CN114324528B - Carbon nano tube field effect transistor biosensor and preparation method thereof - Google Patents
Carbon nano tube field effect transistor biosensor and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biosensors, in particular to a carbon nano tube field effect transistor biosensor and a preparation method thereof. The preparation method provided by the invention comprises the following steps: providing a sensor; after undecyl trimethoxy silane modification is carried out on a sensor channel of the sensor, depositing a carbon nano tube wrapped by polycarbazole to obtain a carbon nano tube layer wrapped by polycarbazole; and after polylysine modification is carried out on the carbon nano tube layer wrapped by the polycarbazole, gold nano particles are deposited on the surface of the obtained polylysine layer, and biological probe modification is carried out on the obtained gold nano particles, so that the carbon nano tube field effect transistor biosensor is obtained. The preparation method can well realize the functional preparation of the biosensor.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a carbon nano tube field effect transistor biosensor and a preparation method thereof.
Background
In recent years, the wide application of biosensors in the fields of clinical medicine, food processing and the like has attracted great attention. Biosensors based on Field Effect Transistors (FETs) have been used in a variety of biological applications to detect different biomolecular targets due to their unique advantages of real-time screening, ultrasensitive detection, low cost, and ease of device miniaturization, integration, etc. These targets are often important biomarkers for clinical diagnosis of heart disease, kidney injury, diabetes, cancer, inflammation, and infectious disease. Other potential applications include virus or bacteria detection for diagnosis of infectious diseases such as 2019 for new coronavirus pneumonia, aids, and hepatitis b, and other important biological assays such as metabolites. As these technologies continue to improve, FET biosensors have been identified as good candidates for next generation point of care testing (POCT). Compared to silicon-based FETs, semiconducting Carbon Nanotube (CNT) FETs have quasi-collisional transmission at low voltages, higher transconductance, higher drive current, higher average carrier velocity, lower heat dissipation, and higher switching speed, and can accommodate higher k gate dielectrics with better sensing characteristics.
CNTs have an ultra-thin one-dimensional structure, superior electronic properties and biocompatibility, and are capable of minimizing short channel effects while achieving high carrier transport, which suggests that CNTs are potential channel materials for future high performance scale technologies. However, although great progress has been made in the related studies of carbon nanotube growth and purification, the purity of the semiconductor carbon nanotube solution is <99.9%, which is insufficient for mass production of FET biosensors of high uniformity and stability. Recently, professor team of Beijing university Zhang Zhiyong (Liu, L, han, J, xu, L, zhou, J, zhao, C, ding, S, shi, H, xiao, M, ding, L, ma, Z, jin, C, zhang, Z, & Peng, L.M. (2020) Aligned, high-density semiconducting carbon nanotube arrays for high-performance electronics.science (New York, N.Y.), 368 (6493), 850-856) reported a polymer sorting technique by which semiconducting carbon nanotube solutions with purities higher than 99.9% could be prepared.
Carbon nanotubes are poorly soluble in most media and are prone to van der Waals forces and pi-pi interactions leading to agglomeration. To achieve good dispersion and strong interfacial interactions, functionalization of carbon nanotubes is considered to be an effective method to prevent carbon nanotube agglomeration and improve load transfer at the carbon nanotube and polymer interface. There are two main methods for functionalization of carbon nanotubes; covalent functionalization and non-covalent functionalization. Covalent functionalization is typically achieved by oxidizing the CNT in an acid to attach carboxyl or hydroxyl groups to the ends or defect sites of the CNT. Non-covalent functionalization is the modification of functional molecules such as various inorganic and organic molecules onto the sidewalls of carbon nanotubes by pi-pi stacking, van der Waals forces, or charge transfer interactions. Among these, pi-pi stacking is the strongest interaction between the CNT wall and the functional molecule. The advantage of non-covalent functionalization is that it does not damage the CNT sidewalls, and the reaction conditions are milder. Thus, it is more attractive than covalent methods in maintaining the original structure and properties of carbon nanotubes. However, the high-purity semiconductor carbon nanotube prepared by the polymer sorting technology is wrapped by the polycarbazole, so that the polycarbazole has large molecular weight, has great influence on the dispersibility of the carbon nanotube, has side chains formed in synthesis, has an undefined molecular structure, has weak interaction between the polycarbazole-wrapped carbon nanotube and small molecules pi-pi, has poor stability, and is difficult to perform functionalization to prepare the biosensor.
Disclosure of Invention
The invention aims to provide a carbon nano tube field effect transistor biosensor and a preparation method thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a carbon nano tube field effect transistor biosensor, which comprises the following steps:
providing a sensor; the sensor comprises a substrate and a sensor array arranged on the surface of the substrate, wherein the substrate comprises a silicon matrix and a silicon dioxide layer which are sequentially stacked; the sensor array is positioned on the surface of the silicon dioxide layer; forming a sensor channel between the sensor arrays;
after undecyl trimethoxy silane modification is carried out on a sensor channel of the sensor, depositing a carbon nano tube wrapped by polycarbazole to obtain a carbon nano tube layer wrapped by polycarbazole;
and after polylysine modification is carried out on the carbon nano tube layer wrapped by the polycarbazole, gold nano particles are deposited on the surface of the obtained polylysine layer, and biological probe modification is carried out on the obtained gold nano particles, so that the carbon nano tube field effect transistor biosensor is obtained.
