A preparation method and application of a molecular crystal thin film organic field effect transistor biosensor based on an ultra-thin sensing layer

Abstract

The present invention discloses a preparation method and application of a molecular crystal film organic field effect transistor biosensor based on an ultra-thin sensing layer, and belongs to the technical field of biosensors. The present invention uses a layer definition strategy to prepare a C6-DPA organic molecular crystal film with controllable number of layers, and the thinnest single layer thickness can be as low as 2.8nm. In order to efficiently fix the biosensitive probe on the biosensing layer, ADDA with a ‑COOH functional group is deposited as a functional modification layer with a thickness as low as 3nm using a vacuum deposition strategy. Finally, a molecular crystal film organic field effect transistor biosensor with a sensing layer thickness as low as 5.8nm was prepared, which was successfully applied to the field of liquid phase biosensors for the first time, and the charge-induced sensing mechanism of the OFETs biosensor based on macromolecular proteins was preliminarily proved, achieving high-sensitivity sensing of the liver cancer tumor marker AFP.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a preparation method and application of a molecular crystal thin film organic field effect transistor biosensor based on an ultra-thin sensing layer. Background Art

[0002] The monitoring and collection of personalized health information heavily rely on various portable health monitoring sensors that can convert the received biological, chemical or physical signals into readable electrical signals and / or other forms of signals. With the rapid growth of global aging and high mortality from advanced cancer, healthcare management has shown potential economic and social value. With the rapid development of electronic information and artificial intelligence, the field of medical management is moving towards more accurate, efficient and personalized directions. The monitoring and collection of personalized health information mainly rely on various portable health monitoring sensors that can convert biological, chemical or physical signals into readable electrical signals. Biosensors based on organic field effect transistors (OFETs) have become one of the most advanced biosensor platforms due to their inherent signal amplification capabilities. In order to more accurately assess health conditions and collect accurate and reliable data, it is necessary to develop high-performance OFETs-based biosensors. Optimizing biosensor performance requires comprehensive consideration of sensing mechanisms, transistor device structures and working principles. Today, the sensing mechanisms of OFETs biosensors based on macromolecular proteins are mainly speculated to be charge-induced and molecular permeation. Understanding the sensing mechanism is very important for guiding the design of OFETs biosensor materials and efficient immobilization strategies for biosensitive probes.

[0003] Electronic devices based on organic molecular crystal films have experienced rapid development in the past decade. Organic molecular crystal films have the advantages of free grain boundaries, few defects, and minimal traps and impurities. They are the perfect choice for basic and applied research. Compared with polymers, they have better batch stability and are also used as a prototype model for studying charge transport and achieving high performance. In addition, the tightly arranged structure inside the organic molecular crystal film field effect transistor cannot enable macromolecular proteins to achieve sensing through molecular penetration. This important prerequisite also provides new hope for revealing the internal sensing mechanism of OFETs-based biosensors based on macromolecular proteins. Based on the charge induction mechanism of OFETs biosensors, when the sensing surface is stimulated by external stimuli, the electrostatic interaction between it and the OFETs device will induce / quench more hole carriers in the conductive channel, thereby causing changes in the electrical parameters of the device. Therefore, the sensing performance of OFETs-based biosensors will be limited by the induced thickness of the analyte molecules to the semiconductor channel. How to break this limitation and achieve ultra-sensitive sensing is a difficult problem that needs to be solved in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a molecular crystal film organic field effect transistor biosensor based on an ultra-thin sensing layer and its application, so as to solve the above-mentioned problems existing in the prior art, realize high-sensitivity sensing of target analytes and reveal the sensing mechanism of OFETs biosensors based on macromolecular proteins. The present invention uses a layer definition strategy (a layer definition solution self-assembly method of organic molecules from bulk to monolayer limit on the surface of a liquid matrix) to prepare an organic molecular crystal film with controllable number of layers (such as 2,6-bis(4-hexylphenyl)anthracene (C6-DPA)), and the thinnest monolayer thickness can be as low as 2.8nm. In order to efficiently fix the biosensitive probe on the biosensor layer, a vacuum deposition strategy is used to deposit 4,4'-(anthracene-9,10-diyl)dibenzoic acid (ADDA) with a -COOH functional group as a functional modification layer with a thickness as low as 3nm. Finally, a molecular crystal thin film organic field effect transistor biosensor with a sensing layer thickness as low as 5.8nm was prepared. It was successfully applied to the field of liquid phase biosensing for the first time, and the charge-induced sensing mechanism of the OFETs biosensor based on large molecular proteins was preliminarily demonstrated, achieving high-sensitivity sensing of the liver cancer tumor marker AFP (alpha-fetoprotein).

