stations meeting the conditions in laggard areas are rare, and a large amount of dispersedly collected samples need to be transported and detected intensively at low temperature, so that the method has low detection efficiency, poor instantaneity and untimely result feedback caused by long total use time, and cannot meet the requirements of rapidly carrying out large amount of detection of suspected samples and even overall screening of crowds.
At present, the third world countries behind poverty and the poor nations in the poor and rich polarization areas become virus spreading serious disaster areas (India' S COVID-19 epidemic. the Lancet, Vol 397, May 8, 2021, 1683, DOI: 10.1016/S0140-6736 (21)) and the existing detection means are particularly difficult to realize in the areas and greatly obstruct the work of preventing and controlling the new crown epidemic situation.
Therefore, in order to meet the requirement of large-scale screening in the laggard areas, the problem to be solved is that a detection system which is more economical and convenient and can simply implement sample processing and sample analysis without complex equipment needs to be found out urgently. Therefore, the total time is shortened, the instantaneity of result feedback is improved, and the time and manpower and material resources spent on transporting and concentrating samples to a detection center or a hospital with higher equipment conditions are saved.
Disclosure of Invention
The detection system comprises a gyro micro sample centrifugation device and a paper-based microfluidic chip, and serum can be separated easily by using a gyro centrifugation device (such as a pull-string gyro in particular) so as to facilitate subsequent detection of the paper-based microfluidic chip.
Specifically, in order to obtain a trace serum sample which is enough for detection, the invention adopts a trace blood centrifugal device consisting of a gyroscope and a capillary tube, and the gyroscope is driven to continuously rotate at a high speed to generate centrifugal force by manually pulling a pull rope, so that the serum in the capillary tube is separated from the blood.
The paper-based microfluid chip is a device for driving fluid by the capillary principle, and the operation function of the paper-based microfluid chip is mainly determined by the design structure of the paper-based microfluid chip, and the paper-based microfluid chip can be operated without any external pump. Paper-based microfluidic chips have the advantage of allowing convenient and free control of fluids without the need for timed set-up, allowing easy complex multi-step operations even by untrained users, and good sensitivity and repeatability. The paper-based chip is light in weight and small in volume, so that the paper-based chip is easy to transport to an economically laggard area, and the requirement threshold for an operator is lower because complex equipment such as PCR is not needed, so that the paper-based chip is particularly suitable for areas without high-grade detection conditions.
The invention realizes better fluid manipulation by arranging the movable valve in the paper-based microfluid chip detection system. Specifically, the paper-based microfluid chip detection system can connect the detection areas with the hydrophobic areas and the hydrophilic areas in specific shapes and distribution with the washing flow channel part in a laminated mode through rotation, and the opening and closing of the flow channel can be realized through rotating the movable valve, so that the flow speed and flow of fluid can be controlled, the detected marker is not easy to lose, and the marker 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 well linear (R)2More than 0.95), can simultaneously detect three different novel coronavirus antibodies (IgA, IgM and IgG) in human serum, has good sensitivity and improves the accuracy.
Particularly, the method for separating the trace sample serum by the gyro centrifugation realizes the whole process without electricity, avoids the problem of longer total time of collection-transportation-detection in areas without advanced detection conditions, improves the instantaneity of result feedback, and saves the time and manpower and material resources spent on transporting and concentrating the sample to a detection center or a hospital with higher equipment conditions. Such an effect is of great significance when the system is used for detecting rapidly transmitted diseases caused by pathogens, particularly whether or not new coronaviruses are infected, in economically relatively laggard areas.
The present invention includes the following.
1. A detection system comprising a paper-based microfluidic chip device,
the paper-based microfluidic chip device includes a pivot, a detection portion, and a movable valve that is at least horizontally rotatable about the pivot to overlap with the detection portion,
the detection part and the movable valve respectively contain a paper-based micro-fluidic chip,
the detection part is provided with a hydrophobic area (1) and a hydrophilic area (1), the hydrophobic area (1) separates the hydrophilic area (1) into more than one group (preferably 1-10 groups) of sample dripping area and washing area,
the movable valve is provided with a flow channel part, a hydrophobic area (2) and a hydrophilic area (2) are arranged in the flow channel part, the hydrophobic area (2) divides the hydrophilic area (2) into flow channel areas with the number equal to that of the sample dripping areas (1),
when the movable valve rotates to overlap with the detection part, the hydrophobic area (2) and the hydrophilic area (2) on the flow channel part are correspondingly overlapped and contacted with the hydrophobic area (1) and the hydrophilic area (1) on the detection part respectively to communicate the flow path between the sample dripping area and the washing area, 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 at the moment,
the sample dropwise add the hole (preferably the circular port, more preferably the circular port of diameter in 4 ~ 6mm within range) for the cladding to the antigen of marker in the district, and the washing district contains the washing liquid dropwise add hole (preferably the circular port, more preferably the circular port of diameter in 4 ~ 6mm within range).