Preferably, before the undecyltrimethoxysilane modification, the method further comprises the step of preprocessing the sensor by adopting an APM solution;
the APM solution is NH with the volume ratio of 1:1:5 4 OH solution, H 2 O 2 A mixed solution of the solution and water;
the NH is 4 The concentration of the OH solution is 10-15 mol/L, the H 2 O 2 The concentration of the solution is 8-10 mol/L.
Preferably, the pretreatment temperature is 60-70 ℃ and the pretreatment time is 1-2 h.
Preferably, the undecyltrimethoxysilane modification comprises the steps of:
and performing heat treatment after spin coating a trichloroethylene solution of undecyltrimethoxysilane on the surface of a sensor channel of the sensor.
Preferably, the concentration of the trichloroethylene solution of the undecyltrimethoxysilane is 1-5 mmol/L; the spin coating amount is 0.01-0.05 mm 2 /μL；
The temperature of the heat treatment is 100-200 ℃ and the time is 5-30 min.
Preferably, the process of depositing the polycarbazole-coated carbon nanotube includes: immersing the obtained sensor modified by undecyltrimethylsilane in toluene solution of a carbon nano tube wrapped by polycarbazole, and then carrying out annealing treatment;
the annealing treatment temperature is 150-250 ℃, and the heat preservation time is 5-20 min.
Preferably, the polylysine modification comprises:
dripping polylysine solution on the surface of the carbon nano tube layer wrapped by the polycarbazole, and then heating and baking;
the concentration of the polylysine solution is 0.01-0.2 mg/mL, and the polylysine solution is 1mm in length 2 The dropping amount in the solution is 100-500 mu L, and the weight average molecular weight of the polylysine in the polylysine solution is 15-30 ten thousand;
the heating and baking temperature is 50-100 ℃ and the time is 1-4 h.
Preferably, the deposited gold nanoparticles comprise: covering chloroauric acid aqueous solution on the surface of the polylysine layer, and then incubating in a dark place;
the concentration of the chloroauric acid aqueous solution is 5-20 mu mol/L, and the chloroauric acid aqueous solution is 1mm 2 The internal coating amount is 100 to 500. Mu.L.
Preferably, the biological probe is a thiol DNA probe, a thiol PNA probe or a thiol PMO probe.
The invention also provides a carbon nano tube field effect transistor biosensor prepared by the preparation method, which comprises a sensor, wherein the sensor comprises a substrate and a sensor array on the surface of the substrate, and the substrate comprises a silicon matrix and a silicon dioxide layer which are sequentially laminated; the sensor array is positioned on the surface of the silicon dioxide layer;
the sensor also comprises a polycarbazole wrapped carbon nano tube layer filling the sensor channel;
the carbon nano tube layer wrapped by the polycarbazole is bonded with the silicon dioxide layer through the undecyl trimethoxy silane layer;
the surface of the carbon nano tube layer wrapped by the polycarbazole further comprises a polylysine layer, gold nano particles and a biological probe which are sequentially laminated.
The invention provides a preparation method of a carbon nano tube field effect transistor biosensor, which comprises the following steps: providing a sensor; the sensor comprises a substrate and a sensor array arranged on the surface of the substrate, wherein the substrate comprises a silicon matrix and a silicon dioxide layer which are sequentially stacked; the sensor array is positioned on the surface of the silicon dioxide layer; forming a sensor channel between the sensor arrays; after undecyl trimethoxy silane modification is carried out on a sensor channel of the sensor, depositing a carbon nano tube wrapped by polycarbazole to obtain a carbon nano tube layer wrapped by polycarbazole; and after polylysine modification is carried out on the carbon nano tube layer wrapped by the polycarbazole, gold nano particles are deposited on the surface of the obtained polylysine layer, and biological probe modification is carried out on the obtained gold nano particles, so that the carbon nano tube field effect transistor biosensor is obtained. The invention carries out undecyl trimethoxysilane modification on the sensor channel, thereby enhancing the adsorption of the chip to the semiconductor carbon nano tube; polylysine modification is performed on the carbon nanotube by using the pi-pi effect of polylysine amide bond and polycarbazole to functionalize the polycarbazole-wrapped carbon nanotube, and the biocompatibility of the polycarbazole-wrapped carbon nanotube is utilized to improve the efficiency of detecting biomolecules by the carbon nanotube; the nano gold particles are used as biological probe connecting agents, and can be connected with sulfhydrylation molecular probes through Au-S bonds so as to detect corresponding targets.
Compared with the prior art, the preparation method has the following beneficial effects:
the preparation method is simple and the manufacturing cost is low;
the preparation method can realize the wrapping of the polymer on the semiconductor carbon nano tube, further realize the functionalization of the carbon nano tube, reduce the damage to the side wall structure and the performance of the carbon nano tube, retain the intrinsic superior performance of the semiconductor carbon nano tube, and have good biocompatibility, thereby being more beneficial to preparing the biosensor with high sensitivity, good uniformity and stable performance.
