CN112362711B - Microorganism detection device and detection method - Google Patents
Microorganism detection device and detection method Download PDFInfo
- Publication number
- CN112362711B CN112362711B CN202011251977.2A CN202011251977A CN112362711B CN 112362711 B CN112362711 B CN 112362711B CN 202011251977 A CN202011251977 A CN 202011251977A CN 112362711 B CN112362711 B CN 112362711B
- Authority
- CN
- China
- Prior art keywords
- chamber
- microorganism
- membrane
- buffer
- sensitive element
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- G01N27/307—Disposable laminated or multilayered electrodes
-
- 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
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
-
- 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
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- 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
- G01N27/31—Half-cells with permeable membranes, e.g. semi-porous or perm-selective membranes
-
- 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
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- 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
- G01N27/333—Ion-selective electrodes or membranes
- G01N27/3335—Ion-selective electrodes or membranes the membrane containing at least one organic component
-
- 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
- G01N27/36—Glass electrodes
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L2200/0668—Trapping microscopic beads
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
Abstract
The invention discloses a microorganism detection device and a detection method, the device comprises a cover plate, a first substrate, a second substrate and a bottom plate which are fixedly connected from top to bottom in sequence, wherein a sample inlet is formed at one end of the cover plate; the first substrate is provided with a first microfluidic channel and a microbial chamber which are communicated, and the first microfluidic channel is communicated with the sample inlet; a separation chamber, a micro-fluid channel II and a reaction chamber which are communicated are arranged on the substrate II, a microorganism separation film is fixedly connected between the microorganism chamber and the separation chamber, and a microelectrode I is fixedly connected in the reaction chamber; the bottom plate is provided with a buffer chamber, a sensitive element is fixedly connected between the buffer chamber and the reaction chamber, a second microelectrode is fixedly connected in the buffer chamber, a buffer solution inlet and a buffer solution outlet are respectively arranged on two sides of the buffer chamber, and the buffer solution inlet is arranged below the reaction chamber. The device can effectively solve the problems of low sensitivity, complex operation and time consumption of the existing device.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a microorganism detection device and a detection method.
Background
Microorganisms are widely found in nature, and pathogenic microorganisms refer to the general name of microorganisms causing infection, also called pathogens, mainly including viruses, bacteria, fungi, mycoplasma, prions and the like, wherein the viruses and the bacteria have the greatest threat to human beings. The safety events such as food and water sources caused by pathogenic bacteria seriously threaten the life health of human beings, and 54.3 percent of sudden infectious diseases in the world are caused by bacteria or rickettsia.
The detection method of microorganisms adopted in the prior art is mainly a microorganism culture method, and although various microorganism culture devices exist in the market at present, the devices need long culture time and are severely restricted in the field of rapid detection application.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a microorganism detection device and a detection method, which can effectively solve the problems of low sensitivity, complex operation and time consumption of the existing device.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a microorganism detection device comprises a cover plate, a first substrate, a second substrate and a bottom plate which are fixedly connected from top to bottom in sequence, wherein a sample inlet is formed in one end of the cover plate;
the first substrate is provided with a first microfluidic channel and a microbial chamber which are communicated, and the first microfluidic channel is communicated with the sample inlet;
the substrate II is provided with a separation chamber, a micro-fluid channel II and a reaction chamber which are communicated, a microorganism separation film is fixedly connected between the microorganism chamber and the separation chamber, a microelectrode I is fixedly connected in the reaction chamber, and a sample outlet is arranged in the reaction chamber;
the bottom plate is provided with a buffer chamber, a sensitive element is fixedly connected between the buffer chamber and the reaction chamber, a second microelectrode is fixedly connected in the buffer chamber, a buffer solution inlet and a buffer solution outlet are respectively arranged on two sides of the buffer chamber, and the buffer solution inlet is arranged below the reaction chamber.
The beneficial effect that above-mentioned scheme produced does: the cover plate, the first substrate, the microbial separation membrane, the second substrate, the sensitive element and the bottom plate are bonded, pressed and fixed; the first microbial channel and the second microbial channel are used for flowing of a liquid sample, the microbial separation membrane is used for separating microbes to be detected within a certain size range, filtering of particles, biological cells and the like with large volume is preliminarily achieved, the purpose of sample pretreatment is achieved, the reaction chamber and the buffer chamber are used for containing a biological sample to be detected and an electrolyte buffer solution, the sensitive elements are respectively contacted with liquid to be detected in the reaction chamber and the buffer chamber, the first microelectrode and the second microelectrode are respectively arranged in the reaction chamber and the buffer chamber, an electrochemical detection pool is formed between the reaction chamber and the buffer chamber, when the liquid containing the microbes to be detected is contacted with the sensitive elements, a nanometer channel space blocking effect is formed, the change of an electric signal is generated, and the microbial content of the sample to be detected is determined through the change of the electric signal.
