CN111307912A - Field-effect tube biosensor and preparation method thereof - Google Patents
Field-effect tube biosensor and preparation method thereof Download PDFInfo
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- G—PHYSICS
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- 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
- G01N27/403—Cells and electrode assemblies
- G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
- G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
- H01L29/00—Semiconductor devices adapted for rectifying, amplifying, oscillating or switching, or capacitors or resistors with at least one potential-jump barrier or surface barrier, e.g. PN junction depletion layer or carrier concentration layer; Details of semiconductor bodies or of electrodes thereof ; Multistep manufacturing processes therefor
- H01L29/66—Types of semiconductor device ; Multistep manufacturing processes therefor
- H01L29/68—Types of semiconductor device ; Multistep manufacturing processes therefor controllable by only the electric current supplied, or only the electric potential applied, to an electrode which does not carry the current to be rectified, amplified or switched
- H01L29/76—Unipolar devices, e.g. field effect transistors
- H01L29/772—Field effect transistors
- H01L29/78—Field effect transistors with field effect produced by an insulated gate
Abstract
The invention discloses a field effect tube biosensor and a preparation method thereof, wherein the biosensor comprises two-dimensional molybdenum disulfide-based nano films (Vertically-aligned MoS) which are vertically arranged2nanolayers-VAMNs) as a main body, a sensor substrate with a Field Effect Transistor (FET) structure, and a polydimethylsiloxane (polydimethylsiloxane-PDMS) storage tank which is prepared on the sensor substrate and used for sealing a target solution in the channel region of the FET. The invention can detect the concentration of specific protein related to certain specific diseases (such as tumor or hepatitis B) in human serum by the means of measuring the electrical transmission performance of the sensor through the field effect tube biosensor, thereby carrying out early diagnosis of some diseases, and has the advantages of high detection speed, extremely high sensitivity and high accuracy, and is suitable for useIn the field of in vitro assay in real time.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a field-effect tube biosensor and a preparation method thereof.
Background
A biosensor (biosensor) is an instrument that is sensitive to a biological substance and converts its concentration into an electrical signal for detection. It is an analysis tool or system composed of immobilized biological sensitive material as recognition element (including enzyme, antibody, antigen, microbe, cell, tissue, nucleic acid, etc.), proper physicochemical transducer (such as oxygen electrode, photosensitive tube, field effect tube, piezoelectric crystal, etc.) and signal amplification device. Biosensors can be classified into five types according to the difference of the sensing elements, enzyme sensors (enzymes), microbial sensors (microbiological sensors), cell sensors (organonalsensors), tissue sensors (tis-suesensors) and immunosensors (immunolSensors). The sensitive materials used by the method are enzyme, microorganism individuals, organelles, animal and plant tissues, antigens and antibodies in sequence.
The immunosensor is a type of biosensor developed based on antigen-antibody specific recognition function. Because the immunosensor technology has the advantages of high analysis sensitivity, strong specificity, simple and convenient use, low cost and the like, the application of the immunosensor technology at present relates to the wide fields of clinical medicine, biological monitoring technology, food industry, environmental monitoring and processing and the like. Immunosensors can be divided into two broad categories depending on the detection method: non-labeled immunosensors and labeled immunosensors. The non-labeling immunosensor is characterized in that an antibody or an antigen is immobilized on an electrode, when the non-labeling immunosensor is combined with a specific antigen or an antibody to be detected in a solution, the change of the charge density of an electrode surface membrane and the interface of the solution is caused, the change of the membrane potential is generated, and the change degree is proportional to the concentration of the antigen or the antibody to be detected in the solution; the labeled immunosensor is prepared by labeling a specific antigen or antibody with an enzyme, allowing the labeled antigen or antibody to compete with the antigen or antibody to be detected in a reaction solution to bind to the antibody or antigen on an electrode, taking out the electrode, washing to remove free antigen or antibody, and immersing the electrode in a solution containing an enzyme substrate for determination.