Drawings
FIG. 1 is a schematic diagram of a manufacturing process of a carbon nanotube field effect transistor biosensor according to the present invention;
FIG. 2 is a schematic diagram of the structure of the CNT FET biosensor; wherein, the 1-undecyl trimethoxy silane modified layer, the 2-gold nanoparticle, the 7-silicon substrate, the 8-silicon dioxide layer, the carbon nano tube layer wrapped by 9-polycarbazole, the 10-polylysine layer, the 11-source electrode, the 12-drain electrode and the 13-biological probe;
FIG. 3 is a schematic diagram of a sensor array; wherein, 3-electric pad, 4-wire, 5-metal electrode, 6-sensor array;
FIG. 4 is an SEM image of the carbon nanotubes prepared in example 1;
FIG. 5 is a graph showing the Raman characterization of example 1 before and after polylysine modification;
fig. 6 is an SEM image of gold nanoparticles prepared in example 1.
Detailed Description
According to the flow chart shown in fig. 1, the invention provides a preparation method of a carbon nano tube field effect transistor biosensor, which comprises the following steps:
providing a sensor; the sensor comprises a substrate and a sensor array arranged on the surface of the substrate, wherein the substrate comprises a silicon matrix and a silicon dioxide layer which are sequentially stacked; the sensor array is positioned on the surface of the silicon dioxide layer; forming a sensor channel between the sensor arrays;
after undecyl trimethoxy silane modification is carried out on a sensor channel of the sensor, depositing a carbon nano tube wrapped by polycarbazole to obtain a carbon nano tube layer wrapped by polycarbazole;
and after polylysine modification is carried out on the carbon nano tube layer wrapped by the polycarbazole, gold nano particles are deposited on the surface of the obtained polylysine layer, and biological probe modification is carried out on the obtained gold nano particles, so that the carbon nano tube field effect transistor biosensor is obtained.
In the present invention, all the preparation materials are commercially available products well known to those skilled in the art unless specified otherwise.
Providing a sensor; the sensor comprises a substrate and a sensor array on the surface of the substrate, wherein the substrate comprises a silicon base and a silicon dioxide layer (shown in figure 2) which are sequentially stacked; the sensor array is positioned on the surface of the silicon dioxide layer; a sensor channel is formed between the sensor arrays.
In the present invention, the sensor array is preferably constituted by a metal electrode; the metal electrodes are preferably arranged in parallel (as shown in fig. 3); two adjacent metal electrodes form a group of metal electrode pairs, a sensor channel is formed between the two metal electrodes of the metal electrode pairs, one metal electrode of the metal electrode pairs is used as a source electrode shown in fig. 2, and the other metal electrode is used as a drain electrode shown in fig. 2. In the present invention, the number of the metal electrodes is preferably 7, and the number of the corresponding metal electrode pairs is preferably 6.
In the present invention, the surface of the silicon dioxide layer is also preferably provided with an electric pad and a wire; the electrical pad, wire and metal electrode are connected in sequence (as shown in fig. 3). In the present invention, the electric pad is preferably connected with a signal monitoring device.
And after the sensor channel of the sensor is modified by undecyltrimethoxysilane, depositing a carbon nano tube wrapped by polycarbazole to obtain a carbon nano tube layer wrapped by polycarbazole.
The pretreatment of the sensor with APM solution is also preferably included before the undecyltrimethoxysilane modification.
In the present invention, the APM solution is preferably NH in a volume ratio of 1:1:5 4 OH solution, H 2 O 2 A mixed solution of the solution and water; the NH is 4 The concentration of the OH solution is preferably 10 to 15mol/L, more preferably 13.38mol/L, the H 2 O 2 The concentration of the solution is preferably 8 to 10mol/L, more preferably 9.79mol/L.
In the present invention, the temperature of the pretreatment is preferably 60 to 70 ℃, more preferably 65 ℃; the time is preferably 1 to 2 hours, more preferably 1 hour.
In the present invention, the pretreatment process is preferably immersing the sensor in the APM solution.
After the pretreatment, the invention also preferably comprises the steps of cleaning and drying sequentially; the cleaning is preferably performed by pure water; the drying is preferably performed by drying with nitrogen. The specific process of rinsing and drying is not particularly limited in the present invention, and may be performed by a process well known to those skilled in the art.
In the present invention, the pretreatment is performed to remove impurities such as particles attached to the surface.
In the present invention, the undecyltrimethoxysilane modification preferably comprises the steps of:
and performing heat treatment after spin coating a trichloroethylene solution of undecyltrimethoxysilane on the surface of a sensor channel of the sensor.
In the present invention, the concentration of the trichloroethylene solution of undecyltrimethoxysilane is preferably 1 to 5mmol/L, more preferably 2 to 4mmol/L, and most preferably 3mmol/L; the spin coating amount is preferably 0.01 to 0.05mm 2 mu.L, more preferably 0.05mm 2 mu.L. In the present invention, the spin-coating speed is preferably 1000 to 3000rpm, more preferably 1500 to 2500rpm, and most preferably 2000rpm.
In the present invention, the temperature of the heat treatment is preferably 100 to 200 ℃, more preferably 100 to 150 ℃, and most preferably 120 ℃; the time is preferably 5 to 30 minutes, more preferably 10 to 20 minutes, and most preferably 10 minutes. In the present invention, the heat treatment is preferably performed by heat baking.
In the invention, the undecyltrimethoxysilane modification is used for enhancing the adsorption of the chip to the semiconductor carbon nano tube.
