CN109239135B - Portable biological immunoassay method constructed based on flexible air pressure sensor - Google Patents
Portable biological immunoassay method constructed based on flexible air pressure sensor Download PDFInfo
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- CN109239135B CN109239135B CN201811241023.6A CN201811241023A CN109239135B CN 109239135 B CN109239135 B CN 109239135B CN 201811241023 A CN201811241023 A CN 201811241023A CN 109239135 B CN109239135 B CN 109239135B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
- G01N27/06—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a liquid
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
Abstract
The invention discloses a portable biological immunoassay method constructed based on flexible air pressure sensor, wherein the prepared flexible pressure sensor is sensitive to air pressure change and is used for monitoring biological immune reaction to realize sensitive and portable detection of various antibodies. According to the invention, the paper-based electrode modified by the carbon nano tube is used as a flexible substrate to prepare the flexible pressure sensor, and then the immunoreaction is combined to realize the specific recognition of the target object. After the hydrogen peroxide is injected, the platinum nanoparticles introduced by the nano probe can efficiently catalyze the decomposition of the hydrogen peroxide to generate oxygen, so that the air pressure in the closed detection system is increased. The increased air pressure causes the flexible pressure sensor to deform, thereby decreasing its resistance. The change can be monitored by a digital multimeter by detecting the resistance change of the digital multimeter, so that the quantitative detection of the target object is realized. The method has the advantages of simple equipment, portability and simple operation, and provides a simple, sensitive and stable method for portable detection.
Description
Technical Field
The invention relates to a portable biological immunoassay method based on flexible air pressure sensing construction, and belongs to the technical field of construction of sensing instruments and biochemical analysis.
Background
With the development of society, environmental monitoring, food safety and disease diagnosis are more and more emphasized by people, and a plurality of detection methods based on optics, chromatography, mass spectrometry and electrochemistry are established, and have the advantages of accuracy, sensitivity, stability and the like. However, the expensive equipment and the complex operation limit the further application of the equipment in the aspects of rapid detection, daily life detection and the like, especially in remote areas. Therefore, it is important to design a stable and practical detection method with simple equipment and easy operation.
Air pressure, as a traditional physical phenomenon, has applications in many aspects of life. During chemical reactions, there are many chemical reactions that can generate large amounts of gas to increase the pressure of the gas in the container. Among these reactions, the reaction of decomposing hydrogen peroxide into oxygen has a high gas generation rate and is non-toxic and harmless. Meanwhile, the reaction rate can be greatly improved by catalysts such as catalase, platinum nanoparticles and the like, and the method is very suitable for being used as a reaction for detecting signals. In many existing portable bioassays that use air pressure as the detection signal, conventional barometers and capillary columns are commonly used as signal readout devices. In recent years, flexible devices have been studied in electronic skins, electronic textiles, and stress detection, because of their sensitive response to external stress. In order to improve the sensitivity of the portable sensor, the signal generation source and the signal detection device are two of the most important. Based on the above, the sensitivity and the accuracy of detection can be effectively improved by constructing a sensitive flexible air pressure sensor and applying the flexible air pressure sensor to an air pressure type portable immune biosensor.
Disclosure of Invention
The invention aims to provide a preparation method of a flexible pressure sensor, and a portable air pressure immunosensor is constructed by combining antigen-antibody immunoreaction. The technical principle is that platinum nanoparticles are introduced to an immunity probe to catalyze injected hydrogen peroxide to generate oxygen, the generated oxygen can change the air pressure in a detector, and the generated air pressure can be sensitively detected by a flexible pressure sensor. When no target exists, the air pressure is increased a little, the flexible air pressure sensor is deformed little, and the resistance change is small; when the target exists, the air pressure is increased greatly, the flexible air pressure sensor is enlarged, and the resistance change is large. The change of the resistance is in positive correlation with the concentration of the target object within a certain range, and the portable quantitative detection can be realized through a digital multimeter.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a portable biological immunoassay method based on flexible air pressure sensing construction comprises the following steps:
(1) and (3) modifying the paper-based electrode: cutting qualitative filter paper into a certain shape, soaking the qualitative filter paper in 1-5wt% of carbon nanotube solution (containing 1-10wt% of Pluronic F-127 surfactant), drying, and repeating for several times until the resistance reaches 20-70 k omega;
(2) preparing a flexible pressure sensor: connecting the prepared paper-based electrode with copper conductive adhesive by using silver conductive adhesive, and sticking one surface of the paper-based electrode with a BOPP adhesive tape; combining two prepared paper-based electrode surfaces in a face-to-face manner, and sealing an outer layer by using a layer of polydimethylsiloxane PDMS;
(3) constructing an air pressure type portable immunoassay device: the prepared pressure sensor is sealed in a centrifuge tube by PDMS, the tail end of the centrifuge tube is connected with a detection cell by a conduit, a filtering device is arranged in the middle of the conduit to prevent liquid from flowing in, and all interfaces are sealed by PDMS.