2. The detection system of item 1, further comprising a gyroscopic centrifuge for centrifuging a micro sample,
the spinning top centrifugal device comprises a spinning top main body and a bracket containing a spinning top driving part,
the top body has a fixing member for fixing the sampling capillary on the upper surface, 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, the gear B is fixed on the gear C and rotates coaxially with the gear C,
when the top body is attached to the stand, the gear C engages with the gear D on the top body, and thereby the top body is rotated by the external force when the gear a is rotated.
3. The detection system according to item 1 or 2, wherein a pull rope is connected to the gear A,
when the pulling rope is continuously pulled (preferably manually pulled), the pulling rope drives the gear A to rotate, so that the gyro 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 in such a manner as to pass through the center of a circle.
5. The detection system according to item 3, wherein the fixing member is selected from a group consisting of a snap, a bayonet, and a hot melt adhesive (preferably a hot melt adhesive), the sampling capillary tube has an inner diameter of 0.5-2.0 mm and a length of 30-60 mm, and is made of a material selected from a group consisting of glass, plastic, polystyrene, and other organic polymer materials, and preferably made of glass.
6. The detection system of any one of claims 1 to 5, wherein the pivot is selected from a snappable plastic ring, a metal shaft or a pop rivet, more preferably a snappable plastic ring,
the hydrophobic region is preferably formed by performing hydrophobic treatment with a hydrophobic substance (such as polystyrene, paraffin, and paraffin is preferred), the washing region optionally comprises a flow guide region connected with a washing liquid dropping hole, the washing region is preferably symmetrically distributed at two sides of the sample dropping region, and optionally, a color card for judging the detection result is contained in the paper-based microfluidic chip device.
7. The test 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 neo-coronavirus, and the paper-based microfluidic chip device is used to perform ELISA.
8. The detection system of any one of claims 1 to 7, wherein the paper-based microfluidic chip device is prepared by:
in an assembled paper-based microfluid chip device, dripping a chitosan solution (preferably 5 muL 0.25mg/mL) into a detection part under the 'closed' state of a movable valve, airing at room temperature, dripping a glutaraldehyde solution (preferably 5 muL 2.5%), reacting at room temperature for 2 hours, rotating to the 'open' state of the movable valve, washing the detection part with a washing solution (preferably deionized water), and drying at room temperature;
5-10 μ L of 1-4 μ g/mL (preferably 5 μ L,2 μ g/mL) of an antigen (preferably human neocoronavirus RBD antigen) is dropped onto the detection part in a state that the movable valve is closed, and the reaction is carried out for 20-60 minutes at room temperature.
9. Use of the detection system of any one of items 1 to 8 for detecting antibodies in a sample, preferably whole blood, the antibodies being one or more (preferably 3) of antibodies IgA, IgM and IgG that specifically bind to a neocoronavirus RBD protein.
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:
centrifuging 10-30 μ L of a sample (preferably whole blood) at an angular velocity of 1000-500 rad/s (preferably 700rad/s) for 1-20 minutes by using a gyroscopic centrifuge to obtain serum,
performing an ELISA on said paper-based microfluidic chip device to detect antibodies in serum,
the antibody is one or more (preferably 3) of antibodies IgA, IgM and IgG which are specifically combined with the RBD protein of the new coronavirus.
11. The method for producing a detection system according to any one of items 1 to 8, comprising:
designing a pattern with a hydrophobic area and a hydrophilic area distributed by using drawing software, carrying out micro-injection by using filter paper as a base material and a wax-injection printer, heating and baking at 120-180 ℃ for 15-40 seconds after printing is finished so that wax permeates into paper, thus obtaining a paper-based microfluidic chip with the hydrophobic area and the hydrophilic area distributed, cutting each part of the paper-based microfluidic chip, optionally punching, and assembling the paper-based microfluidic chip device.
12. The detection method according to item 10, comprising:
whole blood (preferably 10-30 mu L) is sucked by a sampling capillary tube by utilizing a self-suction 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, so that 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, an upper layer serum sample is sucked from the unsealed end,
dripping chitosan solution into the sample dripping area, dripping glutaraldehyde solution for activation after drying at room temperature,
carrying out novel coronavirus RBD antigen coating, blocking, antibody standard substance or serum dilution sample incubation, secondary antibody incubation, tertiary antibody incubation and substrate chromogenic reaction.