Further, the pore diameter on the microorganism separation membrane is 200nm-2 μm.
Furthermore, the sensing element is made of a macroporous membrane and a small-pore membrane, the pore diameter of the macroporous membrane is 50-900nm, and the pore diameter of the small-pore membrane is 1-500 nm.
The beneficial effect that above-mentioned scheme produced does: the aperture range on the microorganism separation membrane and the sensitive element is wider, so that the device can be suitable for the rapid detection of microorganisms with various sizes, and during specific operation, the microorganism separation membrane and the sensitive element with the required aperture can be selected according to the type of the microorganism to be detected, so that the applicability of the device is improved.
Furthermore, the material of the macroporous film is polysilicon, silicon nitride, anodic aluminum oxide, polycarbonate, carbon nanotube or graphene/graphene oxide.
Furthermore, the material of the small-pore membrane is mesoporous carbon, mesoporous silicon/mesoporous silica or graphene/graphene oxide.
Furthermore, the material of the microorganism separation membrane is polyethylene terephthalate, polycarbonate, porous silicon or polyvinylidene fluoride.
Furthermore, the material of the first microelectrode and the second microelectrode is a metal electrode, a metal/metal oxide electrode, a carbon electrode or a glassy carbon electrode.
Further, the cross-sectional shapes of the first microfluidic channel and the second microfluidic channel are circular, rectangular, semicircular, semi-elliptical or irregular polygonal.
The beneficial effect that above-mentioned scheme produced does: the first microfluidic channel and the second microfluidic channel are used for the circulation of a sample to be detected.
Further, the shapes of the microorganism chamber, the separation chamber, the reaction chamber and the buffer chamber are cylindrical, square or rectangular.
The beneficial effect that above-mentioned scheme produced does: the microorganism chamber, the separation chamber, the reaction chamber and the buffer chamber are used for temporarily storing microorganism samples to be detected.
Further, the preparation method of the sensitive element comprises the following steps:
(1) capturing the small-pore membrane with the supporting medium PMMA in deionized water by using the large-pore membrane, and drying to finish the tight compounding of the small-pore membrane and the large-pore membrane;
(2) and (3) placing the compounded macroporous membrane and the compounded microporous membrane in acetone to remove the supporting medium PMMA on the microporous membrane, thereby completing the preparation of the sensitive element (14).
A microorganism detection method adopts the microorganism detection device for detection, and the detection process comprises the following steps:
(1) modifying an antibody or an aptamer of a microorganism to be detected on a sensitive element, specifically, hydroxylating the sensitive element by hydrogen peroxide, placing the hydroxylated sensitive element in an acetone or ethanol solution of 5% 3-aminopropyltriethoxysilane for silanization and crosslinking to form an aminated sensitive element surface, and incubating the antibody or the aptamer of the microorganism to be detected with a certain concentration to the sensitive element surface to complete the functional modification of the sensitive element;
(2) adding a microbial sample to be detected containing electrolyte buffer solution into the first microfluidic channel through the sample inlet, wherein the microbial sample enters the microbial chamber along the first microfluidic channel and enters the separation chamber after being separated by the microbial separation membrane;
(3) and a microorganism sample in the separation chamber enters the reaction chamber along the micro-fluid channel II to be contacted with the sensitive element and then is discharged through the sample outlet, a buffer solution is injected into the buffer chamber through the buffer solution inlet, the liquid level of the buffer solution in the buffer chamber is contacted with the sensitive element, and then current signals are detected through the microelectrode I and the microelectrode II.
The beneficial effect that above-mentioned scheme produced does: screening and separating a microorganism sample with a certain size into a reaction chamber through a microorganism separation membrane with a specific aperture to realize rapid pretreatment of microorganisms; modifying a microbial antibody or aptamer to be detected on a sensitive element, and when the microbe to be detected in a sample to be detected contacts the sensitive element, selectively identifying and capturing the microbe to be detected by specific molecules on the sensitive element, so that a nano-channel space blocking effect is formed on the sensitive element, the ion mobility or the electron transfer rate is greatly changed, and the content of the microbe in the sample to be detected is determined according to the change of the generated electric signal.