In the present invention, the depositing a polycarbazole-coated carbon nanotube preferably includes: and soaking the obtained sensor modified by undecyltrimethylsilane in a polycarbazole-coated carbon nanotube toluene solution, and then carrying out annealing treatment.
In the present invention, the concentration of the polycarbazole-coated carbon nanotube toluene solution is preferably 10 to 20. Mu.g/mL, more preferably 10 to 15. Mu.g/mL, and most preferably 12.5. Mu.g/mL; the temperature of the impregnation is preferably 4 to 25 ℃, more preferably 15 to 25 ℃, most preferably 20 ℃, and the time is preferably 15 to 60min, more preferably 15 to 40min, most preferably 30min.
The source of the polycarbazole-coated carbon nanotube is not particularly limited, and the polycarbazole-coated carbon nanotube can be prepared by a preparation process well known to those skilled in the art.
After the impregnation is completed, the invention also preferably comprises a cleaning process; the washing preferably includes washing the excess carbon nanotubes with toluene, and the washing with toluene is not particularly limited in the present invention, and may be performed by a process well known to those skilled in the art. In the present invention, the washing also preferably includes washing with a toluene solution of trifluoroacetic acid and toluene in this order at a volume concentration of 0.1% for 2 minutes each. In the present invention, the purpose of the cleaning is to remove excess polycarbazole.
In the present invention, the temperature of the annealing treatment is preferably 150 to 250 ℃, more preferably 180 to 220 ℃, and most preferably 200 ℃; the time is preferably 5 to 20 minutes, more preferably 5 to 10 minutes, and most preferably 5 minutes. In the present invention, the annealing treatment serves to stabilize the carbon nanotube bonding.
After the annealing treatment is completed, the present invention also preferably includes repeating the above process of depositing carbon dioxide. In the present invention, repeating the above process of depositing carbon dioxide can further improve uniformity and density of carbon nanotubes.
In the invention, the preparation process can prepare the high-purity (the purity can reach 99.99995%) semiconductor carbon nano-tube on a large scale.
After a polycarbazole-coated carbon nano tube layer is obtained, the invention carries out polylysine modification on the polycarbazole-coated carbon nano tube layer, deposits gold nano particles on the surface of the obtained polylysine layer, carries out biological probe modification on the gold nano particles, and obtains the carbon nano tube field effect transistor biosensor
In the present invention, the polylysine modification preferably includes:
and (3) dripping a polylysine solution on the surface of the carbon nano tube layer wrapped by the polycarbazole, and then heating and baking.
In the present invention, the concentration of the polylysine solution is preferably 0.01-0.2 mg/mL, more preferably 0.01-0.05 mg/mL, and most preferably 0.01mg/mL; the molecular weight of polylysine in the polylysine solution is preferably 15-30 ten thousand; the solvent in the polylysine solution is preferably double distilled water (ddH 2 O)。
The dropping rate of the polylysine solution is not particularly limited in the present invention, and the polylysine solution may be dropped at a dropping rate well known to those skilled in the art.
In the present invention, the polylysine solution is used in a unit area (1 mm 2 ) The amount to be added is preferably 100 to 500. Mu.L, more preferably 100 to 300. Mu.L, and most preferably 200. Mu.L.
In the present invention, the temperature of the heating and baking is preferably 50 to 100 ℃, more preferably 60 to 80 ℃, and most preferably 80 ℃; the time is preferably 1 to 4 hours, more preferably 1 to 2 hours, and most preferably 2 hours.
After the heating and baking are finished, the invention also preferably comprises cleaning; the cleaning agent used for the cleaning is preferably water. In the present invention, the purpose of the washing is to remove excess polylysine solution.
In the present invention, the process of depositing gold nanoparticles includes: and (3) covering the surface of the polylysine layer with chloroauric acid aqueous solution, and then incubating in a dark place.
In the present invention, the concentration of the aqueous chloroauric acid solution is preferably 5 to 20. Mu. Mol/L, more preferably 5 to 10. Mu. Mol/L, and most preferably 10. Mu. Mol/L. In the present invention, the aqueous chloroauric acid solution was used in a unit area (1 mm 2 ) The amount to be added is preferably 100 to 500. Mu.L, more preferably 100 to 300. Mu.L, and most preferably 200. Mu.L.
In the present invention, the temperature of the light-shielding incubation is preferably room temperature, and the time is preferably 8 to 12 hours, more preferably 8 hours.
In the present invention, the bioprobe in the bioprobe modification is preferably a thiol DNA aptamer probe, a thiol PNA probe, or a thiol PMO probe. In the present invention, the biological probes are purchased from Shanghai Limited of bioengineering.
In the present invention, the biological probe modification process preferably includes:
and (3) dropwise adding the activated aptamer probe solution, and incubating.
In the present invention, the concentration of the activated aptamer probe solution is preferably 5 to 100. Mu. Mol/L, more preferably 5 to 20. Mu. Mol/L, and most preferably 10. Mu. Mol/L. In the present invention, the solvent in the activated aptamer probe solution is preferably ddH 2 O. In the present invention, the activated aptamer probe solution is used in a specific area (1 mm 2 ) The amount to be added is preferably 100 to 500. Mu.L, more preferably 100 to 300. Mu.L, and most preferably 200. Mu.L.