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is: a molecular crystal film organic field effect transistor biosensor based on an ultra-thin sensing layer, comprising a substrate, an organic molecular crystal film, a source-drain electrode and a functional modification layer arranged in sequence; the organic molecular crystal film is 2,6-bis(4-hexylphenyl)anthracene, 2-decyl-7-phenyl[1]benzothieno[3,2-b[1]benzothiophene, perylene, dioctylbenzothiophene or 6,13-bis(triisopropylsilylethynyl)pentacene; the functional modification layer is 4,4'-(anthracene-9,10-diyl)dibenzoic acid; the thickness of the organic molecular crystal film is 2.8-39.6nm, and the thickness of the functional modification layer is 3nm.

[0007] The sensing layer in the molecular crystal thin film organic field effect transistor biosensor refers to the organic molecular crystal thin film + functional modification layer.

[0008] Furthermore, the thickness of the organic molecular crystal film is preferably 2.8-5.7 nm, more preferably 2.8 nm.

[0009] Furthermore, the substrate is a heavy n with a SiO2 layer + Doped silicon wafer (n ++ Si / SiO2); the source and drain electrodes are made of metal materials.

[0010] Furthermore, the metal material is gold.

[0011] Furthermore, the thickness of the source-drain electrode is 150 nm.

[0012] The second technical solution of the present invention: The method for preparing the molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer comprises the following steps:

[0013] The interface of the substrate is modified; an organic molecular crystal film is grown on the surface of the liquid substrate; the organic molecular crystal film grown on the surface of the liquid substrate is transferred to the surface of the substrate after the interface modification, source-drain electrodes are prepared on the surface of the organic molecular crystal film, and a functional modification layer is deposited on the surface of the organic molecular crystal film (the portion not covered by the source-drain electrodes) and the surface of the source-drain electrodes to obtain the molecular crystal film organic field-effect transistor biosensor based on the ultra-thin sensing layer.

[0014] Furthermore, the interface modification of the substrate includes: firstly treating the substrate with plasma in an oxygen environment, and then modifying the substrate with octadecyltrichlorosilane (OTS).

[0015] Furthermore, the time for treating the substrate with plasma is 5-25 minutes, preferably 10 minutes, and the power is 80W; the specific operation of modifying the substrate with octadecyltrichlorosilane is: placing the substrate in a modification solution composed of octadecyltrichlorosilane and n-heptane, and leaving it to stand at room temperature for 2-5 hours.

[0016] Furthermore, the volume ratio of n-heptane to OTS in the modification solution is 1000:1.

[0017] Furthermore, after the static modification is completed, the substrate is subjected to ultrasonic treatment in n-hexane, chloroform and isopropanol in sequence for 10 minutes.

[0018] Furthermore, the process of performing interface modification on the substrate also includes a cleaning operation on the substrate, specifically, cleaning the substrate with deionized water, acetone and isopropanol in sequence.

[0019] Furthermore, the cleaning time is 5-15 minutes, preferably 10 minutes.

[0020] Furthermore, the growth of an organic molecular crystal film on the surface of a liquid substrate comprises: dropping a mixed solution of organic molecules and a surfactant onto the surface of the liquid substrate, placing it under constant temperature and humidity conditions to completely evaporate the solvent, and obtaining an organic molecular crystal film floating on the surface of the liquid substrate (this step is to grow a molecular crystal film using a layer definition strategy); the organic molecule is 2,6-bis(4-hexylphenyl)anthracene, 2-decyl-7-phenyl[1]benzothieno[3,2-b[1]benzothiophene, perylene, dioctylbenzothiophene or 6,13-bis(triisopropylsilylethynyl)pentacene.

[0021] Furthermore, the surfactant is sodium perfluorooctanoate; the solvent of the mixed solution is toluene; the concentration of organic molecules in the mixed solution is 0.2 mg mL -1 , the concentration of surfactant is 0.001 mg mL -1 ; The liquid matrix is ​​glycerol.

