CN114660283B - Immunoassay plate type chip based on electrical acceleration and preparation method thereof - Google Patents
Immunoassay plate type chip based on electrical acceleration and preparation method thereof Download PDFInfo
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- CN114660283B CN114660283B CN202210566054.9A CN202210566054A CN114660283B CN 114660283 B CN114660283 B CN 114660283B CN 202210566054 A CN202210566054 A CN 202210566054A CN 114660283 B CN114660283 B CN 114660283B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Abstract
The invention relates to the technical field of molecular rapid detection, in particular to an immunoassay plate type chip based on electrical acceleration and a preparation method thereof, wherein the immunoassay plate type chip comprises the following steps: accelerating coating of the biological probe: applying an excitation signal to a plate type chip containing a probe solution to obtain a plate type chip with a probe combined on the surface; sample or standard electrical acceleration: applying an excitation signal to the plate chip combined with the probe to obtain a plate chip combined with a sample or a standard substance; antibodies or proteins accelerate electrically: applying an excitation signal to the plate-type chip to which the sample or the standard is bound, to obtain a plate-type chip to which the antibody or the protein is bound. The invention adopts the plate-type chip as a solid phase carrier to replace the traditional polystyrene material, and adopts the principle of electrically accelerating active particle control to complete the preparation of the biopolymer coated by the ELISA plate and the biomedical detection within 1-5 minutes, and the accuracy, the sensitivity and the specificity are not lower than the quality requirement and the standard of the traditional ELISA plate.
Description
Technical Field
The invention relates to the technical field of molecular rapid detection, in particular to an immunoassay plate type chip based on electrical acceleration and a preparation method thereof.
Background
Immunoassay (such as enzyme-linked immunoassay, chemiluminescence assay, etc.) refers to a qualitative and quantitative detection method in which a polymer such as an antibody or an antigen is bound to a solid-phase carrier such as polystyrene and the like and a specific specificity reaction is utilized, is the most widely applied technology in immunoassay, and has great significance in the fields of immunization, drug screening and identification, medical clinical diagnosis and treatment, and the like.
The existing enzyme label plate is used as a solid-phase polystyrene carrier, adopts a high polymer material surface treatment technology, plays an important role in the adsorption of antigens, antibodies or high polymers and reaction compounds thereof, and is an important device in enzyme-linked immunosorbent assay. The microplate is generally divided into 96-well plates, 48-well plates and 384-well plates, with 96-well plates being the most common.
At present, in the detection field of ELISA (enzyme-Linked immuno sorbent assay) ELISA plates based on solid phase carriers such as polystyrene and the like, such as luminescence detection, chemiluminescence detection and fluorescence detection, although sensitivity and specificity accuracy are high, passive adsorption of the solid phase polystyrene carrier to antigens, antibodies or macromolecules and reaction compounds thereof is subjected to incubation or incubation reaction, the combination of the antibodies or the macromolecules and the antibodies or the macromolecules takes a long time, which is dozens of minutes or even more than hours, and the efficiency in production, preparation and detection processes is very low.
Disclosure of Invention
In order to overcome the defect of long time consumption of the traditional ELISA plate detection, the invention aims to adopt a novel plate-type chip capable of being electrically accelerated as a solid phase carrier to replace the traditional polystyrene material, and the production preparation and the corresponding biomedical detection of the biomacromolecule such as antigen antibody coated by the ELISA plate are completed within a few minutes by adopting the electrically accelerated active particle control principle, so that the accuracy, the sensitivity and the specificity are not lower than the quality requirement and the standard of the traditional ELISA plate, and the preparation and detection efficiency is greatly improved.
The invention provides a preparation method of an immunoassay plate type chip based on electrical acceleration, which comprises the following steps:
s1, biological probe acceleration coating: applying an excitation signal to a plate chip containing a biological probe solution to obtain the plate chip with the surface combined with the biological probe, wherein the plate chip comprises an electrode plate which is activated and used for fixing the biological probe;
s2, sample or standard electrical acceleration: adding a sample or standard substance for binding the biological probes on the plate-type chip bound with the biological probes, and then applying an excitation signal on the plate-type chip to obtain the plate-type chip bound with the sample or the standard substance;
s3, antibody or protein electrically accelerates: adding an antibody or protein for binding the sample or the standard substance into the plate-type chip bound with the sample or the standard substance, and then applying an excitation signal to the plate-type chip to obtain the plate-type chip bound with the antibody or the protein;
s4, carrying out fluorescence detection or chemiluminescence detection on the plate-type chip combined with the antibody or the protein.