Comparing the color development of the sample dripping zone with reference color development obtained by using the concentration gradient of the standard substance or a reference color chart prepared in advance, when the color development depth is equal to or deeper than the standard substance (concentration), judging that the subject is infected with the novel coronavirus, otherwise, judging that the subject is not infected.
In one embodiment of the present invention, a spinning top driven by a pull rope and having a gear structure is used as a spinning top centrifugal device, the spinning top may be a spinning top as shown in fig. 2, specifically, a gear structure as shown in fig. 3, and the 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 cord, and the like in the gear structure may be changed as appropriate, or a spring piece or the like for automatically returning the cord after the cord is terminated may be further provided as long as the object of the present invention is achieved.
TABLE 1
| Gear mark | Radius of gear (millimeter) | Number of teeth (pieces) | Weight of gear (gram) |
| A | 13 | 28 | 2.988 |
| |
4 | 8 | 0.106 |
| C | 7 | 13 | 0.391 |
| |
6 | 12 | 26.671 |
| Pull rope | Rope length (millimeter) | Rope weight (gram) | |
| 219 | 0.2957 |
The structure for driving the top body is not limited, and a structure in which a power spring or the like for storing energy is used instead of the pull cord may be employed. Among them, a string top manually pulled by a person is preferable from the viewpoint of lightness and low cost.
In one embodiment of the invention, a capillary tube with a certain length can be used for directly collecting blood of a fingertip of an object to be detected, a hot melt adhesive is sprayed out from the capillary tube through a common hot melt adhesive gun for sealing after blood collection, the capillary tube can be adhered and fixed on a spinning top through the hot melt adhesive, and at the moment, the fixing component is the hot melt adhesive.
Bayonets or buckles and the like which can be clamped into the capillary tubes can be symmetrically arranged on the upper surface of the gyroscope, and the capillary tubes or the extension lines thereof can pass through the circle center after being fixed.
Because a plurality of sampling capillaries can be fixed on the same gyroscope at the same time, the system can centrifuge 1-10 samples at the same time, for example, centrifuge 8 blood (whole blood) samples at the same time, and has good practicability. Accordingly, the size of the gyroscope can be determined according to the number and the length of the sampling capillary tubes, and the diameter of the gyroscope is 20-100 mm.
Hereinafter, the "paper-based microfluidic chip" is sometimes simply referred to as a "paper chip".
In one embodiment, the paper chip is designed to assemble two movable valve snap-fit plastic rings (e.g., commercially available page clips) with the detection portion, enabling flexible flipping and movement around the core. There is no special requirement for the pivot, so long as the flexible and unimpeded rotation of the paper core piece can be realized, and a metal shaft or a hollow rivet and the like can also be used.
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 in the open state of the movable valve, the movable valve may be temporarily fixed to the drain region by using a jig or the like.
The area size of a single sample dripping area is not less than 7-12 mm2。
Although examples in which the washing zones are symmetrically disposed on both sides of the sample addition zone are given in the examples described later, the washing zones may be disposed on only one side of the sample addition zone. In the case of the distribution on both sides, the sample addition region receives a larger volume of washing solution at the same time during washing, which facilitates the removal of the liquid by permeating through the back surface of the paper chip, and therefore, it is preferable to have washing regions on both sides of the sample addition region.
The washing area may be located at an upper or lower position on the plane of the paper chip, and is not necessarily parallel to the sample dripping area, as long as it forms a flow channel with the sample dripping area in the open state of the movable valve.
The paper-based micro-fluidic chip detection system of the invention can also be used for detection of other substances besides the new coronavirus antibody by changing the type of the antigen fixed on the paper chip. The detection object is not particularly limited as long as it can be measured by an immunoassay such as ELISA method or the like, and is preferably used for detection of an antibody against a pathogen of other infectious diseases, for example.
Because the invention can be provided with a plurality of sample pools on the same paper-based microfluid chip device, the invention not only can complete the simultaneous detection of samples of different subjects, but also can simultaneously detect a plurality of novel coronavirus antibodies of the samples of the same or different subjects, thereby improving the accuracy of diagnosing the infection of the new coronavirus.
In one embodiment of the present invention, the paper-based microfluidic chip is a material that allows coating of antigen or antibody, antigen-antibody reaction, affinity reaction and/or color reaction, and includes polydimethylsiloxane, polymethylmethacrylate, polystyrene, filter paper, and the like, and is preferably filter paper.
In one embodiment of the present invention, three antibodies specifically recognizing the new coronavirus surface RBD protein in human serum, namely anti-RBD IgA, anti-RBD IgM, and anti-RBD IgG (IgA, IgM, IgG for short), are detected.