The beneficial effects produced by the invention are as follows:
1. in the device, the microorganism sample to be detected is injected through the sample inlet by using devices such as an injector and the like, and certain pressure is applied, so that the microorganism sample to be detected passes through the microorganism separation membrane, the rapid separation of microorganisms is realized, and the subsequent detection is convenient.
2. The invention introduces a sensitive element containing a porous nano-channel structure into electrochemical sensing detection, the nano-channel structure has typical asymmetry, selectively identifies and captures the microorganism to be detected by modifying a molecule with a specific identification function on the upper part of the nano-channel structure, obviously enhances the nano-space blocking effect, enables the transmembrane current between a reaction chamber and a buffer chamber to change obviously, improves the specificity of microorganism analysis and detection, greatly improves the detection sensitivity, improves the analysis speed of the microorganism, and can meet the requirement of rapid and sensitive analysis and detection application of the microorganism.
Drawings
FIG. 1 is a diagram of an apparatus of the present invention;
FIG. 2 is a cross-sectional view of a first substrate;
FIG. 3 is a cross-sectional view of a second substrate;
FIG. 4 is a current scan graph;
reference numerals: 1. a cover plate; 2. a first substrate; 3. a second substrate; 4. a base plate; 5. a sample inlet; 6. a first microfluidic channel; 7. a microbial chamber; 8. a separation chamber; 9. a second microfluidic channel; 10. a reaction chamber; 11. a microbial separation membrane; 12. a first microelectrode; 13. a buffer chamber; 14. a sensing element; 15. a second microelectrode; 16. a buffer inlet; 17. a buffer outlet; 18. and (4) a sample outlet.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings.
In one embodiment of the present invention, as shown in fig. 1-3, a microorganism detection apparatus is provided, which comprises a cover plate 1, a first substrate 2, a second substrate 3 and a bottom plate 4, which are fixedly connected in sequence from top to bottom, wherein a sample inlet 5 is arranged at one end of the first cover plate 2; a micro-fluid channel I6 and a microorganism chamber 7 which are communicated are arranged on the substrate I2, and the micro-fluid channel I6 is communicated with the sample inlet 5; a separation chamber 8, a micro-fluid channel II 9 and a reaction chamber 10 which are communicated are arranged on the substrate II 3, a microorganism separation membrane 11 is fixedly connected between the microorganism chamber 7 and the separation chamber 8, and optimally, the aperture on the microorganism separation membrane 11 is 200nm-2 mu m; preferably, the material of the microbial separation membrane 11 is polyethylene terephthalate, polycarbonate, porous silicon or polyvinylidene fluoride.
A first microelectrode 12 is fixedly connected in the reaction chamber 10, and a sample outlet 18 is arranged in the reaction chamber 10; the bottom plate 4 is provided with a buffer chamber 13, a sensitive element 14 is fixedly connected between the buffer chamber 13 and the reaction chamber 10, the sensitive element 14 is made of a macroporous membrane and a microporous membrane, the pore diameter on the macroporous membrane is 50-900nm, and the pore diameter on the microporous membrane is 1-500 nm. Optimally, the material of the macroporous film is polysilicon, silicon nitride, anodic aluminum oxide, polycarbonate, carbon nanotube or graphene/graphene oxide. Optimally, the material of the small-pore membrane is mesoporous carbon, mesoporous silicon/mesoporous silica or graphene/graphene oxide. A second microelectrode 15 is fixedly connected in the buffer chamber 13, a buffer solution inlet 16 and a buffer solution outlet 17 are respectively arranged at two sides of the buffer chamber 13, and the buffer solution inlet 16 is arranged below the reaction chamber 10. Preferably, the materials of the first microelectrode 12 and the second microelectrode 15 are metal electrodes, metal/metal oxide motors, carbon electrodes or glassy carbon electrodes.
