CN112694394A - Ultrasensitive multi-output signal biosensor and preparation method and application thereof - Google Patents

Ultrasensitive multi-output signal biosensor and preparation method and application thereof Download PDF

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CN112694394A
CN112694394A CN202010864800.3A CN202010864800A CN112694394A CN 112694394 A CN112694394 A CN 112694394A CN 202010864800 A CN202010864800 A CN 202010864800A CN 112694394 A CN112694394 A CN 112694394A
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程姗姗
胡文平
孙辰芳
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Abstract

The invention discloses an ultrasensitive multiple output signal biosensor and a preparation method and application thereof, wherein the ultrasensitive multiple output signal organic field effect transistor biosensor comprises: the PDVT-8 polymer layer is positioned above the substrate, the source electrode and the drain electrode are clamped between the PDVT-8 polymer layer and the substrate, and the BFPA layer covers the PDVT-8 polymer layerOn the top surface of the compound layer, a sensitive probe is grafted on the BFPA layer, and the change value delta I of source-drain current in the transfer curve of the organic field effect transistor before and after incubating the marker molecules of the cancer to be detected is monitoreddsAnd/or the variation value DeltaV of the threshold voltagethThe concentration of the cancer marker molecules to be detected is detected, and false positive results generated by the concentration determination of the cancer marker molecules to be detected due to environmental factors and instrument errors are avoided during the detection.

Description

Ultrasensitive multi-output signal biosensor and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to an ultrasensitive multi-output signal biosensor and a preparation method and application thereof.
Background
Early diagnosis of cancer can realize early discovery and early treatment of cancer, and greatly improve the cure rate and survival rate of patients at the early stage of cancer. In clinical diagnosis, the detection of the concentration of cancer marker molecules in serum is an effective early diagnosis method of cancer. Based on the advantages that the organic field effect transistor is low in price, can realize large-area low-temperature preparation, can carry out controllable design synthesis on organic molecules, can be combined with a flexible substrate to realize good biocompatibility and the like, the organic field effect transistor is widely applied to construction of an immunosensor to carry out label-free detection on target molecules. However, in the construction process of such sensors, the performance of the device is greatly affected by the multistep liquid phase modification process, so that the stability, sensitivity and specificity of the sensors in detecting cancer markers need to be improved. In addition, due to the influence of environmental factors and the error of the instrument, the concentration of the cancer marker is easily determined by only one output signal to generate a false positive result, so that misdiagnosis and missed diagnosis are caused, and the condition of an illness is delayed. In conclusion, since early cancer diagnosis is relatively complicated, a simpler and faster means is required to be explored to effectively detect the concentration of the cancer marker, which is a key step for pushing the sensing technology to clinical diagnosis, can greatly promote the practical and popularization process of the sensor, and has important scientific significance and potential application value.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a preparation method of 2, 6-di (p-formylphenyl) anthracene.
Based on 2, 6-di (p-formylphenyl) anthracene, the invention also aims to provide an ultrasensitive multi-output signal organic field effect transistor biosensor, which takes an organic field effect transistor as a carrier, obtains an organic field effect transistor with nondestructive modification, good biocompatibility and stability by depositing 2, 6-di (p-formylphenyl) anthracene (BFPA) containing a modification functional group, and further efficiently and stably introduces a sensitive probe to realize the ultrasensitive and ultrahigh specificity detection of a cancer marker molecule to be detected.
It is another object of the present invention to provide a method for preparing the above-mentioned ultrasensitive multiple output signal organic field effect transistor biosensor.
Another object of the present invention is to provide a method for using the above-mentioned ultrasensitive multiple output signal organic field effect transistor biosensor, which improves the accuracy and reliability of detecting a single cancer marker molecule in serum, and is beneficial to the ultimate development of the ultrasensitive multiple output signal organic field effect transistor biosensor into a class of immunosensor that can provide high stability, high sensitivity and multiple output signal detection.
The purpose of the invention is realized by the following technical scheme.
A method for preparing 2, 6-di (p-formylphenyl) anthracene comprises the following steps:
step 1, 2, 6-dibromoanthracene, 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzaldehyde and Pd (PPh)3)4Water, Na2CO3Mixing with toluene, stirring and reacting at 90-120 ℃ for 24-48 hours in a nitrogen or inert gas environment, and cooling to room temperature, wherein the mass fraction of 2, 6-dibromoanthracene, the mass fraction of 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzaldehyde, and Pd (PPh)3)4Part by mass of (3), part by volume of water, Na2CO3The ratio of the mass part of (2) to the volume part of toluene is (500-600): (800-900): (80-90): (5-10): (900-1000): (20-30);
in the step 1, the Na2CO3First, the mixture is mixed with the water uniformly, and then the mixture is added.
And 2, adding cooling water into the system obtained in the step 1 to obtain a precipitate in the system, wherein the ratio of the cooling water to the water in the step 1 is (20-50): (5-10);
in the step 2, the temperature of the cooling water is 0-10 ℃.
And 3, taking out and purifying the precipitate obtained in the step 2 to obtain a light yellow solid, namely 2, 6-di (p-formylphenyl) anthracene (BFPA).
In the step 3, the method for taking out the precipitate obtained in the step 2 comprises the following steps: and (3) filtering the system obtained in the step (2) to obtain solid precipitate, immersing the solid precipitate into dichloromethane for extraction, filtering again, washing the solid precipitate by using a saturated sodium chloride solution, and performing suction filtration under reduced pressure to remove the saturated sodium chloride solution to obtain the precipitate.
In the step 3, the purification is performed by a physical vapor transport method.
In the above technical solution, the unit of the mass part is mg, and the unit of the volume part is mL.
An ultrasensitive multiple output signal organic field effect transistor biosensor, comprising: the PDVT-8 polymer layer is located above the substrate, the source electrode and the drain electrode are clamped between the PDVT-8 polymer layer and the substrate, the source electrode and the drain electrode are arranged in parallel, the bottom surfaces of the source electrode and the drain electrode are both attached to the substrate, the top surfaces of the source electrode and the drain electrode are both attached to the PDVT-8 polymer layer, the BFPA layer covers the top surface of the PDVT-8 polymer layer, and the sensitive probe is grafted on the BFPA layer.
In the above technical scheme, the substrate is SiO2a/Si substrate of, among others, SiO2As an insulating layer and Si as a gate.
In the technical scheme, the thicknesses of the source electrode and the drain electrode are respectively 10-40 nm.
In the technical scheme, the thickness of the PDVT-8 polymer layer is 10-30 nm.
In the technical scheme, the thickness of the BFPA layer is 0-30 nm.
In the above technical scheme, the sensitive probe is an antibody corresponding to a cancer marker molecule to be detected.