The specific protein kit for detecting some specific diseases (such as tumor or hepatitis B) of human body and the detection method of the specific protein adopted by the current clinical medicine generally have the problems of long time consumption, low detection sensitivity and the like.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a field effect tube biosensor with short detection time and high sensitivity. The technical scheme is as follows:
a field effect tube biosensor, comprising:
the sensor comprises a sensor substrate with a Field Effect Transistor (FET) structure and a Polydimethylsiloxane (PDMS) storage tank, wherein the sensor substrate is mainly provided with two-dimensional molybdenum disulfide-based nano films (VAMNs) which are vertically arranged, and the PDMS storage tank is prepared on the sensor substrate and used for sealing a target solution in the channel area of a field effect transistor.
As a further improvement of the invention, both poles of the sensor matrix are made of chromium/gold double-layer materials, and Al is deposited on both pole surfaces of the sensor matrix2O3。
As a further improvement of the invention, the sensor substrate is prepared on a silicon substrate, and an intermediate layer is prepared between the sensor substrate and the silicon substrate.
As a further improvement of the invention, the intermediate layer is SiO2。
As a further improvement of the invention, the device also comprises a reference electrode which is inserted into the storage tank and used as a grid for regulating grid voltage during measurement.
As a further development of the invention, the reference electrode is an Ag/AgCl reference electrode.
The second purpose of the invention is to provide a preparation method of the field effect tube biosensor. The technical scheme is as follows:
a method for preparing a field effect transistor biosensor comprises the following steps:
s10, SiO after plasma enhancement2Layer CVD deposition onto a silicon wafer followed by sputter deposition of a Mo film;
s20, vulcanizing the Mo thin film layer in a CVD system;
s30, preparing a double-layer chromium/gold electrode by adopting electron beam evaporation deposition and photoetching processes;
s40 deposition of Al on the surface of the electrode by ALD2O3A layer, then etching the pattern using an etching solution, leaving openings for the FET channels and metal contact areas;
s50, annealing in a vacuum environment, and ashing the photoresist/solvent residues on the VAMNs;
s60, curing the silicon rubber reagent to form a PDMS stamp, stamping the PDMS stamp to form a PDMS storage tank, and then treating the PDMS storage tank by using oxygen plasma to directly bond the PDMS storage tank with the FET substrate to complete the preparation of the sensor.
As a further improvement of the present invention, the CVD system comprises a three-zone temperature-controlled tube furnace and is equipped with a quartz tube, an argon gas delivery system and a vacuum pump.
As a further improvement of the present invention, the step S20 specifically includes:
s21, loading the Mo film into a downstream high-temperature area of the quartz tube, and putting a sulfur powder precursor into an upstream low-temperature area to evaporate to form sulfur vapor;
s22, flushing the quartz tube by using argon gas, removing air residues, and then keeping the vacuum pressure of 200mTorr in the quartz tube by using a vacuum pump under the constant argon gas flow of 300 sccm;
s23, in the synthesis process, raising the downstream temperature to 750 ℃ within 30 minutes; meanwhile, in the same time, the temperature of the upstream area for evaporating the sulfur powder is increased and kept at 270 ℃ so as to be far higher than the melting point temperature;
and S24, finally, preserving the temperature of the furnace, completing the vulcanization process, naturally cooling to room temperature, and taking out the sample.
As a further improvement of the present invention, the method further comprises the steps of:
s71, adding 0.1mol/L mercaptoundecanoic acid (11-MUA) solution into a PDMS storage tank for incubation;
s72, after the culture is finished, thoroughly washing the PDMS storage tank by deionized water, and removing excessive 11-MUA by using low-frequency ultrasonic treatment;
s73, preparing an NHS/EDC mixture in deionized water, and injecting the mixture into the surface of the oil reservoir for activation;
s74, washing the surface of the equipment three times by using a phosphate buffer solution, introducing a specific protein solution related to certain specific diseases of the human body into the surface of the channel, and fixing probe molecules after incubation;
s75, introducing a phosphate buffer solution into a PDMS storage tank by using a pipette, and washing redundant specific protein by using the pipette for three times;
s76, introducing the standard protein solution into a PDMS storage tank for incubation, and then washing with a phosphate buffer solution.