A microorganism detection method adopts the microorganism detection device for detection, and the detection process comprises the following steps:
(1) modifying an antibody or an aptamer of a microorganism to be detected on a sensitive element, specifically, hydroxylating the sensitive element by hydrogen peroxide, placing the hydroxylated sensitive element in an acetone or ethanol solution of 5% 3-aminopropyltriethoxysilane for silanization and crosslinking to form an aminated sensitive element surface, and incubating the antibody or the aptamer of the microorganism to be detected with a certain concentration to the sensitive element surface to complete the functional modification of the sensitive element;
(2) injecting a to-be-detected microorganism sample containing electrolyte buffer solution into the first microfluidic channel through a sample inlet by using a device such as an injector, and the like, so that the purpose of pressurizing the first microfluidic channel is realized in the injection process, the flow of microorganism sample liquid in the device is promoted, and the microorganism sample enters a microorganism chamber along the first microfluidic channel, is separated by a microorganism separation membrane and then enters a separation chamber;
(3) and the microorganism sample in the separation chamber enters the reaction chamber along the microfluidic channel II and is discharged through the sample outlet after contacting with the sensitive element, a buffer solution is injected into the buffer chamber through the buffer solution inlet, the liquid level of the buffer solution in the buffer chamber contacts with the sensitive element, the sensitive element in the reaction chamber recognizes and captures the microorganism sample to form a nano-channel space blocking effect, and the content of the microorganism in the microorganism sample is quantitatively detected according to the change of the generated electric signal.
The device and the method are adopted to detect microorganisms in different samples to be detected, and because the sensitive elements are made of various materials, the preparation methods of the different sensitive elements are explained in the current embodiment, and the preparation methods are as follows:
example 1
The sensitive element is made of a small-pore membrane and a large-pore membrane, wherein the small-pore membrane is a mesoporous silicon membrane, and the large-pore membrane is an Anodic Aluminum Oxide (AAO);
the preparation method of the small-pore membrane mesoporous silicon membrane comprises the following steps: the preparation method is realized by a solution growth method, and comprises the following steps: selecting ITO glass with the size of 2cm multiplied by 2cm as a growth substrate of a mesoporous silicon film, firstly ultrasonically washing the ITO glass with 1mol/L NaOH ethanol solution, acetone solution, ethanol solution and deionized water for 15 minutes, then immersing the washed ITO glass into a mixed solution containing 70mL of water, 30mL of ethanol, 10 mu L of ammonia water, 0.16g of hexadecyl trimethyl ammonium bromide and 80 mu L of tetraethyl silicate, and reacting for 20 hours at the temperature of 60 ℃; after the reaction is finished, drying the ITO glass at 100 ℃ overnight; then, placing the grown mesoporous silicon film and ITO glass into 0.1mol/L HCl ethanol solution to remove cetyl trimethyl ammonium bromide surfactant in the inner wall of the mesoporous silicon film; spin-coating a drop of PMMA solution (5%) on ITO glass with a mesoporous silicon film, evaporating the solvent at room temperature, and baking in an oven at 115 ℃ for 20 minutes; and then, placing the film in 2mol/L HCl solution overnight to remove the ITO layer to obtain a mesoporous silicon film taking PMMA as a supporting medium, and finally transferring the mesoporous silicon film into deionized water to clean the mesoporous silicon film for later use to obtain the microporous film.
The preparation method of the sensitive element comprises the following steps: anodic aluminum oxide films (AAO) for macroporous films are available on the market in a customized manner. Firstly, putting the macroporous membrane in a boiling 30% hydrogen peroxide solution for hydroxylation treatment so as to form-OH functional groups on the inner wall surface of the macroporous membrane, then putting the macroporous membrane in a 10% APTCS (3-aminopropyltriethoxysilane) acetone solution for reaction overnight, and drying at 120 ℃ for 2 hours to form a crosslinked silane layer; secondly, capturing the small-pore membrane in deionized water by taking the macroporous membrane as a supporting medium, drying at room temperature for 1h, drying at 100 ℃ for 2h to complete the compounding of the macroporous membrane and the small-pore membrane so as to form a nano-channel membrane with a multilayer composite membrane structure, and then dissolving and removing the supporting medium PMMA of the small-pore membrane by using acetone; and finally, incubating the antibody or aptamer of the microorganism to be detected with proper concentration to the inner wall surface of the nano-channel membrane of the multilayer composite membrane structure to form the sensitive element with the specific functional recognition molecule.