A method for preparing the ultrasensitive multi-output signal organic field effect transistor biosensor comprises the following steps:
1) cleaning a substrate, treating the substrate by using plasma in an oxygen environment, drying, and modifying the substrate by using Octadecyltrichlorosilane (OTS);
in the step 1), the method for cleaning the substrate comprises the following steps: firstly, carrying out ultrasonic treatment for 5-15 min by using ultrapure water for removing dust easy to clean on the surface of a substrate, then putting the substrate into a piranha solution and keeping the substrate in a water bath of boiling water for 5-20 min for removing dust difficult to clean on the surface of the substrate, washing redundant piranha solution on the surface of the substrate by using the ultrapure water, and finally carrying out ultrasonic treatment for 5-15 min by using isopropanol.
In the step 1), the substrate is treated by plasma in an oxygen environment for 5-25 min, and the power is 80-100W; the drying temperature is 90-120 ℃, and the drying time is 1-2 hours; the method for modifying the substrate by using the Octadecyl Trichlorosilane (OTS) comprises the following steps: dropping 1-3 uL octadecyl trichlorosilane on a substrate, and keeping the substrate at 120-150 ℃ for 1-2 hours in a vacuum environment.
2) Adhering a mask plate on the substrate obtained in the step 1), evaporating a gold layer to form the source electrode and the drain electrode on the gold layer, and taking down the mask plate;
in the step 2), the deposition rate of the gold layer is 0.1 to
Figure BDA0002649394470000031
s-1。
In the step 2), the mask is a transmission electron microscope copper mesh, and the W/L (width-to-length ratio) of the mask is 8-10.
3) Coating a PDVT-8 polymer layer on the substrate obtained in the step 2);
in the step 3), the coating method is spin coating, and the coating method comprises the following steps: mixing PDVT-8 polymer with chlorobenzene, stirring for 10-20 hours at 70-100 ℃ to obtain a polymer solution, wherein the concentration of the PDVT-8 polymer in the polymer solution is 5-15 mg/mL, spin-coating the polymer solution at 1000-4000 rpm for 30-60 s during coating, and annealing for 10-30 min at 150-200 ℃ after spin-coating.
4) Depositing 2, 6-di (p-formylphenyl) anthracene on the PDVT-8 polymer layer obtained in step 3) and forming a BFPA layer;
in the step 4), the deposition is evaporation plating with an evaporation plating rate of 0.1 to
Figure BDA0002649394470000041
s-1。
5) Grafting a sensitive probe onto the BFPA layer.
In the step 5), the grafting method comprises the following steps: and dripping 5-20 mu L of sensitive probe solution on the BFPA layer, and incubating for 1-2 hours at 20-25 ℃, wherein the sensitive probe solution is a mixture of 1 XPBS and a sensitive probe, and the concentration of the sensitive probe in the sensitive probe solution is 50-150 mu g/mL.
The use method of the ultrasensitive multi-output signal organic field effect transistor biosensor comprises the following steps:
i. dropwise adding 5-20 mu L of Ethanolamine (EA) solution serving as a sealing agent on the ultrasensitive multiple output signal organic field effect transistor biosensor, standing for 1-2 hours, washing the ultrasensitive multiple output signal organic field effect transistor biosensor for 3-5 times by using phosphoric acid buffer solution, drying at room temperature for at least 5s, testing the transfer curve of the ultrasensitive multiple output signal organic field effect transistor biosensor and obtaining output signal current I0And/or voltage V0；
Dripping 10-30 mu L of a solution to be detected containing cancer marker molecules to be detected on the ultra-sensitive multi-output signal organic field effect transistor biosensor, incubating for 1-3 hours at room temperature, drying for at least 5s at room temperature, testing the transfer curve of the ultra-sensitive multi-output signal organic field effect transistor biosensor and obtaining output signal current I and/or voltage V;
ii. Will I0And substituting I into formula (1) and/or substituting V0Substituting the sum V into a formula (2), and calculating to obtain a change value delta I of the source leakage currentdsAnd/or the variation value DeltaV of the threshold voltageth；
△Ids＝(I-I0)/I0Formula (1)
△Vth＝V-V0Formula (2)
The change value delta I of the source-drain currentdsSubstituting the first standard curve and/or the variation value DeltaV of the threshold voltagethSubstituting the concentration of the cancer marker molecules to be detected in the solution to be detected into a second standard curve to obtain the concentration of the cancer marker molecules to be detected in the solution to be detected, wherein the first standard curve is the change value delta I of the source-drain current of the ultra-sensitive multi-output signal organic field effect transistor biosensordsAnd the concentration of the cancer marker molecules to be detected, wherein the second standard curve is the change value delta V of the threshold voltage of the ultra-sensitive multi-output signal organic field effect transistor biosensorthAnd the concentration of the cancer marker molecule to be detected.
In the above technical solution, the method for obtaining the first standard curve and the second standard curve includes: preparing N solutions with different concentrations and containing to-be-detected cancer marker molecules as standard solutions, wherein the standard solutions are mixtures of the same solvents as those in the to-be-detected solutions and to-be-detected cancer marker molecules, and the concentrations of the to-be-detected cancer marker molecules in the N standard solutions are known and are CiI 1 … … N, the following operations were performed for each standard solution: dripping 10-30 mu L of standard solution on the ultrasensitive multi-output signal organic field effect transistor biosensor, incubating for 1-3 hours at room temperature, drying for at least 5s at room temperature, and testing the ultrasensitive multi-output signal organic field effect transistor biosensor and the CiCorresponding transfer curve and obtaining output signal current IiAnd/or voltage ViIs shown byiSubstituting into I (I) in formula (1)0Also substituted) and/or by ViSubstituting V (V) into equation (2)0Also substituted), the variation value DeltaI of the source leakage current is calculateddsAnd/or the variation value DeltaV of the threshold voltageth；
Establishing a coordinate system, and determining the source-drain current variation value delta I of the N standard solutionsdsAnd CiRespectively making Y axis and X axis to obtain the first standard curve and/or change value DeltaV of threshold voltage of N standard solutionsthAnd CiAnd respectively drawing a Y axis and an X axis to obtain the second standard curve.
In the technical scheme, the ethanolamine solution is a mixture of ethanolamine and 1 × PBS, and the concentration of the ethanolamine in the ethanolamine solution is 10-20 nM.
In the technical scheme, the pH value of the 1 multiplied by PBS is 7-8, and the phosphoric acid buffer solution for washing the ultrasensitive multiple output signal organic field effect transistor biosensor is 0.01 multiplied by PBS with the pH value of 7-8.
In the above technical scheme, the solvent in the standard solution is serum.
The invention has the following beneficial effects:
1. the invention synthesizes a novel organic material 2, 6-di (p-formylphenyl) anthracene (BFPA) containing functional groups for nondestructive modification of the surface of a sensing device and effective immobilization of antibody molecules. Specifically, a BFPA layer is modified on the semiconductor surface of Organic Field Effect Transistors (OFETs) to functionalize the surface of the OFETs, so that a stable organic field effect transistor which is nondestructively modified and has good biocompatibility is constructed, and a sensitive probe is efficiently and stably introduced in a large area to realize the detection of the ultrasensitive and ultrahigh specificity of the marker molecules of the cancer to be detected.