In one embodiment, when the concentration of each antibody recognizing the RBD protein on the surface of the new coronavirus is set to be a threshold value of IgA0.6. mu.g/mL, IgM 0.5. mu.g/mL, IgG 1.25. mu.g/mL, the sample is determined to be positive for viral infection by defining the threshold value or more as positive for viral infection and the sample is determined to be positive for viral infection by defining the threshold value or less as negative for viral infection by defining the threshold value or less as positive for at least one of the three antibody results of IgA, IgM and IgG from the same sample.
Drawings
FIG. 1 is a photograph of one embodiment of a paper-based microfluidic chip device for use in the present invention, patterned using Illustrator software, printed and fabricated using a wax printing process. The black areas are hydrophobic wax and the white areas are hydrophilic paper (filter paper). 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 centrifuge of the present invention, wherein (A) is a top body for centrifuging a blood sample and a hand-held holder with a string and gear structure, and (B) is a capillary tube containing a blood sample fixed to the upper surface of the top. (C) To fix a plurality of capillaries simultaneously.
Fig. 3 shows a schematic gear structure of the top, wherein A is a photograph of a bracket with a shell removed and shows the mutual relationship among gears, and B, C and D are photographs of various parts.
FIG. 4 is a calibration graph prepared from the gray scale values of the standards obtained in examples 1, 2 and 3. A, B and C are standard curves of IgA, IgM and IgG of anti-RBD in sequence.
FIG. 5 is the concentration of the corresponding antibody in the raw serum (μ g/mL) calculated from the gray value of each serum dilution sample using the standard curve equation of examples 1, 2, 3. A, B and C are the concentrations of IgA, IgM and IgG of anti-RBD in the original serum in turn.
Detailed Description
One embodiment of the invention comprises a gyro micro sample centrifugation-paper-based microfluidic chip detection system which takes a pull-rope gyro with a gear device as a gyro centrifugation device.
In one embodiment, the string top has, for example, the following gear structure and string coefficient (table 2), and the gear device is as shown in fig. 3.
TABLE 2
| Gear number | Radius of gear (millimeter) | Number of teeth (pieces) | Weight of gear (gram) |
| A | 13 | 28 | 2.988 |
| |
4 | 8 | 0.106 |
| C | 7 | 13 | 0.391 |
| |
6 | 12 | 26.671 |
| Pull rope | Rope length (millimeter) | Rope weight (gram) | |
| 219 | 0.2957 |
The present invention includes a method for detecting antibodies (e.g., novel coronavirus antibodies) in serum using a paper-based microfluidic chip detection system, comprising:
a collection step: collecting fingertip blood of an object to be detected by using a disposable blood collecting 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 tube 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;
a centrifugation step: putting 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 layer serum sample (at least 1 microliter, enough for detection after 40 times dilution);
the preparation method of the paper chip comprises the following steps: designing a pattern which can realize the distribution of the hydrophobic area and the hydrophilic area according with the characteristics of the item 1 by using drawing software (such as Illustrator software), printing the pattern on a common filter paper substrate by adopting a paraffin printing process, and heating and baking after printing to obtain the paper chip with the water conveying functional channel. Cutting each part of the paper chip and then completing assembly;
an activation step: the detection part of the paper chip prepared in the above step was activated. Specifically, under the state that the movable valve is closed, a chitosan solution is dripped into a sample dripping area of a paper chip detection part, the mixture is dried at room temperature, a glutaraldehyde solution is dripped, after the mixture reacts for 1-5 hours at room temperature, the movable valve is rotated to an open state to connect a flow path, a washing solution (such as deionized water, physiological saline, PBS, PBST containing or not containing Tween substances and the like, preferably PBST containing 0.05% Tween 20) is dripped into the washing area, the permeated washing solution is removed from the back side (such as by suction) of the paper chip of the sample dripping area, the process is repeated for a plurality of times, and the drying at room temperature is carried out;
ELISA step: performing enzyme-linked immunosorbent assay (ELISA) using the above paper chip to detect the concentration of an antibody (for example, one or more selected from the group consisting of viral antibodies IgA, IgM, and IgG) in the serum of each test object;
and (3) an analysis step: analyzing the gray value of the paper chip detection part (including the sample dripping area), making a standard curve and a formula thereof by combining the concentration of the standard substance, and calculating the concentration of the antibody of the corresponding standard substance in the serum sample; or by comparing the color developed at the sample application zone to a reference color chart or a pre-prepared reference color chart.
Optionally, the force for pulling the top in the centrifugation step is 1-20 newtons, the top is pulled in a continuous pulling manner, and the rotation time is 3-20 minutes, preferably 10 minutes.