Example 2
The sensitive element is made of a small-pore film of a mesoporous silicon film and a large-pore film of a porous silicon nitride film (p-SiN);
the preparation process of the small-pore membrane mesoporous silicon membrane comprises the following steps: the preparation method is realized by a solution growth method, and comprises the following steps: selecting ITO glass with the thickness of 2cm multiplied by 2cm as a growth substrate of a mesoporous silicon film, firstly ultrasonically washing the ITO glass with 1mol/L NaOH ethanol solution, acetone solution, ethanol solution and deionized water for 15 minutes, then immersing the washed ITO glass multiplied by glass into a mixed solution containing 70mL of water, 30mL of ethanol, 10 mu L of ammonia water, 0.16g of hexadecyl trimethyl ammonium bromide and 80 mu L of tetraethyl silicate, and reacting for 14 hours at the temperature of 60 ℃; after the reaction is finished, drying the ITO glass at the temperature of 100 ℃ overnight; then, placing the grown mesoporous silicon film and ITO glass in 0.1mol/L HCl ethanol solution to remove cetyl trimethyl ammonium bromide surfactant in the inner wall of the mesoporous silicon film; spin-coating a drop of PMMA solution (6%) on ITO glass with a mesoporous silicon film, evaporating the solvent at room temperature, and then placing the ITO glass in an oven at 120 ℃ for baking for 15-20 minutes; and then, placing the film in 2mol/L HCl solution overnight to remove the ITO layer to obtain a mesoporous silicon film taking PMMA as a supporting medium, and finally transferring the mesoporous silicon film into deionized water to clean the mesoporous silicon film for later use to obtain the mesoporous silicon film of the microporous film.
The preparation method of the sensitive element comprises the following steps: porous silicon nitride thin films (p-SiN) for macroporous films are commercially available. Placing the macroporous membrane in a boiling 30% hydrogen peroxide solution for hydroxylation treatment so as to form-OH functional groups on the inner wall surface of the macroporous membrane, then placing the macroporous membrane in a 10% APTCS (3-aminopropyltriethoxysilane) acetone solution for reaction overnight, and drying at 120 ℃ for 2 hours to form a crosslinked silane layer; secondly, capturing the small-pore membrane in deionized water by taking the macroporous membrane as a supporting medium, drying at room temperature for 1h, drying at 100 ℃ for 2h to complete the compounding of the macroporous membrane and the small-pore membrane so as to form a nano-channel membrane with a multilayer composite membrane structure, and then dissolving and removing the supporting medium PMMA of the small-pore membrane by using acetone; and finally, incubating the antibody or aptamer of the microorganism to be detected with proper concentration to the inner wall surface of the nano-channel membrane of the multilayer composite membrane structure to form the sensitive element with the specific functional recognition molecule.
Example 3
The sensitive element is made of a mesoporous carbon film of a small-pore film and an anodic aluminum oxide film of a large-pore film;
the preparation method of the small-pore membrane mesoporous carbon membrane comprises the following steps: the mesoporous carbon film is mainly synthesized on a silicon chip, phenolic resin is used as a carbon precursor, a triblock polymer F127 is used as a template agent, and a solution volatilization self-assembly method is adopted to prepare the ordered mesoporous carbon film, wherein the specific preparation process comprises the following steps: (1) cleaning a silicon wafer (20mm multiplied by 20mm) in 90 ℃ piranha acid (the volume ratio of concentrated sulfuric acid to 30% hydrogen peroxide is 2:1) for 30 minutes to remove organic matters or oxide layers on the surface of the silicon wafer; (2) synthesizing a water-soluble phenolic resin solution by using NaOH as a catalyst and phenol and formaldehyde as raw materials (the mass ratio is 1:6:10), and preparing the phenolic resin solution with the mass fraction of 20% by using absolute ethyl alcohol as a solvent; (3) spin-coating a drop of mixed solution containing 0.06g of phenolic resin solution, 0.03g of Pluronic F127 surfactant and 0.24g of ethanol on the surface of the cleaned silicon wafer, slowly evaporating at room temperature for 6h, and transferring to 105 ℃ for growth for 24 h; (4) the silicon chip after the reaction is placed in a carbonization furnace at 600 ℃ under the protection of nitrogen for reaction for 3 hours (the nitrogen flow rate is 60 cm)3At a heating speed of 1 ℃/min per minute), and finally obtaining the mesoporous carbon film taking the silicon wafer as a supporting medium; (5) spin coating a drop of PMMA solution (8%) on the surface of the mesoporous carbon film grown on the silicon wafer, steaming the solvent at room temperature, then placing the film in a drying oven at 110 ℃ for baking for 20 minutes, then placing the film in a KOH solution with the concentration of 10% for reaction for 6 hours, so as to obtain the mesoporous carbon film taking PMMA as a supporting medium by stripping the mesoporous carbon film from the silicon wafer, and finally transferring the mesoporous carbon film into deionized water for slicing for later use.