The invention has the beneficial effects that:
the invention provides a FET type biosensor which takes vertically arranged two-dimensional molybdenum disulfide-based nano-films (VAMNs) as FET channels and is used for non-labeling and hypersensitivity immunodetection. The field effect tube biosensor has the characteristics of ultrahigh sensitivity, easiness in manufacturing, short detection time and the like, and has an important application prospect in early diagnosis of major dangerous diseases.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood, the present invention may be implemented in accordance with the content of the description, and in order to make the above and other objects, features, and advantages of the present invention more clearly understood, the following preferred embodiments are described in detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of a field effect transistor biosensor according to a preferred embodiment of the present invention;
FIG. 2 is a cross-sectional view of a field effect tube biosensor in a preferred embodiment of the invention;
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example one
As shown in fig. 1-2, a field effect transistor biosensor according to an embodiment of the present invention is directed to detecting specific protein concentrations associated with certain specific diseases in a human body, and comprises a sensor substrate having a Field Effect Transistor (FET) structure and including two-dimensional molybdenum disulfide-based nanomembranes (VAMNs) arranged vertically as a main body, and a Polydimethylsiloxane (PDMS) reservoir formed on the sensor substrate for enclosing a target solution (an analyte) in a channel region of the field effect transistor.
The sensor substrate is prepared on a silicon substrate, and an intermediate layer SiO is prepared between the sensor substrate and the silicon substrate2The two electrodes of the sensor matrix are both made of chromium/gold double-layer materials, and Al is deposited on the surfaces of the two electrodes of the sensor matrix2O3。
The biosensor also includes a reference electrode for insertion into the reservoir tank as a gate to regulate the gate voltage during measurement. In this example, the reference electrode was an Ag/AgCl reference electrode.
Example two
A method for preparing a field effect transistor biosensor, which is used for preparing the biosensor in the first embodiment, the method comprising the following steps:
s10, SiO after plasma enhancement2Layers are CVD deposited onto a silicon wafer and then a Mo film is sputter deposited.
Specifically, the plasma-enhanced 280nm thick SiO2Layer CVD deposition onto a precleaned 3 inch silicon wafer followed by sputter deposition of a 15nm thick Mo film.
S20, vulcanizing the Mo thin film layer in the CVD system.
Wherein a Mo thin film is made into a designed 1mm × 1mm square, and then a vulcanization treatment is performed on the Mo thin film layer in a customized CVD system including a three-zone temperature-controlled tube furnace equipped with a quartz tube, an argon gas delivery system, and a vacuum pump.
Step S20 specifically includes:
s21, loading the Mo film into a downstream high-temperature area of the quartz tube, and putting a sulfur powder precursor into an upstream low-temperature area to evaporate to form sulfur vapor;
s22, flushing the quartz tube by using argon gas, removing air residues, and then keeping the vacuum pressure of 200mTorr in the quartz tube by using a vacuum pump under the constant argon gas flow of 300 sccm;
s23, in the synthesis process, raising the downstream temperature to 750 ℃ within 30 minutes; meanwhile, in the same time, the temperature of the upstream area for evaporating the sulfur powder is increased and kept at 270 ℃ so as to be far higher than the melting point temperature;
and S24, finally, preserving the temperature of the furnace, completing the vulcanization process, naturally cooling to room temperature, and taking out the sample.
And S30, preparing the double-layer chromium/gold electrode by adopting electron beam evaporation deposition and photoetching processes.
Specifically, the double-layer chromium/gold electrode (5 nm/30nm in thickness) is prepared by adopting an electron beam evaporation deposition and photoetching process.
S40 deposition of Al on the surface of the electrode by ALD2O3The layer is then patterned using an etching solution, leaving openings for the FET channels and metal contact areas.
In particular, 30nmAl was deposited using ALD2O3Layer, then using a special solution (H)3PO4Water (1:1)) etch (3 minutes) to pattern, leaving openings for FET channels and metal contact areas.
S50, annealing in a vacuum environment, and ashing the photoresist/solvent residue on the VAMNs.
Specifically, annealing is performed at 200 ℃ for 2 hours in a vacuum environment (0.1mTorr) to reduce contact resistance and ash the photoresist/solvent residue on the VAMNs.
S60, curing the silicon rubber reagent to form a PDMS stamp, stamping the PDMS stamp to form a PDMS storage tank, and then treating the PDMS storage tank by using oxygen plasma to directly bond the PDMS storage tank with the FET substrate to complete the preparation of the sensor.
Specifically, a silicon rubber reagent is cured to form a PDMS stamp (5mm × 5mm), the PDMS stamp is stamped to form a PDMS storage tank with the diameter of 2mm, and then oxygen plasma is used for processing the PDMS storage tank so as to directly combine the PDMS storage tank with the FET substrate, thereby completing the preparation of the device.