In the invention, when the aptamer probe in the activated aptamer probe solution is a sulfhydryl DNA aptamer probe, the activating agent used is preferably 10mmol/L tris (2-chloroethyl) phosphate solution; the solvent of the tris (2-chloroethyl) phosphate solution is preferably ddH 2 O。
In the present invention, when the aptamer probe in the activated aptamer probe solution is a thiol PNA aptamer probe, the activating agent used is preferably 10mmol/L tris (2-chloroethyl) phosphate solution; the solvent in the activator is preferably ddH 2 O。
In the invention, when the aptamer probe in the activated aptamer probe solution is a sulfhydryl PMO aptamer probe, the activating agent adopted is preferably 10mmol/L tris (2-chloroethyl) phosphate solution; the solvent in the activator is preferably ddH 2 O。
The process of activating the aptamer probe with the activator is not particularly limited, and may be performed by any process known to those skilled in the art.
In the present invention, the temperature of the incubation is preferably room temperature; the time is preferably 8 to 24 hours, more preferably 8 to 12 hours, and most preferably 8 hours.
After the incubation is completed, the present invention also preferably includes washing and drying sequentially. In the present invention, the washing is preferably performed by sequentially washing with a phosphate buffer solution having a pH of 7.4 and ultrapure water; the purpose of the washing is to remove the non-immobilized aptamer probes. In the present invention, the drying is preferably performed by drying with nitrogen.
After the biological probe modification process is completed, the method also preferably comprises immersing the obtained sensor modified with the aptamer probe in a Bovine Serum Albumin (BSA) solution with the mass concentration of 1% for 1h, flushing with ultrapure water and drying with nitrogen. In the invention, the bovine serum albumin has the function of blocking redundant active sites on the surface of polylysine and unbound gold nanoparticles, thereby avoiding the generation of nonspecific adsorption.
The invention also provides a carbon nano tube field effect transistor biosensor prepared by the preparation method, which comprises a sensor, wherein the sensor comprises a substrate and a sensor array on the surface of the substrate, and the substrate comprises a silicon matrix and a silicon dioxide layer (shown in figure 2) which are sequentially laminated; the sensor array is positioned on the surface of the silicon dioxide layer;
the sensor array comprises a sensor array channel, a polycarbazole wrapped carbon nano tube layer and a sensor array layer, wherein the sensor array channel is filled with polycarbazole wrapped carbon nano tube layer;
the carbon nano tube layer wrapped by the polycarbazole is bonded with the silicon dioxide layer through the undecyl trimethoxy silane layer;
the surface of the carbon nano tube layer wrapped by the polycarbazole further comprises a polylysine layer, gold nano particles and a biological probe which are sequentially stacked.
In the present invention, the sensor array is preferably constituted by a metal electrode; the metal electrodes are preferably arranged in parallel (as shown in fig. 3); two adjacent metal electrodes form a group of metal electrode pairs, a sensor channel is formed between the two metal electrodes of the metal electrode pairs, one metal electrode of the metal electrode pairs is used as a source electrode shown in fig. 2, and the other metal electrode is used as a drain electrode shown in fig. 2. In the present invention, the number of the metal electrodes is preferably 7, and the number of the corresponding metal electrode pairs is preferably 6.
In the present invention, the surface of the silicon dioxide layer is also preferably provided with an electric pad and a wire; the electrical pad, wire and metal electrode are connected in sequence (as shown in fig. 3). In the present invention, the electric pad is preferably connected with a signal monitoring device.
In the present invention, the thickness of the polylysine layer is preferably 2 to 10nm, more preferably 2 to 6nm.
In the present invention, the gold nanoparticles preferably have a thickness of 10 to 100nm, more preferably 10 to 50nm.
In the present invention, the biological probe is preferably a thiol DNA aptamer probe, a thiol PNA probe, or a thiol PMO probe. The thickness of the biological probe is preferably 5 to 25nm, more preferably 8 to 20nm, and most preferably 10nm.
The carbon nanotube field effect transistor biosensor and the method of manufacturing the same according to the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Providing a sensor; the sensor comprises a substrate and a sensor array on the surface of the substrate, wherein the substrate comprises a silicon base and a silicon dioxide layer (shown in figure 2) which are sequentially stacked; the sensor array is positioned on the surface of the silicon dioxide layer.