[0022] Furthermore, the constant temperature and humidity conditions are specifically: temperature is 15° C., and humidity is 30%.

[0023] Furthermore, before the mixed solution of organic molecules and surfactants is dropped onto the surface of the liquid matrix, the mixed solution is ultrasonicated for 3 hours at a power of 100 W.

[0024] Furthermore, the deposition is vacuum deposition.

[0025] Furthermore, the deposition rate of the functional modification layer is

[0026] Furthermore, the deposition temperature is 25-50°C, preferably 25°C.

[0027] Furthermore, after the solvent is completely evaporated, the process further includes rinsing with deionized water.

[0028] The third technical solution of the present invention: a method for detecting disease markers, using the above-mentioned molecular crystal thin film organic field effect transistor biosensor based on ultra-thin sensing layer.

[0029] Furthermore, the method comprises the following steps:

[0030] A disease marker antibody is grafted onto the surface of the functional modification layer of the molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer as a biosensitive probe; a disease marker standard solution of different concentrations is grafted onto the surface of the disease marker antibody, and the threshold voltage (V TH), the change in threshold voltage of the transfer curve of the biosensor before and after grafting the disease marker standard solution (△V TH ) as the sensor output signal; a standard curve is drawn with the concentration of the disease marker standard solution as the horizontal axis and the change value of the threshold voltage at the corresponding concentration as the vertical axis;

[0031] The test solution is grafted on the surface of the disease marker antibody, and the threshold voltage of the transfer curve of the biosensor before and after grafting the test solution is detected. The change value of the threshold voltage corresponding to the test solution is substituted into the working curve to obtain the concentration of the disease marker in the test solution.

[0032] Furthermore, the disease markers include but are not limited to AFP (liver cancer tumor marker), CEA (carcinoembryonic antigen) or PSA (prostate specific antigen).

[0033] The molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer of the present invention has universal applicability when used to detect disease markers.

[0034] Technical solution 4 of the present invention: Application of the above-mentioned molecular crystal thin film organic field effect transistor biosensor based on ultra-thin sensing layer in the preparation of a biosensor for detecting AFP.

[0035] Furthermore, the application includes: grafting AFP antibody as a biosensitive probe on the surface of the functional modification layer of the molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer to obtain the biosensor for detecting AFP.

[0036] Furthermore, the step of detecting AFP using the biosensor for detecting AFP includes: grafting a test solution containing AFP on the surface of the AFP antibody grafted on the surface of the biosensor, and detecting the threshold voltage (V TH ), the change in threshold voltage of the transfer curve of the biosensor before and after grafting the test solution (△V TH ) as the sensor output signal.

[0037] Furthermore, the grafting of AFP antibodies on the surface of the functional modification layer of the molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer includes: first activating the functional modification layer with EDC and NHS, rinsing it with phosphate buffer solution for 3-5 times after the activation, and then dripping AFP antibody solution on the functional modification layer and incubating it; the grafting of the test solution on the antibody surface includes: dripping the test solution on the surface of the grafted AFP antibody and incubating it.

[0038] Furthermore, the AFP to be detected specifically refers to AFP antigen.

[0039] Furthermore, the activation time is 1 hour.

[0040] Furthermore, the incubation temperature is room temperature, and the incubation time is 1-2 hours, preferably 1 hour.

[0041] Furthermore, the process of detecting the threshold voltage of the transfer curve of the biosensor before and after grafting the test solution (the test solution containing AFP), and taking the change value of the threshold voltage of the transfer curve of the biosensor before and after grafting the test solution (i.e., grafting AFP) as the sensor output signal can be expressed by the following formula:

[0042] △V TH =V-V0, where V is the V after the AFP antibody reacts with AFP (i.e. after grafting AFP). TH , V0 is the V measured before the AFP antibody reacts with AFP (i.e. before grafting AFP) TH .

[0043] Furthermore, before the test solution is grafted onto the antibody surface, the surface of the biosensor is rinsed 3-5 times with a phosphate buffer solution, and then the transfer curve is tested after being dried at room temperature for at least 5 seconds to obtain an output threshold voltage as the threshold voltage of the transfer curve of the biosensor before grafting the test solution, i.e., V0.