Preparation of the sensitive element: anodic aluminum oxide films (AAO) for macroporous films are available on the market in a customized manner. Firstly, putting the macroporous membrane in a boiling 30% hydrogen peroxide solution for hydroxylation treatment so as to form-OH functional groups on the inner wall surface of the macroporous membrane, then putting the macroporous membrane in a 10% APTCS (3-aminopropyltriethoxysilane) acetone solution for reaction overnight, and drying at 120 ℃ for 2 hours to form a crosslinked silane layer; secondly, capturing the small-pore membrane in deionized water by taking the macroporous membrane as a supporting medium, drying at room temperature for 1h, and drying at 100 ℃ for 2h to complete the compounding of the macroporous membrane and the small-pore membrane so as to form a nano-channel membrane with a multilayer composite membrane structure; and finally, incubating the antibody or aptamer of the microorganism to be detected with proper concentration to the inner wall surface of the nano-channel membrane of the multilayer composite membrane structure to form the sensitive element with the specific functional recognition molecule.
Test examples
Taking the preparation method in the embodiment 1 to prepare the sensitive element as an example, the sensitive element and other structures are sequentially assembled to prepare a microorganism detection device, and the pseudomonas aeruginosa is quantitatively detected:
(1) preparation of functionalized sensitive element
Dropwise applying the pseudomonas aeruginosa aptamer with the concentration of 10 mu M to the sensitive element in the embodiment 1 in the volume of 20 mu L, and incubating for 4h at room temperature; after the incubation is finished, washing the sensitive element for three times by using PBS (phosphate buffer solution) (pH 7.4), and then dripping 20 mu L of 1% Bovine Serum Albumin (BSA) PBS solution on the surface of the sensitive element to block the non-specific adsorption sites of the sensitive element; and after the BSA solution is incubated for 1h, washing the sensitive element for three times by adopting a PBS buffer solution, thus finishing the preparation of the functionalized sensitive element.
(2) Quantitative detection of pseudomonas aeruginosa
In the experimental example, the first microelectrode of the microorganism detection device adopts a self-made platinum wire electrode, and the second microelectrode adopts a screen printing electrode (comprising a gold working electrode, a platinum counter electrode and a silver/silver chloride auxiliary electrode) of a three-electrode system; injecting a PBS buffer solution into the buffer chamber through the buffer solution inlet before the microorganism detection; then using PBS solution as electrolyte, [ Ru (NH)3)6]3+Injecting mixed solutions containing pseudomonas aeruginosa with different concentrations to be detected into a sample inlet at the flow rate of 20 mu L/min by a micro pump respectively for an electrochemical probe; the electrochemical quantitative detection of the microorganism adopts a microelectrode I as a counter electrode, a microelectrode II as a working electrode and an auxiliary electrode, and the microorganism to be detected is subjected to quantitative analysis by scanning the curve of ionic current and time when the bias voltage is 0V, and the result is shown in figure 4 (left); at the same time, after 30 minutes of stable permeation by the sensitive element, the working electrode of the second microelectrode, the platinum electrode and the silver/silver chloride are used for electric treatmentThe electrode is scanned with the DPV signal, and the result is shown in fig. 4 (right).
FIG. 4 (left) shows a gradual decrease in ion current with increasing concentration of target bacteria; FIG. 4 (right) shows that after the electrochemical probe stably permeates through the sensing element for 30 minutes, the DPV signal of the electrochemical probe in the buffer chamber is gradually reduced along with the increase of the concentration of the target bacteria captured by the sensing element; fig. 4 shows that the space blocking effect of the sensor is obviously increased after the target bacteria are captured, so that the ion current and the probe DPV signal are gradually reduced, and the target bacteria can be quantitatively analyzed by measuring the ion current and the DPV signal generated by the ions in the solution passing through the sensor.
Claims (8)
1. The microorganism detection device is characterized by comprising a cover plate (1), a first substrate (2), a second substrate (3) and a bottom plate (4) which are fixedly connected from top to bottom in sequence, wherein a sample inlet (5) is formed in the end part of the cover plate (1);
a first micro-fluid channel (6) and a microbial chamber (7) which are communicated are arranged on the first substrate (2), and the first micro-fluid channel (6) is communicated with the sample inlet (5);
a separation chamber (8), a micro-fluid channel II (9) and a reaction chamber (10) which are communicated with each other are arranged on the substrate II (3), a microorganism separation film (11) is fixedly connected between the microorganism chamber (7) and the separation chamber (8), a microelectrode I (12) is fixedly connected in the reaction chamber (10), and a sample outlet (18) is arranged in the reaction chamber (10);
a buffer chamber (13) is arranged on the bottom plate (4), a sensitive element (14) is fixedly connected between the buffer chamber (13) and the reaction chamber (10), a second microelectrode (15) is fixedly connected in the buffer chamber (13), a buffer solution inlet (16) and a buffer solution outlet (17) are respectively arranged on two sides of the buffer chamber (13), and the buffer solution inlet (16) is arranged below the reaction chamber (10); the sensing element (14) is made of a large-pore membrane and a small-pore membrane, the pore diameter of the large-pore membrane is 50-900nm, and the pore diameter of the small-pore membrane is 1-500 nm.