Optionally, in the activation step, specifically, 2 to 30 μ L of 0.25mg/mL chitosan solution is dripped, the mixture is dried at room temperature, 2 to 30 μ L of 2.5% glutaraldehyde solution is dripped, the mixture reacts at room temperature for 1 to 4 hours, preferably 2 hours, a washing channel is connected, the paper chip detection part is washed by deionized water for 3 to 5 times, and the mixture is dried at room temperature.
Optionally, the ELISA step specifically comprises the steps of:
(1) diluting the concentration of the RBD antigen to 2-10 mu g/mL by using PBS buffer solution with pH7.4, dripping 2-20 mu L of the RBD antigen into a sample dripping zone, 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 zone, and washing the paper chip detection part for 3-5 times;
(2) dripping 2-20 mu L of 0.5% -movable valve into the sample dripping area to be in an open state, reacting for 10-60 minutes at room temperature, and washing the paper chip detection part for 3-5 times by using 10-30 mu L of PBST solution; optionally, after drying, storing at the refrigeration temperature of a refrigerator (e.g., 0-4 ℃) for transport or storage until use;
(3) diluting a serum sample by using a PBS (phosphate buffer solution) solution in a ratio of 1: 40-1: 100 to obtain a serum dilution sample;
(4) diluting an antibody (for example, one or more of IgA, IgM and IgG of virus RBD antibodies) standard according to concentration gradients of 100ng/mL,50ng/mL,25ng/mL,12.5ng/mL,6.25ng/mL and 0ng/mL respectively to obtain a standard substance;
(5) dripping 2-20 mu L of serum into the sample dripping area to dilute the sample or the standard substance, reacting for 5-40 minutes at room temperature, rotating the movable valve to an open state, and washing the paper chip detection part for 3-5 times by using PBST solution such as 10-30 mu L;
(6) preparing 500-2000 ng/mL of a secondary antibody (for example, specifically recognizing IgA, IgM or IgG), dripping 2-20 mu L of the 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 for 3-5 times by using 10 mu L of PBST;
(7) when the secondary antibody is not connected with the HRP (for example, the detected virus antibody is IgG), 2-20 mu L of the HRP-connected tertiary antibody is dripped into the sample dripping zone, and after the reaction is carried out for 10-60 minutes, the sample is washed for 3-5 times by 10-30 mu L of PBST;
(8) 10 to 40. mu.L of a reaction substrate TMB solution was dropped onto each detection part, and the depth of blue color appearing in the sample dropping region was observed after 10 minutes.
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: in the embodiment, the washing operation is performed in the open state of the movable valve, the dropping position of the washing solution is the washing solution dropping hole of the washing area, the washing solution is sucked away from the back of the sample dropping area of the chip, and the movable valve is returned to the closed state after the washing is finished.
Example 1 detection of novel coronavirus antibody IgA
The subjects tested were from a group of healthy volunteers and a group of patients diagnosed with a new coronavirus infection, 35 individuals each.
The method comprises the following steps: collecting blood from finger of the test object with disposable blood collecting needle, and sucking 20 μ L of blood with 50mm long capillary;
step two: sealing one end of the capillary tube by using a sol gun (Shanghai Ganchun Utility Co., Ltd., backheating sol gun type No. RJQHAC50-GC113) and a matched hot melt adhesive rod (Shanghai Gangun Utility Co., Ltd.), and fixing the capillary tube on the upper surface of the gyroscope by using hot melt adhesive, wherein as shown in FIG. 2B, the unsealed end 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;
step three: placing the top into the pull rope device shown in FIG. 2A, pulling the rope with a force of 10N newtons to rotate for 10 minutes, removing the capillary tube, and sucking out the upper layer serum sample from the open end by using a Hamilton needle;
step four: the pattern of fig. 1A was designed using Illustrator software, and printed using ashless analysis filter paper as a base material using a paraffin printer (XEROX Phaser 8560DN), and heated and baked at 150 ℃ for 30 seconds to allow wax to penetrate into the paper, thereby obtaining a paper chip master having the distribution of hydrophobic and hydrophilic regions of the pattern of fig. 1A. Cutting the original sheet, punching, and fixing the detecting part and the movable valve together with the page buckle to obtain the state of 1B, C in FIG. 1;
step five: dripping 5 mu L of 0.25mg/mL chitosan solution into the sample dripping area of the paper chip, drying at room temperature, dripping 5 mu L of 2.5% glutaraldehyde solution, reacting at room temperature for 2 hours, rotating to the opening state of a movable valve, dripping 20 mu L of deionized water into a washing area, guiding liquid away from the back of the sample dripping area of the paper chip by using multiple layers of filter paper, discarding, repeatedly washing for three times, and drying at room temperature;
step six: enzyme-linked immunosorbent assay specifically comprises