[0044] Furthermore, a test solution containing AFP is dripped onto the surface of the grafted AFP antibody, incubated, dried at room temperature for at least 5 seconds, and then the transfer curve is tested and the output threshold voltage is obtained as the threshold voltage of the transfer curve of the biosensor after grafting the test solution, i.e., V.

[0045] Furthermore, the phosphate buffer solution is 0.01×PBS (ie, the concentration is 0.01 M) and the pH is 7-8.

[0046] Furthermore, the step of quantitatively detecting AFP using the biosensor for detecting AFP is specifically as follows:

[0047] Step 1: Rinse the surface of the biosensor for detecting AFP with a phosphate buffer solution for 3-5 times, dry at room temperature for at least 5 seconds, test the transfer curve of the sensor and obtain the value V0 of the output threshold voltage;

[0048] Step 2: Add different concentrations of AFP standard solutions to the surface of the biosensor for detecting AFP for incubation, test the transfer curve of the biosensor incubated with different concentrations of AFP standard solutions and obtain the corresponding output threshold voltage value V; calculate the change value △V of the threshold voltage corresponding to the different concentrations of AFP standard solutions TH , with AFP concentration as the horizontal axis, the corresponding concentration △V TH Plot a standard curve for the ordinate;

[0049] Step 3: Add a test solution containing AFP to the surface of the biosensor for detecting AFP and incubate it, test the transfer curve of the biosensor incubated with the test solution containing AFP and obtain the corresponding output threshold voltage value V; calculate the change value △V of the threshold voltage corresponding to the test solution containing AFP TH , substituted into the standard curve to obtain the concentration of AFP in the test solution.

[0050] Furthermore, a new biosensor for detecting AFP is used each time a concentration of an AFP standard solution or a test solution is detected, and the biosensor corresponding to the AFP standard solution or the test solution is rinsed with a phosphate buffer solution before incubating the AFP standard solution or the test solution, and the corresponding V0 is tested.

[0051] The present invention uses the strategy of vacuum deposition of functional modification layers to apply molecular crystal thin film OFETs biosensors with ultra-thin functional modification layers to the field of liquid phase biosensing for the first time. In addition, based on the controllable preparation of the thickness of the molecular crystal film and its compact arrangement structure that prevents large molecular proteins from sensing by molecular penetration, the charge-induced sensing mechanism of the OFETs biosensor based on large molecular proteins is preliminarily demonstrated. Using ADDA material as a functional modification layer, a molecular crystal thin film OFETs biosensor with a sensing layer thickness as thin as 5.8nm was prepared, achieving high-sensitivity sensing of the liver cancer tumor marker AFP with a detection limit as low as aM. Based on the high sensitivity and good stability of the biosensor, it shows good reliability in monitoring the target AFP biomarker in human serum, and can effectively distinguish between liver cancer patients and healthy individuals, and has application potential in monitoring early cancer.

[0052] The present invention discloses the following technical effects:

[0053] (1) The present invention uses a method of vacuum deposition of a functional modification layer on the surface of a molecular crystal film to prepare an ultra-thin molecular crystal film OFETs biosensor with a sensing layer of only 5.8 nm, and applies molecular crystal film field-effect transistors to the field of liquid phase biosensors for the first time, ultimately achieving high-sensitivity detection of AFP as low as aM.

[0054] (2) Based on the advantage that the tightly packed structure inside organic molecular crystal thin film field effect transistors (OFETs) cannot enable large molecular proteins to achieve sensing through molecular penetration, the present invention prepares crystal films with different numbers of molecular layers, thereby revealing the internal sensing mechanism of OFETs-based biosensors based on large molecular proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0056] Figure 1 This is a polarizing microscope characterization image of the C6-DPA molecular crystal film grown in Example 1 of the present invention, wherein a is before rotation and b is after rotation;

[0057] Figure 2 This is an out-of-plane X-ray diffraction (XRD) characterization diagram of the C6-DPA molecular crystal film grown in Example 1 of the present invention;

[0058] Figure 3 This is an atomic force microscope (AFM) characterization image of the C6-DPA molecular crystal film grown in Example 1 of the present invention;

[0059] Figure 4 A schematic diagram of the device structure of C6-DPA molecular crystal thin film OFETs prepared in Example 2 of the present invention;