The invention can well fix the biological probe molecules on the plate-type chip, in particular to well fix the biological probe molecules on the electrode plate of the plate-type chip, so that the prepared chip can be used in a biological chip detection method based on electrical detection, and the development of the biological chip detection method based on the electrical detection is promoted.
The invention creatively adds a probe acceleration coating step on the basis of chemiluminescence detection, utilizes an electrode on a plate-type chip to generate a specific electric field, promotes the local temperature change of liquid under the action of the electric field, can also promote the movement of a biological probe molecule, realizes the fixation of the biological probe molecule on an electrode plate, further accelerates the process of immunodetection, simultaneously assists a sample or standard substance electrical acceleration step and an antibody or protein electrical step, realizes the rapid combination of the biological probe molecule and the sample or standard substance, the sample or standard substance and an antibody or protein (an enzyme-labeled antibody, a protein and an acridinium ester-labeled antibody, a protein), and can finish the detection in a short time.
In the probe accelerating coating step, an excitation signal is applied, an electrophoresis effect is generated to accelerate the speed of probe molecules moving towards the electrode plate direction, an electrothermal effect is generated to enable the chip to form obvious temperature gradient to cause local fluid flow, and the temperature effect and the dielectrophoresis effect are used together to accelerate the binding speed of the probe molecules on the fixed electrode plate. The excitation signal is an alternating current signal capable of promoting the movement of molecules in the chip, and the characteristic parameters include frequency, voltage and the like.
The invention adopts the plate-type chip capable of electrically accelerating as a novel solid phase carrier to replace polystyrene, and adopts electrically accelerating active particle control technology (such as dielectrophoresis, alternating current electrothermal technology, alternating current electroosmosis technology and the like) to replace incubation or incubation reaction of passive adsorption in the method by setting a specific preparation method, so that the reaction time is greatly shortened, the preparation efficiency and the detection efficiency are greatly improved from tens of minutes, hours to minutes, and the invention also has the advantages of easy operation, high sensitivity, specificity, accuracy and the like.
As a preferred embodiment of the preparation method of the immunoassay plate chip based on electrical acceleration, in the steps S1-S3, the excitation signal is at a voltage of 1V-20V and a frequency of 100Hz-10 MHz.
In steps S1-S3 of the present invention, the excitation signal with the above voltage and frequency can achieve the effect of rapid reaction and combination of the biological probe molecule and the sample or the standard substance, and the sample or the standard substance and the antibody or the protein. In addition, the detection sensitivity can be improved by adopting the parameters of the excitation signal of the invention, and the sensitivity is kept at 0.10-0.15 ng/ml.
As a preferred embodiment of the preparation method of the immunoassay plate chip based on the electrical acceleration, the application time of the excitation signal is 0.1-5 min.
The application time of the excitation signal can ensure that the biological probe molecules are fully combined with the sample or the standard substance, and the sample or the standard substance is fully combined with the antibody or the protein, so that the detection effect is better improved.
As a preferred embodiment of the preparation method of the immunoassay plate type chip based on the electrical acceleration, the concentration of the biological probe is 2-20 mug/mL.
When the concentration of the biological probe is in the range of 2-20 mug/mL, the signal-to-noise ratio of detection can be improved by adopting the electrically accelerated active particle control technology.
As a preferred embodiment of the preparation method of the electrically accelerated immunoassay plate chip of the present invention, the types of the biological probes include one of polymers, proteins, DNA, RNA, antigens, and antibodies.
As a preferred embodiment of the preparation method of the immunoassay plate chip based on electrical acceleration, the biological probe acceleration coating step comprises preparing a biological probe solution using 0.01-10 x phosphate buffer as a solvent.
As a preferred embodiment of the preparation method of the immunoassay plate-type chip based on electrical acceleration, the preparation method sequentially comprises a chip activation film-forming step before a biological probe acceleration coating step; in the chip activation film-forming step, the chip electrode sheet is plasma-cleaned, and APTES is deposited on the surface of the chip by using argon carrier gas to improve the probe bonding efficiency and the effective period.
The invention also provides a chip prepared by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the plate-type chip which can be electrically accelerated as a novel solid phase carrier to replace polystyrene, adopts the electrically accelerated active particle control technology (such as dielectrophoresis, alternating current electric heating, alternating current electroosmosis and the like) to replace the incubation or incubation reaction of passive adsorption, and finishes the production preparation of the enzyme label plate coated with biological macromolecules such as antigen antibody and the corresponding biomedical detection within a few minutes (within 1-5 minutes), the accuracy, the sensitivity and the specificity are not lower than the quality requirement and the standard of the traditional ELISA plate, and the preparation and the detection efficiency are greatly improved.