(4) Constructing a nano probe: 10 mL of a chloroplatinic acid solution (0.1-5 mM) and 1 mL of an ascorbic acid solution (0.1-5M) were mixed, heated at 80 ℃ for 30 min, and stored at 4 ℃ until use. 5 mL of the prepared platinum nanoparticle solution was adjusted to pH 9.0 with sodium carbonate, and a target antibody (10-100. mu.L) was added at a concentration of 0.5 mg/mL, followed by incubation at 4 ℃ overnight. Finally, the above solution was centrifuged at 4 ℃ and 14000 g of centrifugal force for 20 minutes, and the pellet obtained by the centrifugation was re-dispersed in 3 mL of PBS (pH =7.4) containing 1.0 wt% BSA, 0.5 wt% Tween 20 and 0.1 wt% sodium azide and stored at 4 ℃ until use.
(5) Construction of immune response: firstly, adding a capture antibody (10-80 mu L) into a 96-hole high-affinity plate for overnight sealing, then sequentially adding a target detection object (10-80 mu L) and a nano probe (10-80 mu L) to form an antibody-antigen-antibody sandwich type immune complex for incubation for 40 min, washing by using a phosphoric acid buffer solution, and then adding 100 mu L of hydrogen peroxide for reaction;
(6) and (3) output electric signal detection: the target concentration was quantified by reading the resistance signal change with a digital multimeter.
More specifically, the present invention is to provide a novel,
the shape specification of the filter paper cut in the step (1) is shown in fig. 2.
The specification of the flexible pressure sensor in the step (2) is 1 multiplied by 1.5 cm2The specific preparation process is shown in fig. 3, the real graph of the prepared flexible pressure sensor is shown as A in fig. 4, and the response range to air pressure is shown as fig. 5 and is 0.08-50 kPa.
And (4) sealing the outer side of the special detection system in the step (3) by PDMS to increase air tightness, and connecting the special detection system with a digital multimeter, wherein the specific shape is shown as B in figure 4.
The hydrogen peroxide concentration in step (4) was 30wt%, and the reaction time was 270 s.
And (5) the resistance signal is delta R (initial resistance minus ending resistance).
The invention has the following advantages:
(1) the invention has the advantages of easily obtained raw materials, simple preparation process and low cost.
(2) The flexible pressure sensor constructed by the invention is sensitive to air pressure, short in response time and wide in linear range, is used for immunoreaction detection, greatly simplifies a detection device, is simple to operate and sensitive in response, and is suitable for portable detection.
(3) The invention adopts air pressure as a detection signal, which can be greatly changed by increasing a small amount of air, and is beneficial to improving the detection sensitivity.
(4) The invention adopts an immune touch method and platinum nanoparticle catalysis as a signal amplification mechanism, and can greatly improve the accuracy and sensitivity of a detection result.
Drawings
FIG. 1 is a schematic diagram of a portable biological immunoassay method based on flexible air pressure sensing construction;
FIG. 2 is a specification for filter paper cutting;
FIG. 3 is a flexible pressure sensor assembly process;
FIG. 4 is a diagram of a flexible pressure sensor and a detection device;
fig. 5 is a graph showing the response range of the flexible pressure sensor to air pressure.
FIG. 6 is a standard working curve for carcinoembryonic antigen as performed in example 1.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific implementation examples, but the scope of the present invention is not limited thereby.
Example 1
1. Modification of paper-based electrodes
Qualitative filter paper was cut to the specifications shown in fig. 2 and soaked in a 2wt% carbon nanotube solution (containing 2wt% Pluronic F-127 surfactant) and then dried, repeated several times, until the resistance reached 60 k Ω.
2. Preparation of flexible pressure sensor
Connecting the prepared paper-based electrode with copper conductive adhesive by using silver conductive adhesive, and sticking one surface of the paper-based electrode with a BOPP adhesive tape; combining the two prepared paper-based electrode faces, sealing the outer layer with a layer of PDMS, wherein the specific assembly process is shown in figure 3, and the assembly process is shown as A in figure 4.
3. Construction of pneumatic portable immunoassay device
The prepared pressure sensor is sealed in a centrifuge tube by PDMS, the tail end of the centrifuge tube is connected with a detection cell by a conduit, and a filtering device is arranged in the middle of the conduit to prevent liquid from flowing in. The whole device structure is sealed by PDMS to ensure air tightness, and the physical diagram is shown as B in figure 4.