2. By monitoring the change value delta I of source-drain current in the transfer curve of the organic field effect transistor before and after incubation of the cancer marker molecules to be detecteddsAnd/or the variation value DeltaV of the threshold voltagethTo detect the concentration of the cancer marker molecule to be detected. The change of any one electric signal can be monitored or the changes of two electric signals can be monitored simultaneously, false positive results generated by the concentration determination of the cancer marker molecules to be detected due to environmental factors and instrument errors are avoided during monitoring, and the change value delta I of the source-drain currentdsAnd/or the variation value DeltaV of the threshold voltagethThe response is in the detection range, the detection limit can reach femtomole (fM) level, and the sensitivity is ultrahigh.
3. Through the comparison of different output signals and different detection methods, the accuracy and the reliability of detecting a single cancer marker molecule to be detected in human serum are improved, and in addition, compared with the traditional enzyme-linked immunosorbent assay (ELISA), the ultrasensitive multi-output signal organic field effect transistor biosensor has higher sensitivity, shorter analysis time and smaller sample size, can be used for directly quantifying the cancer marker in human serum, can distinguish liver cancer patients from healthy donors in clinical diagnosis, and provides a new detection platform for early cancer diagnosis in clinical medicine.
Drawings
FIG. 1 is a schematic diagram of the process for synthesizing 2, 6-bis (p-formylphenyl) anthracene according to examples 1 to 3;
FIG. 2 is a nuclear magnetic and mass spectrum of 2, 6-bis (p-formylphenyl) anthracene prepared in example 2;
FIG. 3 is a schematic diagram of the preparation of PDVT-8 polymer in example 4;
FIG. 4 shows the high temperature gel chromatography results of the PDVT-8 polymer prepared in example 4;
FIG. 5 is a schematic diagram of the structure and the preparation process of the ultrasensitive multiple output signal organic field effect transistor biosensor, wherein FIG. 5(a-b) is a schematic diagram of SiO modified by Octadecyltrichlorosilane (OTS)2Depositing a source electrode and a drain electrode on the Si substrate; FIG. 5(c) is a schematic diagram of spin-coating PDVT-8 polymer on the surface of the substrate to form an organic semiconductor layer; FIG. 5(d) is the prepared 2, 6-di (p-formylphenyl) anthracene deposited on the surface of the PDVT-8 polymer layer as a modifying layer; FIG. 5(e) is a diagram showing the grafting of alpha fetoprotein antibody as a sensitive probe onto the device surface and blocking with ethanolamine to reduce non-specific adsorption; FIG. 5(f) is a schematic diagram of the detection of the cancer marker alpha-fetoprotein (AFP) cancer marker molecule to be tested;
FIG. 6 is an optical microscope photograph of the PDVT-8 polymer layer of the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in examples 5-8;
fig. 7 shows the mobility (7a) and the threshold voltage (7b) after modification of the BFPA layer in the process of preparing the ultra-sensitive multi-output signal organic field effect transistor biosensor in examples 5, 9, 10 and 11;
fig. 8 is a graph representing transfer (a) before and after modification of a BFPA layer and an output (b) after modification of the BFPA layer in a process of manufacturing the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in example 10;
fig. 9 is a Zeiss LSM 800 Confocal Laser Scanning Microscope (CLSM) diagram after modification of the BFPA layer in the process of preparing the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10;
FIG. 10 is an Atomic Force Microscope (AFM) image of an ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in preparation example 10, wherein FIG. 10(a) is the thicknesses of a PDVT-8 polymer layer and a BFPA layer before grafting of an alpha fetoprotein antibody; FIG. 10(b) is the thickness of the PDVT-8 polymer layer and the BFPA layer after grafting of the alpha fetoprotein antibody; FIG. 10(c) is the roughness of the PDVT-8 polymer layer and the BFPA layer before grafting the alpha fetoprotein antibody; FIG. 10(d) is the roughness of the PDVT-8 polymer layer and the BFPA layer after grafting of the alpha fetoprotein antibody;
FIG. 11 is a Zeiss LSM 800 Confocal Laser Scanning Microscope (CLSM) image after grafting alpha fetoprotein antibody in the process of preparing the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10;
FIG. 12 is an X-ray photoelectron spectroscopy (XPS) analysis of grafted alpha fetoprotein antibody in the process of preparing the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in example 10;
FIG. 13 shows the variation Δ I of the source-drain currentdsAnd a change value DeltaV of the threshold voltagethWherein, in the step (A),
FIG. 13(a) is a graph showing the source-drain current variation Δ I for 6 standard solutions in example 12dsWith CiThe relationship of concentration;
FIG. 13(b) is the variation DeltaV of the threshold voltages of 6 standard solutions in example 12thWith CiThe relationship of concentration;
FIG. 13(c) is a graph showing the source-drain current variation Δ I of 3 solutions to be tested in example 12ds；
FIG. 13(d) is a graph showing the threshold voltage variation values Δ V of 3 solutions to be tested in example 12th。
FIG. 13(e) is a graph showing the variation DeltaI of the source-drain current when the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10 is applied to the detection of 1 XPBS buffer solution, alpha-fetoprotein (AFP) cancer marker molecule and human serum albumin marker molecule (HSA) in example 13ds；
FIG. 13(f) is the change value Δ V of the threshold voltage when the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10 in example 13 is applied to the detection of 1 XPBS buffer solution, alpha-fetoprotein (AFP) cancer marker molecule and human serum albumin marker molecule (HSA) respectivelyth。
FIG. 14 shows the stability test of the ultrasensitive multiple output signal organic field effect transistor biosensor prepared in example 10 placed in an atmospheric environment.
Detailed Description
The technical scheme of the invention is further explained by combining specific examples.
The drug sources referred to in the following examples are as follows:
the raw materials used in the invention all adopt commercially available chemical pure reagents, wherein the concentrated H is2SO4And H2O2Purchased from Yuan Li chemical Co., Ltd., Tianjin, concentrated hydrochloric acid and isopropyl alcohol were purchased from Jiang Tian chemical technology Co., Ltd., Tianjin Xiansi Ox Pudc, Anemain (AFP) antigen antibody, carcinoembryonic antigen (CEA) antigen antibody, human serum albumin (HAS) and Ethanolamine (EA) were purchased from Shanghai san Biotech Co., Ltd., and an A-fetoprotein fluorescent-labeled antibody (sensitive probe) and a carcinoembryonic antigen fluorescent-labeled antibody (sensitive probe) were purchased from Jinan Kuntze commercial Co., Ltd., 1, 2- (E) -bis- (5 '-trimethylstannyl-2' -C-thienyl) ethane and 3, 6-bis- (5-bromothien-2-yl) -2, 5-bis- (2-octyl-1-dodecyl) pyrrolo [3, 4-c]Pyrrole-1, 4-dione from Shenzhen Rui Material science and technology Limited, 2, 6-dibromoanthracene, 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzaldehyde and tetrakis (triphenylphosphine) palladium from Shanghai Nature Limited, tris (o-tolyl) phosphine and tris (dibenzylideneacetone)) Dipalladium from Tianjin Haowenshi photoelectric technology, Ltd, for the preparation of devices, containing 300nm SiO2N-doped silicon wafer (SiO) of the layer2Si substrate) from the group of electronics and technology, China, 1 XPBS from 0.2g KCl, 0.2g KH2PO48g NaCl and 3g Na2HPO4The resistivity of the ultrapure water was 18.2 M.OMEGA.cm, and the ultrapure water was diluted 100 times with 0.01 XPBS (1 XPBS) and prepared by dissolving in 1L of ultrapure water. The piranha solution is H2SO4And H2O2In parts by volume, H2SO4：H2O2＝7：3。
The apparatus referred to in the following examples is as follows:
in the examples of the invention, nuclear magnetic resonance spectroscopy (NMR) was recorded by Bruker ADVANCE 400NMR spectrometer, Elemental analysis was performed on Flash EA 1112 elementary Analyser, molecular weight was determined by AEI-MS50-MS spectrometer, number average molar mass (M) of the polymern) And degree of dispersion (
Figure BDA0002649394470000081
) Measurements were carried out by PL-GPC220 Gel Permeation Chromatography (GPC) in trichlorobenzene at 150 ℃, Atomic Force Microscopy (AFM) using Tapping mode, X-ray photoelectron spectroscopy (XPS) performed on Axis Ultra DLD Ultra high vacuum photoelectron spectroscopy using monochromatic Al Ka radiation (1486.6eV), Confocal Laser Scanning Microscopy (CLSM) obtained by Zeiss LSM 800, and the instrument used in the probe station was Keithley 4200 SCS.
Room temperature: 20 to 25 ℃.
Examples 1 to 3
A method for preparing 2, 6-di (p-formylphenyl) anthracene (BFPA), comprising the following steps:
step 1, adding M1(mg)2, 6-dibromoanthracene, M2(mg)4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzaldehyde and 86mg of Pd (PPh)3)4Into a 100mL two-necked round bottom flask, 950mg of Na dissolved in 8mL of deionized water under an argon atmosphere2CO3And 25mL of toluene were sequentially injected into the two-necked round bottomThe reaction was stirred in a flask at 90 ℃ for 36 hours and cooled to room temperature, where M1And M2The values are shown in Table 1.
Step 2, adding 20mL of cooling water with the temperature of 0 ℃ into the system obtained in the step 1 to obtain a precipitate in the system;
and 3, taking out the precipitate obtained in the step 2, and purifying the precipitate by a physical vapor transport method to obtain a pale yellow solid BFPA, wherein the method for taking out the precipitate obtained in the step 2 comprises the following steps: and (3) filtering the system obtained in the step (2) to obtain solid precipitate, immersing the solid precipitate into dichloromethane for extraction, filtering again, washing the solid precipitate by using a saturated sodium chloride solution (water is used as a solvent), and finally removing the saturated sodium chloride solution by using a reduced pressure suction filtration method to obtain the precipitate.
TABLE 1
Examples M1(mg) M2(mg) Yield of
Example 1 500 800 80％
Example 2 550 850 93％
Example 3 600 900 87％
FIG. 1 is a schematic diagram of the synthesis of 2, 6-bis (p-formylphenyl) anthracene by a Suzuki coupling reaction. The 2, 6-bis (p-formylphenyl) anthracene prepared in example 2 was characterized by nuclear magnetic resonance spectroscopy. [1H NMR (400MHz, CDCl)3)δ(ppm)：10.11(s，2H)，8.56(s，2H)，8.31(s，2H)，8.15-8.17(d，2H)，7.95-8.03(dd，8H)，7.80-7.83(dd，2H)]. The 2, 6-bis (p-formylphenyl) anthracene prepared in example 2 was characterized by mass spectrometry. [ HR-MS (EI) m/z calculated for C28H18O2：386.1307，found：386.1310]. FIG. 2 shows Nuclear Magnetic Resonance (NMR) and Mass Spectra (MS) of 2, 6-bis (p-formylphenyl) anthracene obtained in example 2.
Example 4
The preparation of PDVT-8 polymers used in the examples of the invention is described in the reference: a method for preparing a specific PDVT-8 polymer from Chen H, et al, high π -extended polymers with a high-performance field-effect transistors [ J ]. adv. Mater.,2012,24,4618-4622, comprises the following steps:
step 1: 200mg of 3, 6-bis- (5-bromothien-2-yl) -2, 5-bis- (2-octyl-1-dodecyl) pyrrolo [3, 4-C ] pyrrole-1, 4-dione, 100mg of 1, 2- (E) -bis- (5 '-trimethylstannyl-2' -C-thienyl) ethane, 5mg of trimethylphenylphosphine, 5mg of tris (dibenzylideneacetone) -dipalladium and 20mL of dry chlorobenzene are added to a 100mL Schlenk flask under a nitrogen atmosphere and stirred at 130 ℃ for 72 hours;
step 2: after the reaction was completed and the system was cooled to room temperature, the reaction mixture was poured into an erlenmeyer flask stirred with 100mL of methanol and 8mL of concentrated hydrochloric acid (36.0% -38.0%) to precipitate the PDVT-8 polymer product;
and step 3: in order to remove impurities and low molecular weight oligomers, the reaction product was purified by soxhlet extraction using methanol, ethyl acetate and chloroform as extraction solvents, respectively, each for 12 hours;
and 4, step 4: collecting with organic microporous membrane (0.45 μm) by vacuum filtration, extracting with chloroform, and drying in vacuum oven at 60 deg.C for 12 hr to obtain PDVT-8 polymer;
number average molar mass (M) of PDVT-8 polymer was measured by PL-GPC220 Gel Permeation Chromatography (GPC) using trichlorobenzene as solvent at 150 deg.Cn17.1kDa) and dispersity (c) ((k)
Figure BDA0002649394470000091
) It is demonstrated that the PDVT-8 polymer which is well purified and meets the requirements of the experiment has been prepared by the above method, as shown in FIG. 4.
Synthesis of PDVT-8 Polymer obtained in example 4 by Stille coupling reaction the procedure for the preparation of the polymer is shown in FIG. 3.
Examples 5 to 11
An ultrasensitive multiple output signal organic field effect transistor biosensor, as shown in fig. 5, comprising: the detection device comprises a substrate, a source electrode, a drain electrode, a PDVT-8 polymer layer, a BFPA layer and a sensitive probe for detecting a cancer marker molecule to be detected, wherein the cancer marker molecule to be detected is an Alpha Fetoprotein (AFP) cancer marker molecule, the sensitive probe is an antibody corresponding to the cancer marker molecule to be detected, the PDVT-8 polymer layer is positioned above the substrate, the source electrode and the drain electrode are clamped between the PDVT-8 polymer layer and the substrate, the source electrode and the drain electrode are arranged in parallel, the bottom surfaces of the source electrode and the drain electrode are both attached to the substrate, the top surfaces of the source electrode and the drain electrode are both attached to the PDVT-8 polymer layer, the BFPA layer covers the top surface of the PDVT-8 polymer layer, and the sensitive probe is grafted on the.
The method for preparing the ultrasensitive multi-output signal organic field effect transistor biosensor comprises the following steps:
1) preparation of SiO2The substrate being a/Si substrate (silicon wafer), SiO2And (3) taking an insulating layer and Si as a grid electrode, and cleaning a substrate: firstly, performing ultrasonic treatment with ultrapure water for 10min to remove easily cleaned dust on the surface of the substrate, and then placing into piranha solution in a water bath of boiling waterKeeping for 10min for removing the dust which is difficult to clean on the surface of the substrate, washing the redundant piranha solution on the surface of the substrate with ultrapure water, and finally performing ultrasonic treatment for 10min with isopropanol.
Treating the substrate with plasma in an oxygen environment for 10min at a power of 100W, then drying the substrate in an oven at 90 ℃ for 2 hours in vacuum, removing residual excess moisture and organic solvent on the surface of the substrate, and modifying the substrate with Octadecyltrichlorosilane (OTS): dropping 1uL of octadecyl trichlorosilane on a substrate, and keeping the substrate at 120 ℃ for 2 hours in a vacuum environment of an oven to fill traps existing on the surface of the substrate, thereby being beneficial to preparing high-performance OFETs devices;
2) adhering a mask plate on the substrate obtained in the step 1), evaporating a gold layer to form a source electrode and a drain electrode on the gold layer, and taking down the mask plate, wherein the deposition rate of the gold layer is
Figure BDA0002649394470000101
s-1The mask is a transmission electron microscope copper mesh, the W/L (width-to-length ratio) of the mask is 8.2 (the length L of a deposition channel is 25 μm, the width W of the channel is 205 μm), and the thicknesses of the source electrode and the drain electrode are respectively 30 nm;
3) spin coating a PDVT-8 polymer layer on the substrate obtained in the step 2), wherein the spin coating method comprises the following steps: mixing a PDVT-8 polymer with chlorobenzene, stirring for 12 hours at 90 ℃ on a hot table to fully dissolve the PDVT-8 polymer to obtain a polymer solution, wherein the concentration of the PDVT-8 polymer in the polymer solution is 5mg/mL, spin-coating the polymer solution for 40s at V rpm during spin-coating, and annealing a substrate on the hot table at 170 ℃ for 10min after spin-coating to improve the morphology and the compactness of the PDVT-8 polymer layer;
4) evaporating the 2, 6-di (p-formylphenyl) anthracene prepared in the example 2 on the PDVT-8 polymer layer obtained in the step 3) to form a BFPA layer (modified layer), wherein the evaporation rate is
Figure BDA0002649394470000102
s-1The thickness of the BFPA layer is X nm; stable modification of the BFPA layer was demonstrated by Zeiss LSM 800 Confocal Laser Scanning Microscope (CLSM).
5) And (2) grafting a sensitive probe to the BFPA layer, wherein the grafting method comprises the following steps: and (3) dripping 20 mu L of sensitive probe solution on the BFPA layer, and incubating for 1 hour at 20-25 ℃, wherein the sensitive probe solution is a mixture of 1 XPBS and a sensitive probe, and the concentration of the sensitive probe in the sensitive probe solution is 50 mu g/mL.
TABLE 2
Examples Sensitive probe (20 mu L) V (unit: rpm) X (Unit: nm)
Example 5 Alpha-fetoprotein antibodies 1500 0
Example 6 Alpha-fetoprotein antibodies 2500 0
Example 7 Alpha-fetoprotein antibodies 3000 0
Example 8 Alpha-fetoprotein antibodies 4000 0
Example 9 Alpha-fetoprotein antibodies 1500 10
Example 10 Alpha-fetoprotein antibodies 1500 20
Example 11 Alpha-fetoprotein antibodies 1500 30
FIG. 5 is a schematic flow chart of a method for preparing an ultra-sensitive multi-output signal organic field effect transistor biosensor, wherein FIG. 5(a-b) shows SiO modified by Octadecyltrichlorosilane (OTS)2Depositing a source electrode and a drain electrode on the Si substrate; FIG. 5(c) is a schematic diagram of spin-coating PDVT-8 polymer on the surface of the substrate to form an organic semiconductor layer; FIG. 5(d) is the prepared 2, 6-di (p-formylphenyl) anthracene deposited on the surface of the PDVT-8 polymer layer as a modifying layer; FIG. 5(e) is a diagram showing the grafting of alpha fetoprotein antibody as a sensitive probe onto the device surface and blocking with ethanolamine to reduce non-specific adsorption; FIG. 5(f) is a schematic diagram of the detection of the cancer marker alpha-fetoprotein (AFP) cancer marker molecule to be tested;
FIG. 6 is an optical microscope photograph of the PDVT-8 polymer layer in the process of preparing the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in examples 5-8, from which it can be known that different rotation speed conditions have influence on the morphology of the PDVT-8 polymer layer, and through optimization, the optimal rotation speed for forming the compact and stable PDVT-8 polymer layer is 1500 rpm;
fig. 7 is a graph showing the influence of modifying BFPA layers with different thicknesses on the device mobility (a) and the electrical properties of the threshold voltage (b) in the process of preparing the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in examples 5, 9, 10, and 11, and it can be seen that the optimal thickness is 20nm by optimization, that is, the optimal solution is example 10;
FIG. 8 is a graph showing the transfer (a) curve of OFET devices before and after the modification of BFPA layer and the output (b) curve after the modification of BFPA layer in the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in example 10, the transfer curves showing that the number of carriers can be measured by an externally applied gate voltage VgControl, i.e. with gate-tunable hole-charge transport properties, the inner circle representing the leakage current IgsIt is worth noting that the change of the transfer curves before (square) and after (triangle) modification of the BFPA layer is very small, which proves that the modification of the BFPA layer does not have great influence on the performance of the device; the output curve shows a clear transition from the linear state to the saturated state, the saturation current is almost flat;
fig. 9 is a diagram of a Zeiss LSM 800 Confocal Laser Scanning Microscope (CLSM) after modification of a BFPA layer in a process of manufacturing the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10, from which it can be seen that the device surface exhibits an obvious blue fluorescence after modification of the BFPA layer, demonstrating stable modification of BFPA;
FIG. 10 is an Atomic Force Microscope (AFM) for preparing the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in example 10, wherein successful grafting of the alpha fetoprotein antibody in the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in example 10 is characterized by the AFM, and it can be seen from the graphs (a-b) that the thickness of the grafted alpha fetoprotein antibody is increased from 44.21nm to 49.74nm, and the increased thickness is consistent with the size of the alpha fetoprotein antibody molecule itself; and the roughness in panels (c-d) increased from 3.99nm to 4.30nm, further demonstrating successful grafting of alpha-fetoprotein antibody;
fig. 11 is a graph representing successful grafting of alpha fetoprotein antibodies in the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10 by Confocal Laser Scanning Microscopy (CLSM), from which it can be seen that the device surface exhibits significant green fluorescence after grafting of alpha fetoprotein antibody molecules, further demonstrating successful grafting of alpha fetoprotein antibodies;
FIG. 12 is an X-ray photoelectron spectroscopy (XPS) graph of successful grafting of alpha fetoprotein antibody in the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in example 10, from which it can be seen that the N element on the surface of the device is significantly enhanced after grafting of alpha fetoprotein antibody molecules (dotted line), further demonstrating successful grafting of alpha fetoprotein antibody;
example 12
Based on the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10, the OFET having one source electrode and one drain electrode is used as an ultrasensitive multiple output signal organic field effect transistor biosensor, and the using method of the ultrasensitive multiple output signal organic field effect transistor biosensor comprises the following steps:
i. dripping 20 mu L of Ethanolamine (EA) solution as a sealant on the ultrasensitive multi-output signal organic field effect transistor biosensor, standing for 1 hour to prevent the generation of nonspecific adsorption; the ethanolamine solution was a mixture of ethanolamine and 1 × PBS (pH 7), and the concentration of ethanolamine in the ethanolamine solution was 10 nM. Washing the super-sensitive multi-output signal organic field effect transistor biosensor with phosphoric acid buffer solution (0.01 × PBS, pH 7) for 5 times, drying at room temperature for 5s, testing the transfer curve of the super-sensitive multi-output signal organic field effect transistor biosensor and obtaining output signal current I0And voltage V0；
Preparing serum solutions of 3 volunteers (P1, P2 and P3) as a solution to be detected respectively, dripping 20 mu L of the solution to be detected on the ultrasensitive multi-output signal organic field effect transistor biosensor, incubating for 2 hours at room temperature, drying for 5s at room temperature, testing the transfer curve of the ultrasensitive multi-output signal organic field effect transistor biosensor and obtaining output signal current I and voltage V;
ii. Will I0Substituting I into formula (1) and V0Substituting V into formula (2) to obtainVariation value Delta I of current to source and draindsAnd a change value DeltaV of the threshold voltageth；
△Ids＝(I-I0)/I0Formula (1)
△Vth＝V-V0Formula (2)
The variation value Delta I of the source-drain currentdsSubstituting the first standard curve and the variation value DeltaV of the threshold voltagethSubstituting the second standard curve to obtain the concentration of the cancer marker molecules to be detected in the solution to be detected, wherein the first standard curve is the change value delta I of the source-drain current of the ultra-sensitive multi-output signal organic field effect transistor biosensordsAnd the concentration of the cancer marker molecules to be detected, and the second standard curve is the variation value delta V of the threshold voltage of the ultra-sensitive multi-output signal organic field effect transistor biosensorthAnd the concentration of the cancer marker molecule to be detected.
The method for obtaining the first standard curve and the second standard curve comprises the following steps: 6 solutions containing the cancer marker molecules to be tested at different concentrations were prepared and used as standard solutions, and the 6 standard solutions are detailed in Table 3. The standard solution is a mixture of the same solvent as that in the solution to be detected and the cancer marker molecules to be detected, the solvent in the standard solution is serum, and the concentration of the cancer marker molecules to be detected in the 6 standard solutions is known and is CiI 1 … … 6, the following operations were performed for each standard solution: dripping 20 μ L of standard solution on the ultra-sensitive multi-output signal organic field effect transistor biosensor, incubating at room temperature for 1 hr, drying at room temperature for 5s, and testing the ultra-sensitive multi-output signal organic field effect transistor biosensor and the sample CiCorresponding transfer curve and obtaining output signal current IiAnd voltage Vi(ii) a Then adding IiSubstituting into I (I) in formula (1)0Also substituted) and ViSubstituting V (V) into equation (2)0Also substituted), the variation value DeltaI of the source leakage current is calculateddsAnd a change value DeltaV of the threshold voltageth；
Obtaining the source-drain current variation value delta I of 6 standard solutionsdsAnd threshold voltageChange value of (A) Δ VthThen, a coordinate system is established, and the change value delta I of the source-drain current of 6 standard solutionsdsAnd CiRespectively making Y axis and X axis to obtain first standard curve and variation value DeltaV of threshold voltage of 6 standard solutionsthAnd CiAnd respectively drawing a Y axis and an X axis to obtain a second standard curve.
TABLE 3
- i＝1 i＝2 i＝3 i＝4 i＝5 i＝6
Ci(ng/mL) 10-2 10-1 100 101 102 103
△Ids(％) -3.37 -7.62 -11.13 -16.15 -21.92 -32.08
△Vth(V) -0.84 -1.11 -1.91 -2.30 -3.08 -3.72
FIG. 13(a-b) shows the values of the source-drain current changes Δ I for 6 standard solutions, respectivelydsAnd a change value DeltaV of the threshold voltagethThe detection limits of a good linear fitting curve of the electric signal and the concentration of the cancer marker molecules to be detected are 45fM and 53fM respectively, and the sensitivity is ultrahigh;
detecting and comparing the concentration of alpha-fetoprotein (AFP) cancer marker molecules in a human serum sample: the detection result of the ultrasensitive multiple output signal organic field effect transistor biosensor obtained by using the solution to be detected in the embodiment 10 of the invention is compared with the detection results of a commercially available enzyme-linked immunosorbent assay kit (ELISA) and a hospital chemiluminescence method, the detection results of different methods on Alpha Fetoprotein (AFP) cancer marker molecules are the same, and the accuracy of the ultrasensitive multiple output signal organic field effect transistor biosensor on early liver cancer diagnosis is shown, and the details are shown in Table 4.
TABLE 4
The result of the detection OFET(△Ids) OFET(△Vth) ELISA Hospital
Volunteer
1 High risk High risk High risk High risk
Volunteer
2 Normal level of Normal level of Normal level of Normal level of
Volunteer 3 High risk High risk High risk High risk
The risk is high: the concentration of the alpha-fetoprotein (AFP) cancer marker molecule is overproof, and the risk of cancer is extremely high.
Normal level: the concentration of the alpha-fetoprotein (AFP) cancer marker molecule is at normal levels.
For further explanation, FIG. 13(c-d) shows the source-drain current variation values Δ I of 3 solutions to be tested for 3 volunteers in Table 4dsAnd a change value DeltaV of the threshold voltagethThe electric signal value is obtained by measuring the variation value Delta I of the obtained source-drain currentdsAnd a change value DeltaV of the threshold voltagethThe concentration of the Alpha Fetoprotein (AFP) cancer marker molecules in the solution to be detected corresponding to the first standard curve and the second standard curve is obtained by substituting the first standard curve and the second standard curve respectively, the concentration of the Alpha Fetoprotein (AFP) cancer marker molecules contained in a cancer patient body is far higher than the level of a normal person, namely the Alpha Fetoprotein (AFP) cancer marker molecules in a human serum sample can be quickly detected by the ultrasensitive multiple output signal organic field effect transistor biosensor, the detection time is 40min and is far lower than the detection time of a commercial enzyme-linked immunosorbent assay kit (ELISA) and a hospital chemiluminescence method, and efficient and quick convenient conditions are provided for diagnosis of the cancer marker molecules;
example 13
To further investigate the specificity of the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10 of the present invention, the solution to be tested in example 12 was replaced with 1 × PBS buffer solution, Alpha Fetoprotein (AFP) cancer marker molecular solution, and Human Serum Albumin (HSA) marker molecular solution, and the change value Δ I of the source-drain current was testeddsAnd a change value DeltaV of the threshold voltagethThe test results are shown in FIG. 13 (e-f). Wherein, the Human Serum Albumin (HSA) marker molecule solution is a mixture of Human Serum Albumin (HSA) molecules and 1 XPBS, and the concentration of the Human Serum Albumin (HSA) marker molecule solution is 1 ug/mL; the alpha-fetoprotein (AFP) cancer marker molecule solution is a mixture of alpha-fetoprotein (AFP) cancer marker molecules and 1 XPBS, and the concentration of the alpha-fetoprotein (AFP) cancer marker molecule solution is 1 ug/mL.
The change value Delta I of the source-drain current can be seen from the figuredsAnd a change value DeltaV of the threshold voltagethOnly responds to the alpha-fetoprotein (AFP) cancer marker molecule, but responds to the non-specifically bound Human Serum Albumin (HSA) marker molecule and the blank 1 x PBS buffer solution to a very weak degree, and shows specificity.
To further investigate the stability of the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10 of the present invention, the ultrasensitive multiple output signal organic field effect transistor biosensor was placed in an atmospheric environment for 20 days, and the change in device performance (mobility) was measured every few days, and the test results are shown in fig. 14.
It can be seen from the graph that as the number of days of exposure to the atmospheric environment of the ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 10 increases, the mobility of the device does not change significantly, and stability is exhibited.
Example 14
To further investigate the universality of the ultra-sensitive multi-output signal organic field effect transistor biosensor of the present invention, the sensitive probe in example 10 was replaced with a carcinoembryonic antigen (CEA) antibody, and the other contents of the preparation method were the same as those of example 10, to obtain the ultra-sensitive multi-output signal organic field effect transistor biosensor obtained in example 14.
The ultrasensitive multiple output signal organic field effect transistor biosensor obtained in example 14 was used to measure the concentration of carcinoembryonic antigen (CEA) cancer marker molecules contained in serum solutions of 3 volunteers (P4, P5, P6) according to the method used in example 12, and the detection results of the ultrasensitive multiple output signal organic field effect transistor biosensor were compared with the detection results of a commercially available enzyme-linked immunosorbent assay kit (ELISA) and a hospital chemiluminescence method, and the measurement results are shown in table 5.
TABLE 5
The result of the detection OFET(△Ids) OFET(△Vth) ELISA Hospital
Volunteer
4 High risk High risk High risk High risk
Volunteer
5 Normal level of Normal level of Normal level of Normal level of
Volunteer 6 High risk High risk High risk High risk
The risk is high: the concentration of carcinoembryonic antigen (CEA) cancer marker molecules is overproof, and the risk of suffering from cancer is extremely high.
Normal level: carcinoembryonic antigen (CEA) cancer marker molecule concentrations are at normal levels.
The ultrasensitive multiple-output signal organic field effect transistor biosensor can realize the high-efficiency grafting of the sensitive probe on the basis of not influencing the performance of the organic field effect transistor device, thereby realizing the detection of the ultrahigh sensitivity of the marker molecules of the cancer to be detected. Meanwhile, through comparison of different output signals and different detection methods, the accuracy and the reliability of detection of a single cancer marker molecule in human serum are improved, and the immunosensor which can provide high stability, high sensitivity and multi-output signal detection is beneficial to being finally developed, so that the accuracy and the reliability of a detection result are greatly improved.
Compared with the existing cancer diagnosis technology, the ultrasensitive multi-output signal organic field effect transistor biosensor can meet the real-time detection requirements of low price, no identification, rapidness, convenience and the like, and provides a new idea for solution phase detection. By using BFPA as a modification layer, the high-efficiency grafting of the sensitive probe can be realized on the basis of not influencing the performance of the organic field effect transistor device. Most importantly, the ultrasensitive multiple-output organic field effect transistor biosensor has excellent reliability due to the benefit of multiple-output signal detection, can monitor to-be-detected cancer marker molecules in a complex serum sample, and can distinguish a cancer patient from a healthy donor by quantifying the concentration of the cancer marker in the serum sample. Based on all the advantages, the ultrasensitive multi-output signal organic field effect transistor biosensor can be used as a reliable multi-signal biosensor, and the sensing sensitivity, stability and selectivity are greatly improved.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

Claims (10)

1. A method for preparing 2, 6-di (p-formylphenyl) anthracene is characterized by comprising the following steps:
step 1, 2, 6-dibromoanthracene, 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzaldehyde and Pd (PPh)3)4Water, Na2CO3Mixing with toluene, stirring and reacting at 90-120 ℃ for 24-48 hours in a nitrogen or inert gas environment, and cooling to room temperature, wherein the mass fraction of 2, 6-dibromoanthracene, the mass fraction of 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzaldehyde, and Pd (PPh)3)4Part by mass of (3), part by volume of water, Na2CO3The ratio of the mass part of (2) to the volume part of toluene is (500-600): (800-900): (80-90): (5-10): (900～1000)：(20～30)；
And 2, adding cooling water into the system obtained in the step 1 to obtain a precipitate in the system, wherein the ratio of the cooling water to the water in the step 1 is (20-50): (5-10);
and 3, taking out and purifying the precipitate obtained in the step 2 to obtain a light yellow solid, namely the 2, 6-di (p-formylphenyl) anthracene.
2. The method according to claim 1, wherein in the step 1, the Na is2CO3Mixing with the water uniformly, and then adding;
in the step 2, the temperature of the cooling water is 0-10 ℃;
in the step 3, the method for taking out the precipitate obtained in the step 2 comprises the following steps: filtering the system obtained in the step 2 to obtain solid precipitate, immersing the solid precipitate into dichloromethane for extraction, filtering again, washing the solid precipitate by using a saturated sodium chloride solution, and performing suction filtration under reduced pressure to remove the saturated sodium chloride solution to obtain the precipitate;
in the step 3, the purification is performed by a physical vapor transport method;
the unit of the mass part is mg, and the unit of the volume part is mL.
3. An ultrasensitive multiple output signal organic field effect transistor biosensor, comprising: the PDVT-8 polymer layer is located above the substrate, the source electrode and the drain electrode are clamped between the PDVT-8 polymer layer and the substrate, the source electrode and the drain electrode are arranged in parallel, the bottom surfaces of the source electrode and the drain electrode are both attached to the substrate, the top surfaces of the source electrode and the drain electrode are both attached to the PDVT-8 polymer layer, the BFPA layer covers the top surface of the PDVT-8 polymer layer, and the sensitive probe is grafted on the BFPA layer.
4. According to the claims3 the ultrasensitive multiple output signal organic field effect transistor biosensor is characterized in that the substrate is SiO2a/Si substrate of, among others, SiO2Making an insulating layer and Si as a grid electrode;
the thicknesses of the source electrode and the drain electrode are respectively 10-40 nm;
the thickness of the PDVT-8 polymer layer is 10-30 nm;
the thickness of the BFPA layer is 0-30 nm;
the sensitive probe is an antibody corresponding to a cancer marker molecule to be detected.
5. Method for preparing an ultrasensitive multiple output signal organic field effect transistor biosensor according to claim 3 or 4, comprising the steps of:
1) cleaning a substrate, treating the substrate by using plasma in an oxygen environment, drying, and modifying the substrate by using octadecyl trichlorosilane;
2) adhering a mask plate on the substrate obtained in the step 1), evaporating a gold layer to form the source electrode and the drain electrode on the gold layer, and taking down the mask plate;
3) coating a PDVT-8 polymer layer on the substrate obtained in the step 2);
4) depositing 2, 6-di (p-formylphenyl) anthracene on the PDVT-8 polymer layer obtained in step 3) and forming a BFPA layer;
5) grafting a sensitive probe onto the BFPA layer.
6. The method according to claim 5, wherein in the step 1), the substrate is cleaned by: firstly, carrying out ultrasonic treatment for 5-15 min by using ultrapure water for removing dust easy to clean on the surface of a substrate, then putting the substrate into a piranha solution and keeping the substrate in a water bath of boiling water for 5-20 min for removing dust difficult to clean on the surface of the substrate, washing redundant piranha solution on the surface of the substrate by using the ultrapure water, and finally carrying out ultrasonic treatment for 5-15 min by using isopropanol;
in the step 1), the substrate is treated by plasma in an oxygen environment for 5-25 min, and the power is 80-100W; the drying temperature is 90-120 ℃, and the drying time is 1-2 hours; the method for modifying the substrate by using the octadecyl trichlorosilane comprises the following steps: dripping 1-3 uL of octadecyl trichlorosilane on a substrate, and keeping the substrate at 120-150 ℃ for 1-2 hours in a vacuum environment;
in the step 2), the deposition rate of the gold layer is
Figure FDA0002649394460000022
In the step 2), the mask is a transmission electron microscope copper mesh, and the W/L of the mask is 8-10;
in the step 3), the coating method is spin coating, and the coating method comprises the following steps: mixing a PDVT-8 polymer with chlorobenzene, stirring for 10-20 hours at 70-100 ℃ to obtain a polymer solution, wherein the concentration of the PDVT-8 polymer in the polymer solution is 5-15 mg/mL, spin-coating the polymer solution at 1000-4000 rpm for 30-60 s during coating, and annealing for 10-30 min at 150-200 ℃ after spin-coating;
in the step 4), the deposition is evaporation coating, and the evaporation coating rate is
Figure FDA0002649394460000021
In the step 5), the grafting method comprises the following steps: and dripping 5-20 mu L of sensitive probe solution on the BFPA layer, and incubating for 1-2 hours at 20-25 ℃, wherein the sensitive probe solution is a mixture of 1 XPBS and a sensitive probe, and the concentration of the sensitive probe in the sensitive probe solution is 50-150 mu g/mL.
7. The method of using the ultrasensitive, multiple output signal, organic field effect transistor biosensor of claim 5 or 6, comprising the steps of:
i. dropwise adding 5-20 mu L of ethanolamine solution serving as a sealant on the ultrasensitive multi-output signal organic field effect transistor biosensor, standing for 1-2 hours, and washing the ultrasensitive multi-output signal organic field effect transistor biosensor 3 by using a phosphoric acid buffer solutionDrying at room temperature for at least 5s for 5 times, testing transfer curve of the ultra-sensitive multi-output signal organic field effect transistor biosensor and obtaining output signal current I0And/or voltage V0；
Dripping 10-30 mu L of a solution to be detected containing cancer marker molecules to be detected on the ultra-sensitive multi-output signal organic field effect transistor biosensor, incubating for 1-3 hours at room temperature, drying for at least 5s at room temperature, testing the transfer curve of the ultra-sensitive multi-output signal organic field effect transistor biosensor and obtaining output signal current I and/or voltage V;
ii. Will I0And substituting I into formula (1) and/or substituting V0Substituting the sum V into a formula (2), and calculating to obtain a change value delta I of the source leakage currentdsAnd/or the variation value DeltaV of the threshold voltageth；
△Ids＝(I-I0)/I0Formula (1)
△Vth＝V-V0Formula (2)
The change value delta I of the source-drain currentdsSubstituting the first standard curve and/or the variation value DeltaV of the threshold voltagethSubstituting the concentration of the cancer marker molecules to be detected in the solution to be detected into a second standard curve to obtain the concentration of the cancer marker molecules to be detected in the solution to be detected, wherein the first standard curve is the change value delta I of the source-drain current of the ultra-sensitive multi-output signal organic field effect transistor biosensordsAnd the concentration of the cancer marker molecules to be detected, wherein the second standard curve is the change value delta V of the threshold voltage of the ultra-sensitive multi-output signal organic field effect transistor biosensorthAnd the concentration of the cancer marker molecule to be detected.
8. Use according to claim 7, wherein the method of obtaining the first and second standard curves is: preparing N solutions with different concentrations and containing to-be-detected cancer marker molecules as standard solutions, wherein the standard solutions are the same solvent as the to-be-detected solutions and a mixture of the to-be-detected cancer marker molecules, and the to-be-detected cancer in the N standard solutionsThe concentration of the marker molecule is known and is CiI 1 … … N, the following operations were performed for each standard solution: dripping 10-30 mu L of standard solution on the ultrasensitive multi-output signal organic field effect transistor biosensor, incubating for 1-3 hours at room temperature, drying for at least 5s at room temperature, and testing the ultrasensitive multi-output signal organic field effect transistor biosensor and the CiCorresponding transfer curve and obtaining output signal current IiAnd/or voltage ViIs shown byiSubstituting I into formula (1) and/or substituting ViSubstituting V in formula (2) to obtain the variation value Delta I of the source leakage currentdsAnd/or the variation value DeltaV of the threshold voltageth；
Establishing a coordinate system, and determining the source-drain current variation value delta I of the N standard solutionsdsAnd CiRespectively making Y axis and X axis to obtain the first standard curve and/or change value DeltaV of threshold voltage of N standard solutionsthAnd CiAnd respectively drawing a Y axis and an X axis to obtain the second standard curve.
9. The use method according to claim 7, wherein the ethanolamine solution is a mixture of ethanolamine and 1 XPBS, and the concentration of ethanolamine in the ethanolamine solution is 10-20 nM.
10. The use method of claim 7, wherein the pH of the 1 XPBS is 7-8, and the phosphate buffer solution for washing the ultrasensitive multiple output signal organic field effect transistor biosensor is 0.01 XPBS with pH 7-8;
the solvent in the standard solution is serum.
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