Drawings
FIG. 1 is a detail view of a plate chip;
FIG. 2 is a schematic view of a plate chip;
FIG. 3 is a graph of a standard curve for electrical accelerated testing in accordance with the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In an enzyme-linked immunoassay, antigens, antibodies and other biomolecules are adsorbed to the surface of a carrier through a plurality of mechanisms, including passive adsorption through hydrophobic bonds, hydrogen bonds/ionic bonds and other functional bonds, covalent bonding of other active groups such as amino groups and carbon groups can be introduced, and the bonding strength is improved through hydrophilic bonds after surface modification. In any way, the purity, concentration and proportion of the antigen, antibody, labeled antibody or antigen involved in the immunological reaction, and the conditions such as the type, concentration and ionic strength of the buffer solution, pH value, reaction temperature, time and the like play a key role in the efficiency of finally completing the combination, especially the reaction temperature and the incubation time, and if the reaction balance is completely achieved, the reaction can be completed within dozens of minutes or even hours.
The invention adopts the chip material capable of electrically accelerating as a novel solid phase carrier to replace polystyrene, and adopts the electrically accelerating active particle control technology (such as dielectrophoresis, alternating current electrothermal technology, alternating current electroosmosis technology and the like) to replace the incubation or incubation reaction of passive adsorption through the designed high-flux plate-type chip, the chip accelerator, the related clamp and the matched device, so that the reaction time is greatly shortened, the reaction time is from tens of minutes, hours to a few minutes, the preparation efficiency and the detection efficiency are greatly improved, and the invention also has the advantages of easy operation, high sensitivity, specificity, accuracy and the like.
In the following examples and comparative examples, the experimental methods used were conventional ones unless otherwise specified, and the materials, reagents and the like used were commercially available ones unless otherwise specified.
Example 1
In this example, the chip coated with the antibody is processed on the basis of the chip on which the coated molecule (antibody, antigen or other affinity molecule) is not immobilized.
The chip used in this example is referred to in our prior patent CN104965081B (mobile device based antibody antigen detection method), in which: an antibody antigen detection test system comprises at least one reaction unit, wherein the reaction unit comprises a reaction cavity with an opening at the top, the bottom of the reaction cavity is provided with a detection plate, at least one pair of electrode plates are paved on the detection plate, and the wiring terminals of the electrode plates penetrate through and are fixed on a box body of the reaction cavity; the detection plate is also fixed with a target antibody or a corresponding antigen or antibody of the antigen. In this embodiment, the chip specifically refers to the reaction unit in CN104965081B, and the structure of the detection plate with the electrode pads laid thereon can be referred to the paper published earlier by the inventor (Development of an AC electronics-based immunoassay system for on-site diagnostics of electrical diseases, Xiaozhu Liu, Sensors and Actuators a, 171 (2011) 406. 413, fig. 3 (b)). Usually, the reaction chamber and the detection plate are made of insulating materials (the surface is SiO) 2 Glass, in this example SiO 2 ) The electrode plate is made of metal (aluminum, gold or copper, and is made of aluminum), and is very easy to be corroded gradually in the processing process or the storage process after the processing is finished, so that the whole chip is invalid.
The scheme is improved on the process of the method for manufacturing the finished chip from the chip.
1. Pretreatment:
observing the surface of the chip without coating by a metallographic microscope under a 10-fold ocular lens, determining whether the electrode plates of the chip have broken strips and continuous strips (the electrode plates are parallel to each other) and other adhered impurities, and selecting the chip without the broken strips and the adhered impurities to continue a subsequent experiment (called as primary microscopic examination). The judgment basis of the adhered impurities is as follows: spots, particles, dirt or dust particles with the particle diameter or the length larger than 0.5 mu m do not exist at the interdigital part of the electrode plates (namely gaps among the electrode plates), and if the spots, the particles, the dirt or the dust particles are judged to be unqualified.
2. Chip cleaning
The chip is placed in a beaker filled with absolute ethyl alcohol, cleaned for 10 minutes by an ultrasonic cleaner, rinsed for 10 seconds by ultrapure water and finally dried by nitrogen.