4. Preparation of nanoprobes
10 mL of a chloroplatinic acid solution (1 mM) and 1 mL of an ascorbic acid solution (0.4M) were mixed, heated at 80 ℃ for 30 min, and stored at 4 ℃ until use. 5 mL of the prepared platinum nanoparticle solution was adjusted to pH 9.0 with sodium carbonate, and 50. mu.L of carcinoembryonic antigen antibody having a concentration of 0.5 mg/mL was added thereto, followed by incubation at 4 ℃ overnight. Finally, the above solution was centrifuged at 4 ℃ and 14000 g of centrifugal force for 20 minutes, and the pellet obtained by the centrifugation was re-dispersed in 3 mL of PBS (pH =7.4) containing 1.0 wt% BSA, 0.5 wt% Tween 20 and 0.1 wt% sodium azide and stored at 4 ℃ until use.
5. Detection of target carcinoembryonic antigen CEA (carcinoembryonic antigen as a model target analyte in this example)
FIG. 1 is a schematic diagram of the process of the analysis method of the present invention. First, 50. mu.L of CEA antibody at a concentration of 10. mu.g/mL was added to a 96-well high affinity plate, incubated overnight at 4 ℃, and blocked for 1 hour by adding 300. mu.L of BSA at a concentration of 1.0 wt%. And after washing with a PBS solution, sequentially adding 50 muL of CEA standard sample and 50 muL of nanoprobe, and reacting for 40 min respectively. After removing the supernatant, 100 muL of H with the concentration of 30wt% is added2O2Reaction 270 s, reading the resistance change of the digital multimeter for quantification. The standard working curve of the resistance change to CEA obtained by the result is shown in FIG. 6, the linear range is 0.5-60 ng/mL, and the detection limit is 0.167 ng/mL.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
1. A portable biological immunoassay method constructed based on flexible air pressure sensing is characterized in that: the method comprises the following steps:
(1) preparing a paper-based electrode modified by carbon nano tubes;
(2) preparing a flexible pressure sensor and constructing an immunoassay device;
(3) synthesizing platinum nanoparticles and preparing a nano probe marked by the platinum nanoparticles;
(4) forming an antibody-antigen-nano probe immune sandwich structure on a high-affinity 96-well enzyme label plate;
(5) the hydrogen peroxide added is catalyzed by the marked platinum nanoparticles to generate oxygen, so that the pressure in the sealed immunoassay device is increased, the conductivity of the flexible pressure sensor is changed, and the purpose of portable immunoassay is realized by using a digital multimeter as a reading device;
the diameter of the carbon nano tube in the step (1) is 10-20 nm, and the length is 1-2 μm; the paper-based electrode is modified by adopting a direct soaking-drying method, soaked in 1-5wt% of carbon nanotube solution containing 1-10wt% of Pluronic F-127 surfactant, and then dried until the resistance reaches 20-70 k omega;
the flexible pressure sensor in the step (2) is assembled by the prepared paper-based electrode, copper conductive adhesive and polydimethylsiloxane PDMS; the immunoassay device is assembled by a detection pool, an air duct, a flexible pressure sensor and a digital multimeter.
2. The portable biological immunoassay method based on the flexible air pressure sensing construction of claim 1, which is characterized in that: in the step (3), the platinum nano-particles are reduced by ascorbic acid, and the particle size is 35 nm; the nano-probe is prepared by adsorbing an antibody on platinum nano-particles by virtue of static electricity.
3. The portable biological immunoassay method based on the flexible air pressure sensing construction of claim 1, which is characterized in that: the high-affinity 96-hole enzyme label plate in the step (4) is a detection pool in an immunoassay device; the detection and recognition process is realized by forming an antibody-antigen-nano probe immune sandwich structure compound.
4. The portable biological immunoassay method based on the flexible air pressure sensing construction of claim 1, which is characterized in that: the process of the step (4) is specifically as follows: 10-80 mul of capture antibody is added into a high-affinity 96-well enzyme label plate for overnight sealing, and then 10-80 mul of target detection object and 10-80 mul of nano probe are sequentially added to form an antibody-antigen-nano probe immune sandwich structure compound for incubation for 40 min.
5. The portable biological immunoassay method based on the flexible air pressure sensing construction of claim 1, which is characterized in that: the concentration of the hydrogen peroxide in the step (5) is 30wt%, and the catalytic reaction time is 270 s; the measured resistance change value of the flexible pressure sensor decreases as the concentration of the target substance increases.
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| CN106441646A (en) * | 2016-10-27 | 2017-02-22 | 江苏科技大学 | Flexible pressure sensor and preparation method thereof |
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| CN107898463A (en) * | 2017-11-13 | 2018-04-13 | 深圳大学 | A kind of flexible electronic pressure sensor and preparation method thereof |
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