In the invention, the sensor array comprises 7 metal electrodes which are arranged in parallel; two adjacent metal electrodes form a group of metal electrode pairs, a sensor channel is formed between the two metal electrodes of the metal electrode pairs, one metal electrode of the metal electrode pairs is used as a source electrode shown in fig. 2, and the other metal electrode is used as a drain electrode shown in fig. 2; the 7 parallel metal electrodes are respectively connected with a wire and an electric pad in sequence (as shown in figure 3); the electric pad is connected with a signal monitoring device;
immersing the sensor inAPM solution (NH) 4 OH solution/H 2 O 2 solution/H 2 The volume ratio of O is 1:1:5, the NH 4 The concentration of the OH solution was 13.38mol/L, the H 2 O 2 The concentration of the solution was 9.79 mol/L), after treatment at 65℃for 1 hour, rinsing with pure water, drying with nitrogen, spin coating at 2000rpm was carried out at 0.05mm 2 1 mu L of undecyl trimethoxysilane trichloroethylene solution with the concentration of 3mmol/L is internally coated in the area unit of the (B), and then the mixture is heated and baked for 10min at 120 ℃; immersing the carbon nano tube into toluene solution of which the concentration is 12.5 mu g/mL and coating the carbon nano tube with polycarbazole for 30min, washing off redundant toluene solution of the polycarbazole coated carbon nano tube by toluene, washing off redundant coating polymer by toluene solution of trifluoroacetic acid with volume percentage of 0.1% and toluene for 2min, annealing at 200 ℃ for 5min, and repeating the processes of immersing and washing in the toluene solution of the polycarbazole coated carbon nano tube for 1 time so as to improve the uniformity and density of the carbon nano tube;
at 0.05mm 2 10 mu L of polylysine solution (ddH solvent) with molecular weight of 15-30 ten thousand and concentration of 0.01mg/mL is dripped on the carbon nano tube layer coated by polycarbazole in the area unit 2 O), baking at 80 ℃ for 2 hours, and washing with pure water to remove excessive polylysine solution; covering 10 mu L of chloroauric acid aqueous solution with the concentration of 10mmol/L in a sensor array, and incubating for 8 hours at room temperature in a dark place to obtain gold nanoparticles;
10mmol/L tris (2-chloroethyl) phosphate solution (ddH as solvent 2 O) after the thiol DNA aptamer probe was activated, a thiol DNA aptamer probe solution (ddH as solvent) was prepared at a concentration of 10. Mu. Mol/L 2 O)；
After 10. Mu.L of the activated thiol DNA aptamer probe solution is dripped on the surface of the gold nanoparticle, the gold nanoparticle is incubated at room temperature for 8 hours, the unfixed probe is washed away by phosphate buffer solution with pH of 7.4 and ultrapure water in sequence, and after the nitrogen is blown dry, the gold nanoparticle is immersed in bovine serum albumin solution with the mass concentration of 1% (the solvent is ddH) 2 O) for 1h to block superfluous active sites on the surface of polylysine and unbound gold nanoparticles, avoiding the generation of nonspecific adsorption by using superwavesWashing with pure water, and drying with nitrogen to obtain the carbon nanotube field effect transistor biosensor;
FIG. 4 is an SEM image of the polycarbazole-coated carbon nanotube layer, and FIG. 4 shows that the carbon nanotubes are uniformly distributed in the sensing channel;
FIG. 5 is a Raman spectrum of the carbon nanotube and the polylysine layer prepared by the method, and as can be seen from FIG. 5, polylysine is successfully modified on the carbon nanotube;
fig. 6 is an SEM image of gold nanoparticles prepared by the above method, and as can be seen from fig. 6, the gold nanoparticles are uniformly modified on the polylysine layer.
Example 2
Providing a sensor; the sensor comprises a substrate and a sensor array on the surface of the substrate, wherein the substrate comprises a silicon base and a silicon dioxide layer (shown in figure 2) which are sequentially stacked; the sensor array is positioned on the surface of the silicon dioxide layer.
In the invention, the sensor array comprises 7 metal electrodes which are arranged in parallel; two adjacent metal electrodes form a group of metal electrode pairs, a sensor channel is formed between the two metal electrodes of the metal electrode pairs, one metal electrode of the metal electrode pairs is used as a source electrode shown in fig. 2, and the other metal electrode is used as a drain electrode shown in fig. 2; the 7 parallel metal electrodes are respectively connected with a wire and an electric pad in sequence (as shown in figure 3); the electric pad is connected with a signal monitoring device;
immersing the sensor in APM solution (NH 4 OH solution/H 2 O 2 solution/H 2 The volume ratio of O is 1:1:5, the NH 4 The concentration of the OH solution was 13.38mol/L, the H 2 O 2 The concentration of the solution was 9.79 mol/L), after treatment at 65℃for 1 hour, rinsing with pure water, drying with nitrogen, spin coating at 2000rpm was carried out at 0.05mm 2 1 mu L of undecyl trimethoxysilane trichloroethylene solution with the concentration of 3mmol/L is internally coated in the area unit of the (B), and then the mixture is heated and baked for 10min at 120 ℃; then immersing the carbon nano tube in toluene solution with the concentration of 12.5 mug/mL for 30min, and then washing the carbon nano tube with tolueneRemoving redundant toluene solution of the carbon nano tube wrapped by the polycarbazole, respectively washing for 2min by using toluene solution of trifluoroacetic acid and toluene with volume percentage of 0.1%, washing off redundant wrapping polymer, annealing for 5min at 200 ℃, and repeating the processes of soaking and washing in the toluene solution of the carbon nano tube wrapped by the polycarbazole for 1 time so as to improve the uniformity and density of the carbon nano tube;
at 0.05mm 2 10 mu L of polylysine solution (ddH solvent) with molecular weight of 15-30 ten thousand and concentration of 0.01mg/mL is dripped on the carbon nano tube layer coated by polycarbazole in the area unit 2 O), baking at 80 ℃ for 2 hours, and washing with pure water to remove excessive polylysine solution; covering 10 mu L of chloroauric acid aqueous solution with the concentration of 10mmol/L in a sensor array, and incubating for 8 hours at room temperature in a dark place to obtain gold nanoparticles;
10mmol/L tris (2-chloroethyl) phosphate solution (ddH as solvent 2 O) after the thiol DNA aptamer probe was activated, a thiol DNA aptamer probe solution (ddH as solvent) was prepared at a concentration of 10. Mu. Mol/L 2 O)；
After 10. Mu.L of the activated thiol DNA probe solution is dripped on the surface of the gold nanoparticle, the gold nanoparticle is incubated for 10 hours at room temperature, the unfixed probe is washed out by phosphate buffer solution with pH of 7.4 and ultrapure water in sequence, and after the nitrogen is blown dry, the gold nanoparticle is immersed in bovine serum albumin solution with the mass concentration of 1 percent (the solvent is ddH 2 And O) for 1h, so as to seal redundant active sites on the surface of polylysine and unbound gold nanoparticles, avoid the generation of nonspecific adsorption, flush with ultrapure water, and blow-dry with nitrogen, thereby obtaining the carbon nanotube field effect transistor biosensor.