[0060] Figure 5 The transfer (a) and output curves (b) of the C6-DPA molecular crystal thin film OFETs prepared in Example 2 of the present invention;

[0061] Figure 6 The AFM characterization diagram of OFETs with different molecules as functional modification layers in Example 3 of the present invention, wherein a, d, and g are respectively the structural formula of a molecule with a functional group of -CHO, the roughness characterization after deposition, and the height characterization after deposition; b, e, and h are respectively the structural formula of a molecule with a functional group of -NH2, the roughness characterization after deposition, and the height characterization after deposition; c, f, and i are respectively the structural formula of a molecule with a functional group of -COOH, the roughness characterization after deposition, and the height characterization after deposition;

[0062] Figure 7 Atomic force microscope (AFM) characterization images of molecular crystal thin film OFETs obtained by depositing an ADDA layer under different deposition conditions in Example 3 of the present invention, wherein af represents different deposition rates or deposition temperatures respectively;

[0063] Figure 8 The transfer curves of molecular crystal thin film OFETs deposited with ADDA layers of different thicknesses in Example 3 of the present invention;

[0064] Fig. 9The figure is a schematic diagram of the process of constructing a molecular crystal thin film OFETs biosensor according to the present invention and the process of sensing AFP using the molecular crystal thin film OFETs biosensor; wherein, a is an OTS-modified substrate, b is the growth of a C6-DPA molecular crystal film, c is the stamping of source and drain electrodes, d is the deposition of an ADDA layer, e is the grafting of an AFP antibody, f is the recognition of an AFP antigen, g is a schematic diagram of the structure of an ADDA molecule (wherein blue balls represent carbon atoms, red balls represent oxygen atoms, and white balls represent hydrogen atoms), h is an AFP antigen-antibody pair, and i is a schematic diagram of antibody grafting on an ADDA functional modification layer;

[0065] Fig.10 The transfer (a) and output curve (b) of the molecular crystal thin film OFETs biosensor based on the ultra-thin sensing layer prepared in Example 4 of the present invention;

[0066] Fig.11 The atomic force microscope (AFM) characterization images of the molecular crystal thin film OFETs biosensor surface before and after AFP antibody incubation in Example 5 of the present invention, wherein a and c are height characterizations before and after incubation, and b and d are roughness characterizations before and after incubation;

[0067] Fig.12 This is a characterization diagram of the grafting of the biosensitive probe AFP antibody to the device after ADDA modification by confocal laser scanning microscopy (CLSM) in Example 5 of the present invention, wherein a is before grafting, and b is after grafting;

[0068] Fig.13 The X-ray photoelectron spectroscopy (XPS) N element (a) and O element (b) characterization diagrams of the molecular crystal thin film OFETs biosensor surface before and after AFP antibody incubation in Example 5 of the present invention;

[0069] Fig.14 is the ΔV of the molecular crystal thin film OFETs biosensor in Example 6 of the present invention exposed to different concentrations of AFP cancer markers TH (variation of threshold voltage) linear fitting curve (standard curve);

[0070] Fig.15 is the V of the OFETs biosensor with different number of molecular crystal thin films for the AFP biomarker in Example 7 of the present invention TH Sensing behavior test results of electrical signals;

[0071] Fig.16 The selectivity test results of the molecular crystal thin film OFETs biosensor for AFP cancer marker sensing in Example 8 of the present invention;

[0072] Fig.17The results of the anti-interference test of the molecular crystal thin film OFETs biosensor in Example 9 of the present invention for sensing AFP cancer markers. DETAILED DESCRIPTION