(1) Human neocoronaviruse RBD antigen (for example, preparation method see Weihong Zeng, et al, 2021) was diluted with PBS buffer solution of ph7.4 to a concentration of 2 μ g/mL, and 5 μ L was added dropwise to the sample addition zone, and after reaction at room temperature for 30 minutes, the sample addition zone was washed 3 times with 10 μ L of PBST solution by the procedure described above;
(2) 5 mu L of PBS containing 0.5% BSA is dripped into the sample dripping area, after the reaction is carried out for 20 minutes at room temperature, 10 mu L of PBST solution is used for washing for 3 times;
(3) diluting each serum sample of two groups of detection objects by using PBS solution in a ratio of 1: 100 to serve as a serum dilution sample;
(4) diluting a virus antibody IgA standard (for example, the preparation method is shown in Weihong Zeng, et al, 2021) according to the concentration gradient of 100ng/mL,50ng/mL,25ng/mL,12.5ng/mL,6.25ng/mL and 0ng/mL respectively to obtain a series of gradient standards;
(5) 5 mu L of serum diluted sample or IgA standard substance is dripped into the sample dripping area, after the reaction is carried out for 10 minutes at room temperature, 10 mu L of PBST solution is used for washing for 3 times in the same way;
(6) preparing a specificity recognition IgA secondary antibody (HRP-Goat Anti-human IgA (HRP Conjugated affinity liquid Goat Anti-human IgA) manufacturer Boster biological, a product number BA1066) with a final concentration of 500ng/mL according to the instruction, dripping 5 mu L of the secondary antibody into a sample dripping area, reacting for 10 minutes at room temperature, and washing 3 times by using 10 mu L of PBST;
(7) mu.L of the reaction substrate TMB solution (Biyun day, cat # P0209) was added dropwise to the sample addition zone and reacted for 1 minute.
Step seven: and (3) photographing a sample dripping area of the detection part, analyzing the gray value corresponding to each sample, making a standard curve and a formula thereof by combining the concentration of the IgA standard substance, and calculating the concentration of the virus antibody IgA in the original serum sample.
The standard curve and its formula and the corresponding color development are shown in fig. 4A, and the calculated IgA antibody concentration in the original serum sample is shown in fig. 5A.
The results show that the paper-based microfluidic chip detection system of the present invention, when detecting a standard with a specific concentration, the obtained standard curve is good in linearity (R) as shown in FIG. 4A2> 0.975) indicating good response of the system used in the present invention.
As shown in fig. 5, when the upper limit of the concentration of healthy human serum IgA detected was set to 0.6 μ g/mL, and the upper and lower thresholds were set positive for viral infection and negative for viral infection, the detection rate of IgA in the new corona positive group was 97.1% (34/35), and no false positive occurred in the healthy group. Therefore, the method for detecting the specific antibody of the novel coronavirus RBD protein by ELISA by using the paper-based microfluid chip detection system is practical and effective.
The inventor carries out three times of repeatability tests under the same condition, and the error of the paper-based microfluid chip detection system between repeated measurements is smaller, and the error of a healthy subject is less than 0.05 mu g/mL; the error of a new crown infected person is less than 1.0 mu g/mL, and the repeatability is good.
Example 2 detection of novel coronavirus antibody IgM
In this example, the groups were divided into a healthy group (number: 35) and an infection-positive group (number: 35), and the sources of the two groups of test subjects were the same as in example 1.
The procedure of example 1 was repeated except that a virus antibody IgM standard (see, for example, Weihong Zeng, et al, 2021 for a preparation method) was used as the standard, and a secondary antibody was used (HRP-Goat Anti-Human IgM secondary antibody (HRP Conjugated affinity Goat Anti-Human IgM. mu. Chain) manufacturer Boster Biological product No. BA 1077). The standard curve and its formula and the corresponding color development are shown in FIG. 4B, and the calculated IgM antibody concentration in the original serum sample is shown in FIG. 5B.
The results show that the paper-based microfluidic chip detection system of the present invention, when detecting a standard with a specific concentration, the obtained standard curve is good in linearity (R) as shown in FIG. 4A2> 0.956), indicating a good response of the system used in the present invention.
As shown in fig. 5B, when the concentration threshold of the new coronavirus IgM antibody was set to 0.5 μ g/mL based on the detected upper limit of the healthy human serum IgM concentration, and when the virus infection was positive when the threshold was not lower than the threshold and negative when the virus infection was negative, the detection rate of the new corona infection positive group IgM was 91.4% (32/35). Therefore, the method for detecting the specific antibody of the novel coronavirus RBD protein by ELISA by using the paper-based microfluid chip detection system is practical and effective.