In order to realize specific sensing detection, after the preparation is completed, VAMNs are required to be biologically functionalized in advance, and specific proteins related to certain specific diseases of a human body are combined with VAMNs probe molecules, and the method comprises the following steps:
s71, adding 0.1mol/L mercaptoundecanoic acid (11-MUA) solution into a PMDS storage tank for incubation.
Specifically, 0.1mol/L mercaptoundecanoic acid (11-MUA) solution was added to the PMDS reservoir and incubated for 24 h.
S72, after the culture is finished, the PMDS storage tank is thoroughly washed clean by deionized water, and excessive 11-MUA is removed by low-frequency ultrasonic treatment.
S73, preparing NHS/EDC mixture in deionized water, and injecting into the surface of the reservoir for activation.
Specifically, a 1:1NHS/EDC mixture was prepared in deionized water (0.1mol/L NHS and 0.1mol/L EDC) and injected into the reservoir surface for 30 minutes of activation.
S74, washing the surface of the equipment three times by using phosphate buffer solution, introducing specific protein solution related to certain diseases of the human body into the surface of the channel, and fixing probe molecules after incubation.
Specifically, the surface of the device is washed three times with phosphate buffer solution, 50 μ l of 10 μ g/ml specific protein solution related to some specific diseases of human body is introduced to the surface of the channel, and the probe molecules are immobilized after 1 hour of incubation.
S75, introducing a phosphate buffer solution into a PDMS storage tank by using a pipette, and washing redundant specific protein by using the pipette for three times;
s76, introducing the standard protein solution into a PDMS storage tank for incubation, and then washing with a phosphate buffer solution.
Specifically, 50. mu.l of 0.1% standard protein solution was taken, incubated for 3 minutes by tubing into a reservoir, and then washed with phosphate buffered saline.
Due to the fact that the variety of compounds in human serum is large, the interference degree of high-abundance proteins is high, and the concentration of salts is high, analysis of specific proteins is a relatively challenging and difficult task, and serum albumin which accounts for more than half of proteins in serum can cover detection of sparse biomarkers, and performance of the sensor is reduced. Therefore, before the detection, the serum of the human body to be detected needs to be processed. The treatment process is as follows:
(1) purifying the serum fraction using a microfilter;
(2) human serum was desalted using a centrifugal filter and mixed with a Phosphate Buffered Saline (PBS) solution after desalting was completed.
Before formal detection, the electrical transmission characteristics of the biosensor need to be calibrated. Specific proteins related to certain specific diseases of human bodies are added into treated serum samples of healthy human bodies to enable the concentration of the solutions to be in a gradient (0, 10fg/ml,1pg/ml,100pg/ml and 10ng/ml), then the samples are contacted with biologically functionalized VAMNs channels and incubated for 30 minutes, and the storage tanks are sealed by using Ag/AgCl electrodes during the incubation process. The VAMNsFET biosensor source-drain current (I) is then measured using a dual source measurement deviceds) The measurement is performed. To measure the electrical transfer characteristics of the sensor, the source-drain voltage (V) is measuredd) Set to 0.1V, solution gate voltage (V)g) The scanning range was-0.4V, the scanning step of the gate voltage was changed to 100mV, and the pulse time of the gate voltage was set to 2s to stabilize the source-drain current IdsThe reliability of the transmission curve is ensured, so that a scanning rate of 50mV/s is achieved. After incubation is finished, corresponding data are recorded, and I with different concentration gradients is drawnds-VgAnd (6) fitting a curve.
After calibration, the biosensor can be used for detecting a human serum sample, the detection process is the same as the calibration process, the serum sample is also treated and then is in contact with a biologically functionalized VAMNs channel for incubation for 30 minutes, a double-source measuring device is also used for measuring the electrical transmission performance of the sensor according to the same set parameters, corresponding data is recorded, and a fitting curve is drawn. After the test is finished, the fitted curve is compared with the calibration curve, and the specific protein concentration related to certain specific diseases in the tested sample can be obtained.