[0073] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0074] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0075] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0076] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0077] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0078] All raw materials used in the specific implementation of the present invention are commercially available chemically pure reagents, among which 2,6-bis(4-hexylphenyl)anthracene (C6-DPA, structural formula: ) was purchased from Shanghai Daran Chemical Co., Ltd.; 4,4'-(anthracene-9,10-diyl)dibenzoic acid (ADDA), 4,4'-(anthracene-9,10-diyl)dibenzaldehyde, and 4,4'-(anthracene-9,10-diyl)diphenylamine were purchased from Bid Pharmaceuticals; toluene, acetone, isopropanol, chloroform, and n-hexane were purchased from Lianlong Bohua Co., Ltd.; octadecyltrichlorosilane (OTS) was purchased from Beijing Bailingwei Technology Co., Ltd.; human serum albumin (HSA), carcinoembryonic antigen (CEA), prostate antigen (PSA), mouse monoclonal AFP antibody (anti-AFP antibody) and alpha-fetoprotein antigen (AFP) were all purchased from MyBioSource, USA; sodium perfluorooctanoate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), n-hydroxysuccinimide (NHS) and bovine serum albumin (BSA) were purchased from Shanghai Aladdin Co., Ltd., China; n ++ Si / SiO2 was purchased from the 46th Institute of China Electronics Technology Group Corporation; phosphate buffer solution (pH = 7) was prepared in the laboratory with 137.00 mM NaCl, 8.10 mM Na2HPO4-12H2O, 2.70 mM KCl and 1.50 mM KH2PO4 at room temperature. All reagents were used without further purification.

[0079] In a specific embodiment of the present invention, the cross-polarized optical microscope image is obtained by a Nikon ECLIPSE Ci-POL polarized optical microscope; the Fourier transform infrared spectrum is obtained by a Vertex70 Fourier transform infrared spectrometer; the atomic force microscope (AFM) image is captured in tapping mode in air using a Bruker Dimension Icon; the X-ray photoelectron spectroscopy (XPS) measurement is performed using an Axis Ultra DLD (Kratos, UK) ultrahigh vacuum photoelectron spectrometer under Al Kα radiation (1486.6 eV); the contact angle test is performed using an optical contact angle tension meter LSA / OSA-100 / 200 / 60; the confocal laser scanning microscope (CLSM) image is obtained by a Zeiss LSM 800; the electrochemical properties of the biosensor are analyzed using a Keithley 4200SCS semiconductor parameter analyzer; the carrier mobility (μ) is calculated based on the transfer IV curve under saturation, and the calculation formula is I DS =μC i W(V G -V TH ) 2 / 2L, where I DS is the source-drain current; μ is the charge carrier mobility; C i is the capacitance per unit area of ​​the insulating layer; W is the channel width; V G is the gate voltage; V THis the threshold voltage; L is the channel length.

[0080] The room temperature involved in the specific implementation manner of the present invention specifically refers to 20-25°C.

[0081] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0082] Example 1

[0083] The preparation of organic molecular crystal thin film (growing organic molecular crystal thin film using layer definition strategy) is as follows:

[0084] Step 1: Use toluene as solvent to prepare C6-DPA with a concentration of 0.2 mg mL -1 , surfactant sodium perfluorooctanoate concentration is 0.001 mg mL -1 The prepared mixed solution was ultrasonicated at a power of 100 W for 3 h;

[0085] Step 2: Add 25 mL of glycerol to a 75 mm × 35 mm weighing bottle as a liquid matrix for crystal growth; slowly drop 50 μL of the mixed solution after ultrasonication in step 1 onto the surface of the glycerol in the weighing bottle, and then place it in a constant temperature and humidity cabinet (typical temperature is 15° C., humidity is 30%);

[0086] Step 3: After the solvent is completely evaporated, C6-DPA molecular crystal films of various thicknesses floating on the glycerol surface are obtained.

[0087] The C6-DPA molecular crystal film prepared in this example was characterized by polarized light microscopy (POM). Figure 1 As shown, when the sample is rotated 45° (a is before rotation, b is after rotation), the color of the entire molecular crystal film changes uniformly from light blue to dark blue, which indicates the single crystal nature of the film;

[0088] The C6-DPA molecular crystal film prepared in this example was characterized by out-of-plane X-ray diffraction (XRD). Figure 2 As shown in the XRD spectrum, the first diffraction peak appears at about 2θ = 2.66°, indicating that the interlayer spacing of the C6-DPA crystal film is (3.22nm);

[0089] The surface of the C6-DPA molecular crystal film (the thinnest single-layer film) prepared in this example was randomly scanned by atomic force microscopy (AFM). The results are as follows: Figure 3 As shown, it can be seen that the film surface is flat, the root mean square (RMS) roughness is 0.29nm, and the film thickness is 2.8nm.