2. The microorganism detection apparatus according to claim 1, wherein the pore size on the microorganism separation membrane (11) is 200nm to 2 μm.
3. The microorganism detection apparatus according to claim 1, wherein the material of the macroporous film is polysilicon, silicon nitride, anodized aluminum, polycarbonate, carbon nanotube, or graphene/graphene oxide.
4. The microbial detection device of claim 3, wherein the material of the porous membrane is mesoporous carbon, mesoporous silicon/mesoporous silica, or graphene/graphene oxide.
5. The microorganism detection apparatus according to claim 1 or 2, wherein the material of the microorganism separation membrane (11) is polyethylene terephthalate, polycarbonate, porous silicon, or polyvinylidene fluoride.
6. The microorganism detection apparatus according to claim 1, wherein the material of the first microelectrode (12) and the second microelectrode (15) is a metal electrode, a metal/metal oxide electrode, a carbon electrode or a glassy carbon electrode.
7. The microbial detection apparatus according to claim 1, wherein the sensor (14) is prepared by:
(1) capturing the small-pore membrane with the supporting medium PMMA in deionized water through the large-pore membrane, and drying to complete the close compounding of the small-pore membrane and the large-pore membrane;
(2) and (3) placing the compounded macroporous membrane and the compounded microporous membrane in acetone to remove the supporting medium PMMA on the microporous membrane, thereby completing the preparation of the sensitive element (14).
8. A method for detecting microorganisms, which comprises the steps of using the microorganism detection apparatus according to any one of claims 1 to 7 to perform detection, wherein the detection comprises:
(1) modifying an antibody or an aptamer of a microorganism to be detected on a sensitive element (14);
(2) adding a microbial sample to be detected containing electrolyte buffer solution into the first microfluidic channel (6) through the sample inlet (5), wherein the microbial sample enters the microbial chamber (7) along the first microfluidic channel (6), is separated by the microbial separation membrane (11) and then enters the separation chamber (8);
(3) a microbial sample in the separation chamber (8) enters the reaction chamber (10) along the second microfluidic channel (9) to be in contact with the sensitive element (14) and then is discharged through the sample outlet (18), a buffer solution is injected into the buffer chamber (13) through the buffer solution inlet (16), the liquid level of the buffer solution in the buffer chamber (13) is in contact with the sensitive element (14), and then a current signal is detected through the first microelectrode (12) and the second microelectrode (15).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011251977.2A CN112362711B (en) | 2020-11-11 | 2020-11-11 | Microorganism detection device and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011251977.2A CN112362711B (en) | 2020-11-11 | 2020-11-11 | Microorganism detection device and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112362711A CN112362711A (en) | 2021-02-12 |
| CN112362711B true CN112362711B (en) | 2021-08-17 |
Family
ID=74514533
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011251977.2A Active CN112362711B (en) | 2020-11-11 | 2020-11-11 | Microorganism detection device and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112362711B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113325054B (en) * | 2021-05-31 | 2022-03-01 | 浙江大学 | Fully-integrated portable carbon fiber microelectrode electrochemical sensor and detection system |
| CN116024067A (en) * | 2022-12-19 | 2023-04-28 | 深圳职业技术学院 | Circulating fetal cell separation device and preparation method thereof |
Family Cites Families (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100494360C (en) * | 2001-03-22 | 2009-06-03 | 博奥生物有限公司 | Process for separating cells and its application |
| JP2005245317A (en) * | 2004-03-04 | 2005-09-15 | Mitsubishi Electric Corp | Measuring instrument for microorganism and measuring method |
| US20060019406A1 (en) * | 2004-07-23 | 2006-01-26 | Ning Wei | Lateral flow device for the detection of large pathogens |
| CN1912625A (en) * | 2006-08-25 | 2007-02-14 | 清华大学 | Application of microdrop control in virus detection and detection method and chip |