3. Chip activation film formation
The Dinner plasma equipment is used for APTES deposition film formation. The sample is initially subjected to an activation pretreatment (argon flow: 300sccm, 10-30% of nominal power, pressure 0.10-0.3mbar, duration 5-20 mins). The gas mixture then contains argon Ar and a carrier gas Ar carrying APTES injected into the reaction chamber. The APTES film-forming deposition is carried out by using 10 to 30 percent of rated power and gas pressure. The pressure is 0.10-0.3mbar and the duration is 5-20 mins. Observing the surface of each film-formed chip by using a metallographic microscope under a 10-fold eyepiece, photographing to record the surface condition of each chip, and if the surface is damaged or polluted, abandoning the chip (called as secondary microscopic examination), wherein the judging method is the same as that of the first microscopic examination.
The plate-type chip structure comprises a chip, a silica gel pad, a gasket hole and a chip upper cover, wherein the silica gel pad is attached to an interdigital part of the chip, and the gasket hole is arranged right above the interdigital part and is covered with the chip upper cover (refer to fig. 1-2).
4. Probe acceleration coating
Adding 10-20 μ L (preferably 10 μ L) and 20 μ g/mL (2-20 μ g/mL) of cloth disease antigen (bioprobe comprises one of macromolecule, protein, DNA, RNA, antigen, and antibody) into each well of the plate-type chip, wherein the solvent is 1 XPBS (0.01 XPBS-10 XPBS), placing the plate-type chip into an accelerator acceleration tank, and accelerating for 1min (0.1-5 min) under the conditions of 3V (1V-20V) and 100kHz (100 Hz-10 MHz) to obtain a plate-type chip combined with the probe; wherein the above probe was dispersed and dissolved using 1 × PBS (phosphate buffered saline) as a solvent.
5. Cleaning, sealing and cleaning after sealing after coating: taking out the plate-type chip from the acceleration tank, washing each hole for 10 seconds by PBS (phosphate buffer solution), washing for 2 times, and drying by nitrogen in the last time; adding 10-20 mu L of SuperBlock dissolved by PBS into each hole, and sealing for 0.5-1 hour at room temperature; each chip was rinsed with PBS for 10 seconds, 2 times, and finally blown dry with nitrogen.
6. Sample or standard electrical acceleration: 0.1-1000ng/ml of mouse antibody (1% BSA-PBS) was added to each well of the plate-type chip obtained in step 8, and accelerated for 1min (0.1-5 min) under the conditions of 3V (1V-20V) and 100kHz (100 Hz-10 MHz) using an accelerator.
7. Cleaning: each well was rinsed with PBS for 10 seconds, 2 times, and the last time blown dry with nitrogen.
8. Electrical acceleration of antibodies or proteins: 10-20. mu.L of a secondary HRP-labeled goat anti-mouse IgG antibody (diluted 1:1000 in 1% BSA-PBS) was added to each well of the plate-type chip obtained in step 11, and accelerated by an accelerator at 3V (1V-20V) and 100kHz (100 Hz-10 MHz) for 1min (0.1-5 min).
9. Cleaning: each well was rinsed twice with 0.1% PBST (Tween-20 in 1 XPBS) for 10 seconds, and finally blown dry with nitrogen.
10. And (3) detection: add 10-20. mu.L of luminescent or chemiluminescent substrate to each well and read the value of each well with a microplate reader.
Examples 2 to 4 were substantially the same as example 1 except that the differences were as shown in Table 1.
TABLE 1
| Group of | Probe needle | Detecting target | Sensitivity (ng/ml) |
| Example 1 | Brucellosis antigen | Brucellosis antibody | 0.10-0.15 |
| Example 2 | Myocardial infarction antibody | Myocardial infarction antigen | 0.06-0.07 |
| Example 3 | Carrier proteins | Aflatoxin | 0.01-0.02 |
| Example 4 | Aptamer DNA | Viral DNA | 0.004-0.005 |
Examples 5-6 and comparative examples 1-4 are essentially the same as example 1, except that the electrical acceleration parameters of the probe acceleration coating step are different, as shown in table 2.
TABLE 2
| Group of | Electrical acceleration parameter | Sensitivity (ng/ml) | CV(%) |
| Example 1 | 3V，100KHz | 0.10-0.15 | 3.7 |
| Example 5 | 1V，5KHz | 0.10-0.15 | 4.9 |
| Example 6 | 20V，500KHz | 0.10-0.15 | 4.5 |
| Comparative example 1 | 3V，50Hz | 0.27 | 5.8 |
| Comparative example 2 | 3V，12MHz | 0.58 | 6.7 |
| Comparative example 3 | 0.5V，100KHz | 0.49 | 8.5 |
| Comparative example 4 | 30V，100KHz | 0.36 | 7.8 |
Comparative examples 5 to 6 are substantially the same as example 1 except for the acceleration time; comparative example 7 is a conventional incubation coating, as shown in table 3.