Example 3 detection of novel coronavirus antibody IgG
In this example, the groups were divided into a healthy group (number: 35) and an infection-positive group (number: 35), and the sources of the two groups of test subjects were the same as in example 1.
Wherein, as the standard, a virus Antibody IgG standard (for example, see Weihong Zeng, et al, 2021 for preparation method) is used, the Secondary Antibody is (Goat Anti-Human IgG Secondary Antibody (Goat Anti-Human IgG-Fc Secondary Antibody) manufacturer Sino Biological product number SSA015), and the following steps are added: after the secondary antibody reaction and washing, 5. mu.L of a HRP-linked tertiary antibody (HRP-conjugated Rabbit anti-Goat IgG, product number D110117) manufacturer organism was dropped to the sample dropping region, reacted for 20 minutes, and then washed 3 times with 10. mu.L of PBST.
Except for this, the same operation as in example 1 was performed. The standard curve and its formula and the corresponding visualization are shown in fig. 4C, and the calculated IgM antibody concentration in the original serum sample is shown in fig. 5C.
The results show that the paper-based microfluidic chip detection system of the present invention, when detecting a standard with a specific concentration, the obtained standard curve is good in linearity (R) as shown in FIG. 4C2> 0.966) indicating a good response of the system used in the present invention.
As shown in fig. 5C, when the concentration threshold of the new coronavirus IgG antibody was set to 1.25 μ g/mL based on the upper limit of the serum IgG concentration of the healthy human obtained by the detection, and when the virus infection was positive at a value higher than the threshold and negative at a value lower than the threshold, the detection rate of IgM in the new corona infection positive group was 85.7% (30/35), and no false positive occurred in the healthy group.
It is very noteworthy that since the system of the present invention allows for multiple channels, simultaneous determination of multiple analytes from a sample of the same subject can be achieved, and by combining the results of multiple analytes, the detection accuracy of the detection method can be improved.
The inventors comprehensively analyzed the results of the three antibodies of examples 1, 2 and 3, and found that if at least one of the results of the three antibodies IgA, IgM and IgG in the same sample is positive, the sample is determined to be positive for viral infection, and then: the detection rate of the paper-based microfluid chip detection system for the antibodies in the serum of the new crown infection sample is 99.9%, and the missed detection of infected people can be successfully avoided. Therefore, the method for carrying out ELISA detection by using the paper-based microfluid chip detection system is practical and effective, and is particularly suitable for detecting the specific antibody of the novel coronavirus RBD protein.
In addition, as can be seen from the color photographs in fig. 4A, B, and C, the colors between the wells of the respective concentration gradient standards appear blue with increasing depth, and the difference is significant. Therefore, from the practical point of view, it is considered that the color developing corresponding to the specific concentration of the specific analyte is printed as a color chart and attached to the system package box or the specification, so that the work of repeatedly performing the establishment and calculation of the standard curve can be saved, and the untrained personnel can intuitively obtain the result.
In addition, when the colorimetric card is not used, the detection result pictures can be collected and transmitted to a computer with common software for analysis in a data form, and the transmission can be completed instantly. Compared with the direct transportation of serum samples, the instantaneity of 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 manpower and material cost saving is realized.
Industrial applicability
The invention realizes the effect of simply implementing sample processing and sample analysis processes by utilizing the paper-based microfluid chip detection system comprising the centrifugal device, is particularly suitable for the condition that serum needs to be obtained from blood centrifugation, and is particularly suitable for the identification of antibody infection of various infectious pathogens (particularly novel coronavirus), the rapid screening of crowds and the like.
The system and the method have the characteristics of easily obtained detection tools, low cost, easiness in carrying, no need of expensive large-scale instruments, rapidness, convenience and difficulty in being limited by space in the detection process and the like, and are particularly suitable for being used in regions with undeveloped public health conditions so as to rapidly screen a large number of suspected samples. In addition, the paper-based microfluid chip detection system has good accuracy, and can simultaneously detect a plurality of different analytes in body fluid samples such as whole blood, thereby realizing comprehensive judgment of multiple indexes and further improving the detection accuracy.