| CN101008594B (en) * | 2007-01-25 | 2010-05-26 | 重庆大学 | Production method of micro-fluidic chip containing sample pre-treatment film |
| US10316348B2 (en) * | 2014-09-16 | 2019-06-11 | Northeastern University | Diagnostic system and process for rapid bacterial infection diagnosis |
| CN104560711B (en) * | 2015-01-27 | 2017-02-01 | 哈尔滨工业大学 | Cell-culture micro-fluidic chip with self-antibacterial function |
| CN107267382B (en) * | 2017-07-26 | 2024-04-23 | 南方科技大学 | Microfluidic chip based on dielectrophoresis and preparation method and application thereof |
| CN108048296B (en) * | 2017-11-02 | 2021-06-11 | 暨南大学 | Microorganism real-time separation detection method and application of separation detection device in microorganism separation detection identification |
| CN207423878U (en) * | 2017-11-06 | 2018-05-29 | 四川工商职业技术学院 | The electrophoretic apparatus of microbial DNA in a kind of quick separating extraction wine cellar fermentation substrate |
| CN109966642B (en) * | 2018-12-03 | 2023-01-31 | 东南大学 | Multifunctional micro-fluidic heart chip and application thereof |
| CN110734853B (en) * | 2019-11-05 | 2021-07-30 | 中国农业大学 | Microorganism detection device and detection method |
| CN211463197U (en) * | 2019-12-14 | 2020-09-11 | 南京岚煜生物科技有限公司 | Microfluid detection chip adopting immune electrode |
| CN111514947B (en) * | 2020-04-20 | 2021-07-09 | 南京航空航天大学 | Micro-fluidic chip for cell electrical impedance spectroscopy measurement |
-
2020
- 2020-11-11 CN CN202011251977.2A patent/CN112362711B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112362711A (en) | 2021-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11764047B2 (en) | General-purpose nanochip for mass spectrum analysis, preparation method therefor, and application thereof | |
| CN112362711B (en) | Microorganism detection device and detection method | |
| CN110146580B (en) | Method for detecting l, 5-anhydroglucitol based on persimmon tannin composite nano material | |
| CN101526493B (en) | Electrochemical biosensor based on chitosan-immobilized acetylcholinesterase and application thereof | |
| CN107389755B (en) | Electrochemical sensor for detecting mercury, and preparation method and application thereof | |
| CN101140257B (en) | Biologic sensor enzyme functional susceptivity film containing nickel and aluminum hydrotalcite nano piece and method of producing the same | |
| CN113842901B (en) | Sea urchin-shaped MOFs@COFs core-shell structure material and preparation method and application thereof | |
| CN111974370A (en) | Electric field driven solid phase micro-extraction fiber and preparation method and application thereof | |
| CN108387424B (en) | Preparation method and application of porous silicon material for biological sample pretreatment | |
| CN109331798A (en) | A kind of preparation method of solid phase microextraction material | |
| Su et al. | A non-enzymatic uric acid sensor utilizing ion channels in the barrier layer of a porous anodic alumina membrane | |
| CN109856206B (en) | Method for detecting concentration of dopamine | |
| CN102539486A (en) | Chip and bacterium analysis instrument for food safety detection | |
| CN102495118A (en) | Enzyme electrode for all-solid ethanol gas biosensor and method for manufacturing enzyme electrode | |
| JP3881731B2 (en) | Enzyme reaction sensor and manufacturing method thereof | |
| CN109771988B (en) | Diethyl phthalate solid-phase micro-extraction method based on molecular imprinting technology | |
| CN201083712Y (en) | Biosensor and detection device adopting same | |
| CN111693593B (en) | Silicon dioxide uniform pore membrane for H in blood 2 Electrochemical luminescence detection method of S | |
| Ratchagar et al. | Sensor for continuous and real-time monitoring of biomolecule permeation through ultrathin silicon nanoporous membranes | |
| CN114062449A (en) | Molecularly imprinted electrochemical sensor for detecting L-lysine and application thereof | |
| CN112858407A (en) | Electrochemical sensor for detecting ciprofloxacin and detection method thereof | |
| CN110606463A (en) | Method for preparing nano-fluid diode based on solid-state nano-hole and having dual response to temperature and pH | |
| CN111505075B (en) | Preparation method of graphene-based electrochemical luminescence sensor | |
| CN109706223B (en) | MicroRNA detection method based on nanofluidic | |
| CN111359597A (en) | Adsorption film for selectively fixing trivalent arsenic, trivalent antimony and quadrivalent selenium and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |