CN114354722A - Multichannel field effect transistor nano biosensor and preparation method and application thereof - Google Patents
Multichannel field effect transistor nano biosensor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a multichannel field effect transistor nano biosensor and a preparation method and application thereof, and belongs to the field of analysis and detection. The multichannel field effect transistor nano biosensor realizes the simultaneous and rapid detection of a plurality of bladder cancer markers in a urine sample by establishing the corresponding relation between the concentration of the bladder cancer markers and the electric signals of an Indium Gallium Zinc Oxide (IGZO) field effect transistor sensing device. The field effect transistor nano biosensor can realize simultaneous and rapid detection of a plurality of cancer markers in a urine sample, and well makes up for the defects in the current bladder cancer diagnosis field. The sensor has the advantages of high detection flux, high sensitivity, good selectivity, high detection speed, good reproducibility and the like, and has wide clinical application prospect in the field of tumor marker detection.
Description
Technical Field
The invention relates to the field of analysis and detection, in particular to a multichannel field effect transistor nano biosensor and a preparation method and application thereof.
Technical Field
Bladder cancer is one of the most common malignant tumors of the urinary system, and has the characteristics of strong invasiveness, high recurrence rate and the like. Approximately 75% of patients with new onset bladder cancer are non-muscle invasive bladder cancer and 25% progress to the muscle invasive stage. The early diagnosis has important significance for preventing the patient from developing muscle invasive bladder cancer and improving the survival rate of the patient. Non-muscle invasive bladder cancer does not endanger life, but the five-year recurrence rate after local treatment is as high as 50-70%. In order to prevent the disease from deteriorating, the patient needs to regularly review at any time and keep on long-term follow-up. At present, the clinical means for detecting bladder cancer mainly include gold standard cystoscopy, tissue biopsy and exfoliative urine cytology. Gold standard cystoscopy and tissue biopsy are highly invasive, can bring great pain to patients, can also cause urinary tract injury, infection and the like, and have poor patient compliance. The urine exfoliative cytology belongs to a non-invasive detection means, but has low sensitivity and can not find early canceration. Therefore, there is a need to develop a non-invasive and sensitive detection technique for early screening and prognostic monitoring of bladder cancer.
The bladder acts as a urinary reservoir, and the occurrence and progression of bladder cancer can have a direct impact on the urine composition. Therefore, the development of the urine-based liquid biopsy technology can provide convenient conditions for early diagnosis and postoperative monitoring of bladder cancer, and urine monitoring also has the advantages of being noninvasive and capable of repeatedly sampling. The urine contains multiple bladder cancer markers, such as NMP22, miRNA, bladder tumor antigen, fibrinogen degradation product and bladder cancer cells. Considering the differences among individuals and the heterogeneity of solid tumors, the complexity of tumors is difficult to reflect by using only a single marker as a detection standard, and false positive or false negative results are easy to occur. The types and the contents of the markers expressed by the tumors at different stages are different, and the combined detection of different tumor markers can improve the diagnosis accuracy, realize the staging and grading of the tumors, facilitate the adoption of different treatment means at different development stages of the tumors and achieve the optimal treatment effect.
The field effect transistor sensor has the functions of signal conversion and signal amplification, realizes quantitative detection of a target substance through current signal change caused by combination of target molecules and a semiconductor channel material, and has the advantages of high sensitivity, good selectivity, easiness in integration and the like. Field effect transistor sensors have been widely used in the fields of protein, nucleic acid and bacteria detection. The advantage that the field effect transistor sensor is easy to integrate is utilized, the field effect transistor array is developed, the multi-channel sensor is constructed, the simultaneous detection of multiple bladder cancer markers in urine is hopefully realized, the detection accuracy is improved, and the method has great clinical application value in the fields of cancer diagnosis, postoperative monitoring and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a multichannel field effect transistor nano biosensor and a preparation method and application thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a method for preparing a multichannel field effect transistor nano biosensor, which is characterized by comprising: preparing a gold electrode by using an ultraviolet lithography and metal deposition method, growing an Indium Gallium Zinc Oxide (IGZO) channel material by using a magnetron sputtering method, modifying a recognition molecule by using a chemical modification method, and finally assembling a Polydimethylsiloxane (PDMS) chamber and a device to realize the construction of a high-sensitivity, high-selectivity and high-flux multichannel IGZO field effect transistor sensor, wherein the construction comprises the following steps:
s1: preparing a gold electrode:
s2: preparing an IGZO channel material:
s3: modification of recognition molecules:
s4: the transistor and reservoir are assembled.
Preferably, the gold electrode is prepared in step S1, and specifically, the gold electrode is prepared as follows:
s1.1: designing an electrode pattern consisting of 5 source electrodes, 1 drain electrode and 1 grid electrode by adopting CAD software, and processing the electrode pattern into a corresponding mask for subsequent photoetching operation;
s1.2: selecting a p-type silicon wafer with a 300nm silicon oxide layer as a substrate, uniformly spin-coating a layer of positive photoresist AZ521 on the surface of the p-type silicon wafer, then placing the silicon wafer spin-coated with the photoresist on a heating plate, and heating at 115 ℃ for 4min to solidify the photoresist; then, exposing the pattern on the mask plate by means of an ultraviolet photoetching machine, transferring the electrode pattern onto the spin-coated photoresist, and developing by using a ZX-238 developing solution to expose the electrode position;
s1.3: adopting a thermal evaporation coating instrument to uniformly deposit a 15nm Cr layer and a 50nm Au layer on the surface of the silicon wafer in the step S1.2 in sequence, wherein the Cr layer is used for increasing the adhesive force of Au; and soaking the silicon wafer with the deposited metal in an acetone solution to strip the photoresist, then respectively washing with isopropanol and deionized water, drying with nitrogen, and exposing the gold electrode to complete the preparation of the gold electrode.
Further, the IGZO channel material is prepared in step S2, specifically as follows:
s2.1: preparing an IGZO channel material by adopting a magnetron sputtering method; when the vacuum degree in the cavity of the magnetron sputtering coating instrument meets the requirement, introducing argon, adjusting the power of a radio frequency power supply to be 50W, the total pressure of the cavity to be 0.65Pa, and the temperature of the substrate to be 150 ℃; pre-sputtering for 10min to remove an oxide layer or other pollutants on the surface of the ceramic target, wherein In is In the ceramic target according to mass ratio2O3:Ga2O3ZnO is 1:1: 1; then opening a baffle, and formally sputtering for 25min to finish the preparation of the IGZO channel material;
s2.2: in order to prevent the electric leakage of the field effect transistor, a layer of polymethyl methacrylate (PMMA) passivated gold electrode is spin-coated on the surface of the IGZO transistor device prepared in the step S2.1.
Further, the step S3 of modifying the recognition molecule comprises the following steps:
s3.1: in order to complete the antibody modification of the device, firstly, the surface of the IGZO material in S2.2 is subjected to silanization treatment;
s3.2: placing the silanized transistor in 5% glutaraldehyde phosphate buffer solution (pH 7.4) at the set rotation speed of 180rpm and the temperature of 25 ℃, and incubating for 2 hours in a constant temperature shaking table; finally, washing the residual glutaraldehyde by deionized water, and drying by nitrogen;
s3.3: negation of transistor devices in step S3.2Respectively dripping 30mL of solution with the concentration of 20mg mL into the same channel area–1The different bladder cancer marker antibodies of (1), which comprise nuclear matrix protein 22(NMP22), CA9 recombinant protein (CA9), cytokeratin 8(CK8), cytokeratin 18(CK18), recombinant human CD47 protein (CD47), are then placed in a refrigerator at 4 ℃ for incubation for 12 hours, and antibody modification is completed through the reaction between aldehyde groups of a cross-linking agent glutaraldehyde and protein amino groups; finally, washing the unbound antibody molecules by adopting a phosphate buffer solution with the pH value of 7.4, and drying by using nitrogen;
s3.4: to reduce non-specific adsorption, 0.01g mL was used–1Blocking the active sites of the unmodified antibody molecules in the IGZO channel region in step S3.2 with Bovine Serum Albumin (BSA) solution, and incubating for 1 hour at 4 ℃ in a refrigerator.
Further, the transistor and the reservoir are assembled in step S4, specifically as follows:
s4.1: firstly, preparing a liquid storage tank; weighing Sylgard 184 siloxane prepolymer and curing agent in a mass ratio of 10:1, fully and uniformly mixing the two, and completely removing bubbles in the mixture by vacuumizing; then placing the mixture on a heating plate at 60 ℃ to heat for 2h, accelerating the bonding speed of the prepolymer and the curing agent, and forming PDMS; finally, punching the prepared PDMS out of a required liquid storage tank by using a puncher;
s4.2: treating the liquid storage tank for 3min by adopting oxygen plasma to enable the surface of the liquid storage tank to carry oxygen-containing functional groups; and (4) tightly contacting the liquid storage tank treated by the oxygen plasma with the transistor to finally finish the preparation of the IGZO field effect transistor sensor.
Furthermore, in step S2.2, in order to prevent the leakage of the field effect transistor, a layer of PMMA passivated gold electrode is spin-coated on the surface of the IGZO transistor device prepared in step S2.1; the method comprises the following specific steps:
spin coating by using a spin coater, setting the rotation speed of the spin coater to be 500rpm, pre-spin coating for 5s, and then increasing the rotation speed to 3000rpm for spin coating for 60 s; placing the IGZO device subjected to spin coating on a heating plate for hot drying for 5 min; repeating the operation once to achieve the purpose of completely curing the PMMA; finally, an electron beam exposure system (EBL) and development are used to expose the IGZO channel material covered by PMMA.
Furthermore, in the step S3.1, in order to complete antibody modification of the device, the surface of the IGZO material in S2.2 is first silanized, and the specific steps are as follows:
washing the IGZO FET prepared in S2.2 with ethanol, and soaking in 5% 3-Aminopropyltriethoxysilane (APTES) in ethanol; setting the rotation speed to be 180rpm, setting the temperature to be 25 ℃, and incubating for 1 hour in a constant-temperature shaking table; and finally, placing the glass substrate in a drying oven at 110 ℃ for 30min to finish the silanization treatment on the surface of the IGZO material.
In a second aspect, the present invention provides a multichannel field effect transistor nano biosensor, which is characterized in that: the multichannel field effect transistor nano biosensor is prepared by the preparation method.
In a third aspect, the present invention provides an application of the above multichannel fet nanobiosensor in simultaneous detection of multiple bladder cancer markers, wherein the multichannel fet nanobiosensor is characterized in that:
firstly, testing a background signal of a solution gate of a transistor sensor; secondly, incubating the solution to be detected and the field effect transistor sensor for 30min by utilizing the antigen-antibody specificity recognition function, and respectively capturing corresponding antigen molecules in the solution through NMP22, CA9, CK8, CK18 and CD47 antibody molecules modified on the surface of IGZO; then, washing away the uncombined antigen molecules, testing a current signal by using a solution grid, and influencing the charge quantity of the surface of the IGZO channel material by the captured antigen molecules through electrostatic interaction so as to influence the carrier density of the IGZO and cause the change of the current signal of the sensor; the content of the marker can be analyzed by comparing the background signal with the signal change after protein incubation; different proteins have different charges and different influences on channel materials, so that the analysis of the content of different markers is realized.
The invention has the following advantages and effects:
(1) the IGZO field effect transistor sensor constructed by the invention has excellent electrical performance and has the advantage of high sensitivity in bladder cancer marker monitoring.
(2) The IGZO field effect transistor sensor constructed by the invention can be used for directly detecting urine samples, and the samples do not need to be preprocessed, so that the detection steps are greatly simplified.
(3) The multi-marker simultaneous detection method improves the detection flux, greatly saves time and labor cost and obviously improves the detection efficiency.
(4) The IGZO field effect transistor sensor has the advantage of miniaturization, can be further integrated into portable detection equipment, is applied to family medical treatment and instant diagnosis, and has great application prospect in the fields of cancer diagnosis and postoperative monitoring.
Drawings
Fig. 1 shows a multi-channel IGZO field effect transistor sensor device manufactured in example 1 of the present invention.
Fig. 2 is a schematic diagram of a multi-channel IGZO field effect transistor sensor device manufactured in embodiment 1 of the present invention used for detecting a bladder cancer marker.
FIG. 3 is a transfer characteristic curve diagram of simultaneous detection of multiple bladder cancer markers according to application example 1 of the present invention.
FIG. 4 is a data statistical chart of simultaneous detection of 5 markers on urine samples of 20 patients with bladder cancer and 20 healthy persons according to application example 2 of the present invention.
Detailed Description
For a better understanding of the present invention, the following examples are set forth to provide a further description of the invention, and are intended as a selection of the best mode, rather than the full scope of the invention. Other embodiments, which can be made by those skilled in the art without inventive step, are within the scope of the present invention. The present invention will be described in further detail below with reference to specific embodiments and the accompanying drawings.
Example 1:
construction of a multi-channel IGZO field effect transistor sensor:
s1 preparation of gold electrode:
s1.1, designing an electrode pattern consisting of 5 source electrodes, 1 drain electrode and 1 grid electrode by adopting CAD software, and processing the electrode pattern into a corresponding mask for subsequent photoetching operation.
S1.2, selecting a p-type silicon wafer with a 300nm silicon oxide layer as a substrate, uniformly spin-coating a layer of positive photoresist AZ521 on the surface of the p-type silicon wafer, then placing the silicon wafer spin-coated with the photoresist on a heating plate, and heating at 115 ℃ for 4min to solidify the photoresist. And then, exposing the pattern on the mask plate by using an ultraviolet photoetching machine, transferring the electrode pattern onto the spin-coated photoresist, and developing by using a ZX-238 developing solution to expose the position of the electrode.
S1.3, uniformly depositing a 15nm Cr layer and a 50nm Au layer on the surface of the silicon wafer in S1 by using a thermal evaporation coating apparatus, wherein the Cr layer is used for increasing the adhesive force of Au. And soaking the silicon wafer with the deposited metal in an acetone solution to strip the photoresist, then respectively washing with isopropanol and deionized water, drying with nitrogen, and exposing the gold electrode to complete the preparation of the gold electrode.
S2 preparation of an IGZO channel material:
s2.1 preparing the IGZO channel material by adopting a magnetron sputtering method. When the vacuum degree in the cavity of the magnetron sputtering coating instrument meets the requirement, argon is introduced, the power of a radio frequency power supply is adjusted to be 50W, the total pressure of the cavity is 0.65Pa, and the temperature of the substrate is 150 ℃. Pre-sputtering for 10min, removing ceramic target (In)2O3:Ga2O31:1:1) oxide layer or other contaminants on the surface. And then opening the baffle, and formally sputtering for 25min to finish the preparation of the IGZO channel material. The multi-channel field effect transistor prepared at this time is shown in fig. 1. As shown in fig. 1, yellow is the electrode area, which is composed of 5 sources (sensing channels), 1 drain, and 1 gate, and the blue area between the source and drain represents the channel material.
S2.2 in order to prevent the electric leakage of the field effect transistor, a layer of PMMA passivated gold electrode is coated on the surface of the IGZO transistor device prepared in the S2.1 in a spinning mode. The method comprises the following specific steps: spin coating using a spin coater, the spin speed of the spin coater was set to 500rpm, pre-spin coated for 5s, and then spin-coated for 60s with the spin speed raised to 3000 rpm. The spin-coated IGZO device was placed on a hot plate and baked for 5 min. The above operation is repeated once to achieve the purpose of completely curing PMMA. And finally, exposing the IGZO channel material covered by PMMA by adopting EBL and developing.
In order to improve the stability of the grid, Ag/AgCl is used as a reference electrode. First, uniform on the circular gridCoating a layer of Ag glue, and heating at 70 ℃ for 20min to solidify the Ag glue. Then FeCl is continuously dripped into the solidified Ag glue3The solution was reacted for 4min to form AgCl. And finally, dripping a polyvinyl butyral (PVB) solution to form a PVB film and protecting the formed Ag/AgCl electrode.
Constructing a multi-channel IGZO field effect transistor sensing unit:
s3 modified recognition molecule:
s3.1 the prepared multichannel IGZO field effect transistor is washed by ethanol and then soaked in 5% APTES ethanol solution. The rotation speed was set at 180rpm, the temperature was 25 ℃ and the incubation was carried out for 1 hour in a constant temperature shaker. And finally, placing the glass substrate in a drying oven at 110 ℃ for 30min to finish the silanization treatment on the surface of the IGZO material. The silanized transistors were further incubated in 5% glutaraldehyde phosphate buffer (pH 7.4) at 25 ℃ at 180rpm for 2 hours in a constant temperature shaker. Finally, the remaining glutaraldehyde was washed clean with phosphate buffer (pH 7.4) and dried with nitrogen.
S3.2 continuously and respectively dripping 30mL of solution with the concentration of 20mg mL into different channel regions of the transistor device–1Including NMP22, CA9, CK8, CK18 and CD47, and then incubated in a refrigerator at 4 ℃ for 12 hours, and antibody modification was accomplished by reaction between aldehyde groups of glutaraldehyde, which is a cross-linking agent, and amino groups of proteins. Finally, unbound antibody molecules were washed clean with phosphate buffered saline (pH 7.4) and dried with nitrogen.
S3.3 to reduce nonspecific adsorption, 0.01g mL was used–1Blocking the active sites of the unmodified antibody molecules in the IGZO channel region in step (3), and incubating for 1 hour at 4 ℃ in a refrigerator.
S4 assembling the transistor and the reservoir:
s4.1, firstly, preparing a liquid storage tank. Weighing Sylgard 184 siloxane prepolymer and curing agent in a mass ratio of 10:1, mixing the two uniformly, and completely removing bubbles in the mixture by vacuumizing. Then the mixture is placed on a heating plate at 60 ℃ and heated for 2h, so that the bonding speed of the prepolymer and the curing agent is increased, and the PDMS is formed. And finally, punching the prepared PDMS out of the required liquid storage tank by using a puncher.
And S4.2, treating the liquid storage tank for 3min by adopting oxygen plasma to enable the surface of the liquid storage tank to carry oxygen-containing functional groups. And (4) tightly contacting the liquid storage tank treated by the oxygen plasma with the transistor to finally finish the preparation of the IGZO field effect transistor sensor.
Application example 1: IGZO field effect crystal sensor for simultaneously detecting multiple markers
The field effect transistor sensor that is not bound to the antibody is first tested for background signal. Dropping Phosphate Buffer (PB) into the liquid storage tank, and setting source-drain voltage Vds0.2V, gate voltage VgThe transfer characteristic curve of the solution grid was recorded as a background signal at-0.6V to 1.5V.
Formulation 10–12g mL–1NMP22, CA9, CK8, CK18 and CD47 antigen standard solutions. NMP22, CA9, CK8, CK18 and CD47 antigen standard solutions are respectively dripped into the field effect transistor liquid storage tanks prepared in the example 2, and the combination of the antigen and the antibody is completed after incubation for 30 min. The remaining protein solution was then aspirated and washed 3 times with PB to remove unbound protein molecules, and the PB was added again for the solution grid test. And setting the same test parameters as the background signal, and recording transfer characteristic curves of different channels. FIG. 3 shows the transfer characteristics curves for the background and incubation different channels. It can be seen that the reduction in current is due to the electrostatic shielding effect of antigen-antibody binding to the channel material. The influence degree on the current is different because different proteins have different charges.
Application example 2: IGZO field effect crystal sensor for detecting 5 markers in urine
Morning urine from 20 patients with bladder cancer and 20 healthy persons was stored in a freezer at-80 ℃ for testing. Firstly, setting source-drain voltage Vds0.2V, gate voltage VgThe transfer characteristic curve of the solution gate background of the field effect transistor was recorded in the range-0.6V to 1.5V. And then, dropwise adding the thawed urine into a liquid storage tank, incubating for 30min to complete the combination of various markers in the urine and corresponding antibodies, washing for 3 times by using PB (PB), washing away unbound protein molecules, and adding PB again to perform a solution grid test. In the same testUnder these conditions, the transfer specificity curve of the marker was recorded. To achieve accurate analysis of marker content, V is usedgAnalysis of protein concentration at a relative value of the change in current signal at 0.6V, i.e. background signal I0Difference to signal I after incubation of urine compared to background signal I0The larger the relative value of the current change, the more the marker content. As shown in FIG. 4, the relative values of the current changes of 5 markers in 20 patients with bladder cancer and 20 healthy people can be seen, and the content of the 5 markers in the patients with bladder cancer is obviously higher than that in the healthy people. The multichannel field effect transistor constructed by the invention can sensitively detect the content change of different markers and has the advantage of high sensitivity. Through further integrated design, the multi-channel IGZO field effect transistor is expected to be developed into an instant detection technology for bladder cancer diagnosis and postoperative monitoring.
Claims (9)
1. A preparation method of a multichannel field effect transistor nano biosensor is characterized by comprising the following steps: preparing a gold electrode by an ultraviolet lithography and metal deposition method, growing an IGZO channel material by a magnetron sputtering method, modifying a recognition molecule by a chemical modification method, and finally assembling Polydimethylsiloxane (PDMS) namely a PDMS chamber and a device to realize the construction of the multi-channel IGZO field effect transistor sensor with high sensitivity, high selectivity and high flux, wherein the method comprises the following steps:
s1: preparing a gold electrode:
s2: preparing an IGZO channel material:
s3: modification of recognition molecules:
s4: the transistor and reservoir are assembled.
2. The method for preparing a multi-channel FET nanobiosensor according to claim 1, wherein: the gold electrode is prepared in step S1, specifically as follows:
s1.1: designing an electrode pattern consisting of 5 source electrodes, 1 drain electrode and 1 grid electrode by adopting CAD software, and processing the electrode pattern into a corresponding mask for subsequent photoetching operation;
s1.2: selecting a p-type silicon wafer with a 300nm silicon oxide layer as a substrate, uniformly spin-coating a layer of positive photoresist AZ521 on the surface of the p-type silicon wafer, then placing the silicon wafer spin-coated with the photoresist on a heating plate, and heating at 115 ℃ for 4min to solidify the photoresist; then, exposing the pattern on the mask plate by means of an ultraviolet photoetching machine, transferring the electrode pattern onto the spin-coated photoresist, and developing by using a ZX-238 developing solution to expose the electrode position;
s1.3: adopting a thermal evaporation coating instrument to uniformly deposit a 15nm Cr layer and a 50nm Au layer on the surface of the silicon wafer in the step S1.2 in sequence, wherein the Cr layer is used for increasing the adhesive force of Au; and soaking the silicon wafer with the deposited metal in an acetone solution to strip the photoresist, then respectively washing with isopropanol and deionized water, drying with nitrogen, and exposing the gold electrode to complete the preparation of the gold electrode.
3. The method for preparing a multi-channel FET nanobiosensor according to claim 2, wherein: the IGZO channel material is prepared in step S2, specifically as follows:
s2.1: preparing an IGZO channel material by adopting a magnetron sputtering method; when the vacuum degree in the cavity of the magnetron sputtering coating instrument meets the requirement, introducing argon, adjusting the power of a radio frequency power supply to be 50W, the total pressure of the cavity to be 0.65Pa, and the temperature of the substrate to be 150 ℃; pre-sputtering for 10min to remove an oxide layer or other pollutants on the surface of the ceramic target, wherein In is In the ceramic target according to mass ratio2O3:Ga2O3ZnO is 1:1: 1; then opening a baffle, and formally sputtering for 25min to finish the preparation of the IGZO channel material;
s2.2: in order to prevent the electric leakage of the field effect transistor, a layer of polymethyl methacrylate (PMMA) passivated gold electrode is coated on the surface of the IGZO transistor device prepared in the step S2.1 in a spin mode.
4. The method for preparing the multi-channel field effect transistor nano biosensor according to claim 3, wherein: the step S3 of modifying the recognition molecule comprises the following specific steps:
s3.1: in order to finish the antibody modification of the device, the surface of the IGZO material prepared in the step S2.2 is subjected to silanization treatment;
s3.2: placing the silanized transistor in 5% glutaraldehyde phosphate buffer solution (pH 7.4) at the set rotation speed of 180rpm and the temperature of 25 ℃, and incubating for 2 hours in a constant temperature shaking table; finally, washing the residual glutaraldehyde by deionized water, and drying by nitrogen;
s3.3: in step S3.2, 30mL of the solution with a concentration of 20mg mL is respectively added to different channel regions of the transistor device–1The different bladder cancer marker antibodies comprise nuclear matrix protein 22, namely NMP22, CA9 recombinant protein, namely CA9, cytokeratin 8, namely CK8, cytokeratin 18, namely CK18, and recombinant human CD47 protein, namely CD47, and then the antibodies are placed in a refrigerator at 4 ℃ for incubation for 12 hours, and the antibody modification is completed through the reaction between aldehyde groups of a cross-linking agent glutaraldehyde and protein amino groups; finally, washing the unbound antibody molecules by adopting a phosphate buffer solution with the pH value of 7.4, and drying by using nitrogen;
s3.4: to reduce non-specific adsorption, 0.01g mL was used–1The active sites of the unmodified antibody molecules in the IGZO channel region of step S3.2 were blocked with BSA solution, and the process was incubated at 4 ℃ for 1 hour in a refrigerator.
5. The method for preparing the multi-channel field effect transistor nano biosensor according to claim 4, wherein the method comprises the following steps: the transistor and the reservoir are assembled in the step S4, which is as follows:
s4.1: firstly, preparing a liquid storage tank; weighing Sylgard 184 siloxane prepolymer and curing agent in a mass ratio of 10:1, fully and uniformly mixing the two, and completely removing bubbles in the mixture by vacuumizing; then placing the mixture on a heating plate at 60 ℃ to heat for 2h, accelerating the bonding speed of the prepolymer and the curing agent, and forming PDMS; finally, punching the prepared PDMS out of a required liquid storage tank by using a puncher;
s4.2: treating the liquid storage tank for 3min by adopting oxygen plasma to enable the surface of the liquid storage tank to carry oxygen-containing functional groups; and (4) tightly contacting the liquid storage tank treated by the oxygen plasma with the transistor to finally finish the preparation of the IGZO field effect transistor sensor.
6. The method for preparing a multi-channel FET nanobiosensor according to any one of claims 2 to 5, wherein: in the step S2.2, in order to prevent the electric leakage of the field effect transistor, a layer of PMMA passivated gold electrode is spin-coated on the surface of the IGZO transistor device prepared in the step S2.1; the method comprises the following specific steps:
spin coating by using a spin coater, setting the rotation speed of the spin coater to be 500rpm, pre-spin coating for 5s, and then increasing the rotation speed to 3000rpm for spin coating for 60 s; placing the IGZO device subjected to spin coating on a heating plate for hot drying for 5 min; repeating the operation once to achieve the purpose of completely curing the PMMA; and finally, exposing the IGZO channel material covered by the PMMA by adopting an electron beam exposure system (EBL) and developing.
7. The method for preparing the multi-channel field effect transistor nano biosensor according to claim 6, wherein: in step S3.1, in order to complete antibody modification of the device, the surface of the IGZO material in step S2.2 is subjected to silanization, which specifically includes the following steps:
washing the IGZO FET prepared in S2.2 with ethanol, and soaking in 5% 3-aminopropyltriethoxysilane APTES in ethanol; setting the rotation speed to be 180rpm, setting the temperature to be 25 ℃, and incubating for 1 hour in a constant-temperature shaking table; and finally, placing the glass substrate in a drying oven at 110 ℃ for 30min to finish the silanization treatment on the surface of the IGZO material.
8. A multi-channel field effect transistor nano biosensor is characterized in that: the multichannel field effect transistor nano biosensor is prepared by the preparation method of any one of claims 1 to 7.
9. Use of the multi-channel FET nanobiosensor of claim 8 in the simultaneous detection of multiple bladder cancer markers, wherein:
firstly, testing a background signal of a solution gate of a transistor sensor; secondly, incubating the solution to be detected and the field effect transistor sensor for 30min by utilizing the antigen-antibody specificity recognition function, and respectively capturing corresponding antigen molecules in the solution through NMP22, CA9, CK8, CK18 and CD47 antibody molecules modified on the surface of IGZO; then, washing away the uncombined antigen molecules, testing a current signal by using a solution grid, and influencing the charge quantity of the surface of the IGZO channel material by the captured antigen molecules through electrostatic interaction so as to influence the carrier density of the IGZO and cause the change of the current signal of the sensor; the content of the marker can be analyzed by comparing the background signal with the signal change after protein incubation; different proteins have different charges and different influences on channel materials, so that the analysis of the content of different markers is realized.
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| CN115032255A (en) * | 2022-06-15 | 2022-09-09 | 武汉大学 | Portable analysis device for detecting protein in urine, preparation method and application |
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