The invention can detect the concentration of specific protein related to certain specific diseases in human serum by means of measuring the electrical transmission performance of the sensor through the field effect tube biosensor, thereby carrying out early diagnosis on certain diseases, and the invention has the advantages of high detection speed, extremely high sensitivity and high accuracy, and is suitable for the field of instant in vitro detection.
The above embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A field effect tube biosensor, comprising:
two-dimensional molybdenum disulfide-based nano-film (Vertically-aligned MoS) in vertical arrangement2nanolayers-VAMNs) as a main body, a sensor substrate with a Field Effect Transistor (FET) structure, and a polydimethylsiloxane (polydimethylsiloxane-PDMS) storage tank which is prepared on the sensor substrate and used for sealing a target solution in the channel region of the FET.
2. The FET biosensor of claim 1, wherein both electrodes of the sensor body are made of a Cr/Au bilayer material, and Al is deposited on both electrode surfaces of the sensor body2O3。
3. The fet biosensor of claim 1, wherein the sensor substrate is fabricated on a silicon substrate with an interlayer fabricated therebetween.
4. The FET biosensor of claim 3, wherein the intermediate layer is SiO2。
5. The fet biosensor of claim 1, further comprising a reference electrode for gate regulation of gate voltage inserted into the reservoir during measurement.
6. The field effect tube biosensor of claim 5, wherein the reference electrode is an Ag/AgCl reference electrode.
7. A method for preparing a field effect transistor biosensor comprises the following steps:
s10, SiO after plasma enhancement2Depositing the layer on a silicon wafer by a Chemical Vapor Deposition (CVD) method, and then sputtering and depositing a Mo film;
s20, vulcanizing the Mo thin film layer in a CVD system to form VAMNs;
s30, preparing a double-layer chromium/gold electrode by adopting electron beam evaporation deposition and photoetching processes;
s40, depositing Al on the surface of the electrode by utilizing an Atomic Layer Deposition (ALD) technology2O3A layer, then etching the pattern using an etching solution, leaving openings for the FET channels and metal contact areas;
s50, annealing in a vacuum environment, and ashing the photoresist/solvent residues on the VAMNs;
s60, curing the silicon rubber reagent to form a PDMS stamp, stamping the PDMS stamp to form a PDMS storage tank, and then treating the PDMS storage tank by using oxygen plasma to directly bond the PDMS storage tank with the FET substrate to complete the preparation of the sensor.
8. The method for producing a field-effect tube biosensor as claimed in claim 7, wherein said CVD system comprises a three-zone temperature-controlled tube furnace and is equipped with a quartz tube, an argon gas delivery system and a vacuum pump.
9. The method for preparing a field effect transistor biosensor as claimed in claim 8, wherein the step S20 specifically comprises:
s21, loading the Mo film into a downstream high-temperature area of the quartz tube, and putting a sulfur powder precursor into an upstream low-temperature area to evaporate to form sulfur vapor;
s22, flushing the quartz tube by using argon gas, removing air residues, and then keeping the vacuum pressure of 200mTorr in the quartz tube by using a vacuum pump under the constant argon gas flow of 300 sccm;
s23, in the synthesis process, raising the downstream temperature to 750 ℃ within 30 minutes; meanwhile, in the same time, the temperature of the upstream area for evaporating the sulfur powder is increased and kept at 270 ℃ so as to be far higher than the melting point temperature;
and S24, finally, preserving the temperature of the furnace, completing the vulcanization process, naturally cooling to room temperature, and taking out the sample.
10. The method of manufacturing a field effect tube biosensor as claimed in claim 7, the method further comprising the steps of:
s71, adding 0.1mol/L mercaptoundecanoic acid (11-MUA) solution into a PDMS storage tank for incubation;
s72, after the culture is finished, thoroughly washing the PDMS storage tank by deionized water, and removing excessive 11-MUA by using low-frequency ultrasonic treatment;
s73, preparing a mixture of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in deionized water, and injecting the mixture into the surface of the oil reservoir for activation;
s74, washing the surface of the equipment three times by using a phosphate buffer solution, introducing a specific protein solution related to a specific disease into the surface of the channel, and fixing probe molecules after incubation;
s75, introducing a phosphate buffer solution into a PDMS storage tank by using a pipette, and washing redundant specific protein by using the pipette for three times;
s76, introducing the standard protein solution into a PDMS storage tank for incubation, and then washing with a phosphate buffer solution.
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