Test example 1
The carbon nanotube field effect transistor biosensor prepared in example 1 is subjected to multiple groups of parallel detection, and the specific detection process is as follows:
1) Firstly, dropwise adding 10 mu L of 0.01 XPBS (100 times diluted standard PBS) buffer solution at a sensor array, enabling the buffer solution to cover 6 groups of metal electrode pairs, inserting a silver wire on the buffer solution drop to serve as a grid electrode, respectively connecting two metal electrodes of each group of metal electrode pairs with an electric pad, connecting the electric pad, the grid electrode and an outer side signal detection device to form an independent current path, and measuring current corresponding to each group of non-protein-combined sulfhydryl DNA aptamer probes;
2) Then, dropwise adding 10 mu L of reaction solution (MUC 1 protein solution prepared by molecular biological water) containing a sample to be detected at a sensing array, enabling the reaction liquid drop to cover 6 groups of metal electrode pairs, after the protein in the sample is incubated with a sulfhydryl DNA aptamer probe on a carbon nano tube for 0.5 hour, washing and drying, dropwise adding 10 mu L of 0.01 xPBS buffer solution at the sensing array, enabling the buffer solution to cover 6 groups of metal electrode pairs, inserting a silver wire on the buffer liquid drop as a grid electrode, connecting two metal electrodes of each group of metal electrode pairs with an electric pad respectively, and connecting the electric pad, the grid electrode and an outer side signal detection device to form an independent current path, and measuring currents corresponding to the sulfhydryl DNA aptamer probe and the protein after combination, thereby forming 6 independent current paths to realize 6 groups of parallel detection of the sample to be detected.
The detection results were that the aqueous solution current response values of MUC1 protein at a concentration of 1nmol/L were 6.44, 7.38, 7.31, 7.07, 6.87 and 7.01. Mu.A.
Test example 2
The carbon nanotube field effect transistor biosensor prepared in example 2 is subjected to multiple groups of parallel detection, and the specific detection process is as follows:
1) Firstly, dropwise adding 10 mu L of 0.01 XPBS (100 times diluted standard PBS) buffer solution at a sensor array, enabling the buffer solution to cover 6 groups of metal electrode pairs, inserting a silver wire on the buffer solution drop to serve as a grid electrode, respectively connecting two metal electrodes of each group of metal electrode pairs with an electric pad, connecting the electric pad, the grid electrode and an outer side signal detection device to form an independent current path, and measuring current corresponding to each group of mercapto DNA probes which are not combined with miRNA 21;
2) Then, dropwise adding 10 mu L of miRNA21 aqueous solution at a sensing array, enabling the reaction liquid drop to cover 6 groups of metal electrode pairs, washing and drying after miRNA21 in a sample and a sulfhydryl DNA probe on a carbon nano tube are incubated for 0.5 hour, and dropwise adding 10 mu L of 0.01 XPBS buffer solution at the sensing array, so that the buffer solution covers 6 groups of metal electrode pairs, and carrying out multi-group parallel detection on the carbon nano tube field effect transistor biosensor prepared in the embodiment 1, wherein the specific detection process comprises the following steps:
1) Firstly, dropwise adding 10 mu L of 0.01 XPBS (100 times diluted standard PBS) buffer solution at a sensor array, enabling the buffer solution to cover 6 groups of metal electrode pairs, inserting a silver wire on the buffer solution drop to serve as a grid electrode, respectively connecting two metal electrodes of each group of metal electrode pairs with an electric pad, connecting the electric pad, the grid electrode and an outer side signal detection device to form an independent current path, and measuring current corresponding to each group of mercapto DNA probes which are not combined with miRNA 21;
2) Then, a drop of 10 mu L miRNA21 aqueous solution is dripped at the sensing array, the reaction drop covers 6 groups of metal electrode pairs, after miRNA21 in a sample is incubated with a sulfhydryl DNA probe on a carbon nano tube for 0.5 hour, washing and blow-drying are performed, 10 mu L of 0.01PBS buffer solution is dripped at the sensing array, the buffer solution covers 6 groups of metal electrode pairs, a silver wire is inserted into the buffer solution drop as a grid electrode, two metal electrodes of each group of metal electrode pairs are respectively connected with the electrode pads, and then the electrode pads, the grid electrode and an outer side signal detection device are connected to form an independent current path, and currents corresponding to the combined sulfhydryl DNA probe and miRNA21 are measured, so that 6 independent current paths are formed to realize 6 groups of parallel detection of the sample to be detected.