TABLE 3
| Group of | Coating mode and time | Coating temperature (. degree. C.) | Coating effect% | Sensitivity (ng/ml) |
| Example 1 | Electric acceleration (1 min) | At normal temperature | Greater than 90 | 0.10-0.15 |
| Comparative example 5 | Electric accelerator (5 s) | At normal temperature | 80 | 0.50 |
| Comparative example 6 | 150s incubation | At |
50 | 1.20 |
| Comparative example 7 | 120min (or 720 min) incubation | 37 deg.C (or 4 deg.C) | Greater than 90 | 0.10-0.15 |
The standard curve of the test using the above method (example 1) is shown in fig. 3 when the concentration of bremia antigen is 2-20 μ g/mL, the signal-to-noise ratio of the test is highest when the concentration of bremia antigen is 20 μ g/mL, and the effect of the present invention using the electrical acceleration method is similar to that of the conventional incubation test under the condition that the test time is significantly shortened, as shown in table 4.
TABLE 4
| Group of | Detection time (minutes) | Signal-to-noise ratio (S/N) | Sensitivity (ng/ml) |
| Example 1 | 5 | 210-220 | 0.10-0.15 |
| |
150 | 210-220 | 0.10-0.15 |
Test examples
Negative and positive samples of brucellosis obtained from university of Chongqing and Chongqing City animal epidemic disease prevention control center after sterilization were tested, and the statistical values of the test results as negative or positive are shown in Table 5. The electric acceleration method of the invention can achieve the effect consistent with the traditional incubation detection on the premise of obviously shortening the detection time.
TABLE 5
| Group of | Negative sample detection | Positive sample detection | Total percent line (%) |
| Example 1 | 70/70 | 100/100 | 100 |
| Traditional incubation detection | 70/70 | 100/100 | 100 |
The invention adopts the plate-type chip which can be electrically accelerated as a novel solid phase carrier to replace polystyrene, adopts the electrically accelerated active particle control technology (such as dielectrophoresis, alternating current electric heating, alternating current electroosmosis and the like) to replace the incubation or incubation reaction of passive adsorption, and finishes the production preparation of the enzyme label plate coated with biological macromolecules such as antigen antibody and the corresponding biomedical detection within a few minutes (within 1-5 minutes), the accuracy, the sensitivity and the specificity are not lower than the quality requirement and the standard of the traditional ELISA plate, and the preparation and the detection efficiency are greatly improved.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and the protection scope of the technical solutions of the present invention.
Claims (2)
1. A preparation method of an immunoassay plate type chip based on electrical acceleration is characterized by comprising the following steps:
s1, biological probe acceleration coating: applying an excitation signal to a plate-type chip containing a biological probe solution to obtain the plate-type chip with the surface combined with the biological probe, wherein the plate-type chip comprises an electrode slice which is activated and used for fixing the biological probe;
s2, sample or standard electrical acceleration: adding a sample or a standard substance for binding the biological probes on the plate-type chip combined with the biological probes, and then applying an excitation signal on the plate-type chip to obtain the plate-type chip combined with the sample or the standard substance;
s3, enzyme-labeled antibody or protein electrically accelerates: adding an enzyme-labeled antibody or protein for binding the sample or the standard substance into the plate-type chip combined with the sample or the standard substance, and then applying an excitation signal to the plate-type chip to obtain the plate-type chip combined with the antibody or the protein;
s4, carrying out fluorescence detection or chemiluminescence detection on the plate-type chip combined with the enzyme-labeled antibody or protein;
in steps S1-S3, the excitation signal is at a voltage of 3V and a frequency of 100 kHz; the application time of the excitation signal is 1 min; the biological probe accelerating coating step comprises the steps of using 0.01-10 times phosphate buffer solution as a solvent to prepare a biological probe solution;
the concentration of the biological probe is 1-20 mug/mL;
the biological probe comprises one of DNA, RNA, antigen and antibody;
the method comprises the steps of sequentially activating a chip to form a film before the step of accelerating coating of the biological probe; in the step of chip activation film formation, cleaning the electrode plate of the chip by plasma; APTES was deposited on the chip surface with an argon carrier gas.
2. A chip produced by the production method according to claim 1.
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