Reference documents:
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 (10)
1. a detection system comprising a paper-based microfluidic chip device,
the paper-based microfluidic chip device includes a pivot, a detection portion, and a movable valve that is at least horizontally rotatable about the pivot to overlap with the detection portion,
the detection part and the movable valve respectively contain a paper-based micro-fluidic chip,
the detection part is provided with a hydrophobic area (1) and a hydrophilic area (1), the hydrophobic area (1) separates the hydrophilic area (1) into more than one group (preferably 1-10 groups) of sample dripping area and washing area,
the movable valve is provided with a flow channel part, a hydrophobic area (2) and a hydrophilic area (2) are arranged in the flow channel part, the hydrophobic area (2) divides the hydrophilic area (2) into flow channel areas with the number equal to that of the sample dripping areas (1),
when the movable valve rotates to overlap with the detection part, the hydrophobic area (2) and the hydrophilic area (2) on the flow channel part are correspondingly overlapped and contacted with the hydrophobic area (1) and the hydrophilic area (1) on the detection part respectively, a flow path between the sample dripping area and the washing area is communicated, and the movable valve is in an opening state; when the movable valve continues to rotate, the flow path is interrupted, at which point the movable valve is in a "closed" state,
the sample dropwise add the hole (preferably the circular port, more preferably the circular port of diameter in 4 ~ 6mm within range) for the cladding to the antigen of marker in the district, and the washing district contains the washing liquid dropwise add hole (preferably the circular port, more preferably the circular port of diameter in 4 ~ 6mm within range).
2. The detection system of claim 1, further comprising a gyroscopic centrifuge for centrifuging a micro sample,
the spinning top centrifugal device comprises a spinning top main body and a bracket containing a spinning top driving part,
the top body has a fixing member for fixing the sampling capillary on the upper surface, 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, the gear B is fixed on the gear C and rotates coaxially with the gear C,
when the top body is attached to the stand, the gear C engages with the gear D on the top body, and thereby the top body is rotated by the external force when the gear a is rotated.
3. The detection system according to claim 1 or 2, wherein a pull cord is connected to the gear A,
when the pulling rope is continuously pulled (preferably manually pulled), the pulling rope drives the gear A to rotate, so that the gyro body is driven to rotate.
4. The detection system according to claim 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 in a manner of passing through the center of a circle.
5. The detection system according to claim 3, wherein the fixing member is selected from a group consisting of a snap, a bayonet, a hot melt adhesive (preferably a hot melt adhesive), and the sampling capillary tube has an inner diameter of 0.5-2.0 mm and a length of 30-60 mm, and is made of a material selected from a group consisting of glass, plastic, polystyrene and other organic polymer materials, preferably made of glass.
6. A detection system according to any one of claims 1 to 5, wherein the pivot is selected from a snappable plastic ring, a metal shaft or a pop rivet, more preferably a snappable plastic ring,
the hydrophobic region is preferably formed by performing hydrophobic treatment with a hydrophobic substance (such as polystyrene, paraffin, and paraffin is preferred), the washing region optionally comprises a flow guide region connected with a washing liquid dropping hole, the washing region is preferably symmetrically distributed at two sides of the sample dropping region, and optionally, a color card for judging the detection result is contained in the paper-based microfluidic chip device.
7. The test system according to claim 6, wherein the marker is one or more of the human antibodies IgA, IgM and IgG that specifically bind to the RBD protein of the neo-coronavirus, and the paper-based microfluidic chip device is used to perform ELISA.
8. A detection system according to any one of claims 1 to 7, wherein the paper-based microfluidic chip device is prepared by:
in an assembled paper-based microfluid chip device, dripping a chitosan solution (preferably 5 muL 0.25mg/mL) into a detection part under the 'closed' state of a movable valve, airing at room temperature, dripping a glutaraldehyde solution (preferably 5 muL 2.5%), reacting at room temperature for 2 hours, rotating to the 'open' state of the movable valve, washing the detection part with a washing solution (preferably deionized water), and drying at room temperature;
5-10 μ L of 1-4 μ g/mL (preferably 5 μ L,2 μ g/mL) of an antigen (preferably human neocoronavirus RBD antigen) is dropped onto the detection part in a state that the movable valve is closed, and the reaction is carried out for 20-60 minutes at room temperature.
9. Use of a detection system according to any one of claims 1 to 8 for detecting antibodies in a sample, preferably whole blood, said antibodies being one or several (preferably 3) of the antibodies IgA, IgM and IgG that specifically bind to the RBD protein of neocoronavirus.
10. A method for detecting an antibody, which is carried out using the detection system according to any one of claims 1 to 8, comprising:
centrifuging 10-30 μ L of a sample (preferably whole blood) at an angular velocity of 1000-500 rad/s (preferably 700rad/s) for 1-20 minutes by using a gyroscopic centrifuge to obtain serum,
performing an ELISA on said paper-based microfluidic chip device to detect antibodies in serum,
the antibody is one or more (preferably 3) of antibodies IgA, IgM and IgG which are specifically combined with the RBD protein of the new coronavirus.
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