The detection results are that the current response values of the miRNA21 aqueous solution with the concentration of 1fM are 8.98, 9.04, 9.01, 9.04, 8.95 and 8.99 mu A.
Therefore, the preparation method can be used for preparing the field effect transistor biosensor based on the high-purity semiconductor carbon nano tube in a large scale, and is simple and low in manufacturing cost. The preparation method can functionalize the polymer-wrapped semiconductor carbon nanotube under mild conditions, reduces damage to the side wall structure and performance of the carbon nanotube, retains the intrinsic superior performance of the semiconductor carbon nanotube, has good biocompatibility, and is more beneficial to preparing the biosensor with high sensitivity, good uniformity and stable performance. The carbon nano tube field effect transistor biosensor prepared by the embodiment can realize the purpose of simultaneously carrying out multiple groups of parallel detection on the same sample, and has the advantages of high sensitivity, high analysis speed, small sample consumption, low cost and easiness in mass preparation.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A preparation method of a carbon nano tube field effect transistor biosensor comprises the following steps:
providing a sensor; the sensor comprises a substrate and a sensor array arranged on the surface of the substrate, wherein the substrate comprises a silicon matrix and a silicon dioxide layer which are sequentially stacked; the sensor array is positioned on the surface of the silicon dioxide layer; forming a sensor channel between the sensor arrays;
after undecyl trimethoxy silane modification is carried out on a sensor channel of the sensor, depositing a carbon nano tube wrapped by polycarbazole to obtain a carbon nano tube layer wrapped by polycarbazole;
and after polylysine modification is carried out on the carbon nano tube layer wrapped by the polycarbazole, gold nano particles are deposited on the surface of the obtained polylysine layer, and biological probe modification is carried out on the obtained gold nano particles, so that the carbon nano tube field effect transistor biosensor is obtained.
2. The method of preparing of claim 1, further comprising pre-treating the sensor with APM solution prior to performing the undecyltrimethoxysilane modification;
the APM solution is NH with the volume ratio of 1:1:5 4 OH solution, H 2 O 2 A mixed solution of the solution and water;
the NH is 4 The concentration of the OH solution is 10-15 mol/L, the H 2 O 2 SolutionThe concentration of (C) is 8-10 mol/L.
3. The method according to claim 2, wherein the pretreatment is carried out at a temperature of 60 to 70 ℃ for a time of 1 to 2 hours.
4. The method of preparation of claim 1, wherein the undecyltrimethoxysilane modification comprises the steps of:
and performing heat treatment after spin coating a trichloroethylene solution of undecyltrimethoxysilane on the surface of a sensor channel of the sensor.
5. The method according to claim 4, wherein the concentration of the trichloroethylene solution of undecyltrimethoxysilane is 1 to 5mmol/L; the spin coating amount is 0.01-0.05 mm 2 /μL；
The temperature of the heat treatment is 100-200 ℃ and the time is 5-30 min.
6. The method of preparing of claim 1, wherein depositing the polycarbazole-wrapped carbon nanotubes comprises: immersing the obtained sensor modified by undecyltrimethylsilane in toluene solution of a carbon nano tube wrapped by polycarbazole, and then carrying out annealing treatment;
the annealing treatment temperature is 150-250 ℃, and the heat preservation time is 5-20 min.
7. The method of preparation of claim 1, wherein the polylysine modification comprises:
dripping polylysine solution on the surface of the carbon nano tube layer wrapped by the polycarbazole, and then heating and baking;
the concentration of the polylysine solution is 0.01-0.2 mg/mL, and the polylysine solution is 1mm in length 2 The dropping amount in the solution is 100-500 mu L, and the weight average molecular weight of the polylysine in the polylysine solution is 15-30 ten thousand;
the heating and baking temperature is 50-100 ℃ and the time is 1-4 h.
8. The method of preparing according to claim 1, wherein the depositing gold nanoparticles comprises: covering chloroauric acid aqueous solution on the surface of the polylysine layer, and then incubating in a dark place;
the concentration of the chloroauric acid aqueous solution is 5-20 mu mol/L, and the chloroauric acid aqueous solution is 1mm 2 The internal coating amount is 100 to 500. Mu.L.
9. The method of claim 1, wherein the biological probe is a thiol DNA probe, a thiol PNA probe, or a thiol PMO probe.
10. The carbon nanotube field effect transistor biosensor prepared by the preparation method of any one of claims 1 to 9, which is characterized by comprising a sensor, wherein the sensor comprises a substrate and a sensor array on the surface of the substrate, and the substrate comprises a silicon matrix and a silicon dioxide layer which are sequentially laminated; the sensor array is positioned on the surface of the silicon dioxide layer;
the sensor also comprises a polycarbazole wrapped carbon nano tube layer filling the sensor channel;
the carbon nano tube layer wrapped by the polycarbazole is bonded with the silicon dioxide layer through the undecyl trimethoxy silane layer;
the surface of the carbon nano tube layer wrapped by the polycarbazole further comprises a polylysine layer, gold nano particles and a biological probe which are sequentially laminated.
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