[0090] Example 2

[0091] The preparation of molecular crystal thin film OFETs without functional modification layer is as follows:

[0092] Step 1: Substrate cleaning: with a heavy n with thermally grown SiO2 layer + Doped silicon wafer (n ++ Si / SiO2) as the substrate (the thickness of the SiO2 layer is 300nm, n ++ The size of Si / SiO2 was 1 cm×1 cm). First, the easy-to-clean dust on the substrate surface was removed by ultrasonic treatment with deionized water for 10 min, then the difficult-to-clean dust on the substrate surface was removed by ultrasonic treatment with acetone for 10 min, and finally the final cleaning was performed by ultrasonic treatment with isopropanol for 10 min.

[0093] Step 2: Interface modification: First, the substrate was treated with plasma in an oxygen environment for 10 min at a power of 80 W, and then the substrate was placed in an OTS modification solution (volume ratio: n-heptane:OTS=1000:1), and the modification was allowed to stand at room temperature for 3 h. Then, the substrate was taken out and placed in a beaker containing n-hexane, chloroform, and isopropanol for ultrasonic treatment for 10 min.

[0094] Step 3: Formation of organic molecular crystal film: The C6-DPA molecular crystal film floating on the glycerol surface obtained in step 3 of Example 1 was transferred to the OTS-modified n ++ The Si / SiO2 substrate is used as a semiconductor layer. The specific operation is to cover the substrate on the glycerol surface with a C6-DPA molecular crystal film floating on the surface (the SiO2 layer of the substrate faces the C6-DPA molecular crystal film with a thickness of 2.8 nm to be transferred), and then remove the C6-DPA molecular crystal film after it is attached to the substrate surface; after removing it, gently rinse the n with the C6-DPA molecular crystal film with deionized water. ++ Si / SiO2 substrate to remove excess glycerol and surfactant;

[0095] Step 4: Preparation of source and drain electrodes: Bottom-gate top-contact devices (C6-DPA molecular crystal film OFETs, the channel ratio of the device is 200μm×200μm) were manufactured by stamping gold (150nm thick) stripes as source and drain electrodes on the C6-DPA molecular crystal film.

[0096] The electrical properties of the device prepared in this embodiment were characterized by a Keithley 4200SCS semiconductor parameter analyzer. Figure 5 shown.

[0097] Figure 5 Transfer (a) and output (b) curves of C6-DPA molecular crystal thin film OFETs. Figure 5 It can be seen that the C6-DPA molecular crystal thin film OFETs (without functional modification layer) have typical P-type charge carrier transport characteristics, with a maximum mobility of 3.22 cm 2 V -1 s -1 , the threshold voltage is 5.05V. By measuring its output curve, V G The frequency was swept from 0 to -60 V in steps of -10 V, and the results showed a clear transition from the linear state to the saturation state.

[0098] Example 3

[0099] Deposition of functional modification layers

[0100] (1) Selecting functional modification layer molecules: Select three molecules with side chain functional groups of -CHO (4,4'-(anthracene-9,10-diyl) dibenzaldehyde), -NH2 (4,4'-(anthracene-9,10-diyl) diphenylamine), and -COOH (4,4'-(anthracene-9,10-diyl) dibenzoic acid) and main chain of 9,10-diphenylanthracene (9,10-diphenylanthracene) as functional modification layer molecules, and vacuum deposit them (deposition rate is 100 μm). The surface of the semiconductor layer of the device obtained in step 4 of Example 2 (the surface of the source and drain electrodes not covered by the C6-DPA molecular crystal film and the surface of the source and drain electrodes are deposited) was modified by a method of deposition thickness of 3 nm and substrate temperature of 25°C, and atomic force microscopy (AFM) was performed to characterize it. Figure 6 As shown, a, d, and g are respectively the structural formula of a molecule with a functional group of -CHO, the roughness characterization after deposition, and the height characterization after deposition, b, e, and h are respectively the structural formula of a molecule with a functional group of -NH2, the roughness characterization after deposition, and the height characterization after deposition, c, f, and i are respectively the structural formula of a molecule with a functional group of -COOH, the roughness characterization after deposition, and the height characterization after deposition; the results show that the surface roughness of the three molecules with side chains of -CHO, -NH2, and -COOH functional groups after deposition are 20.20nm, 14.50nm, and 1.08nm, respectively. When the roughness is extremely large, the construction of an ultra-thin biosensor layer cannot be achieved (when the roughness is very large, the surface of the functional modification layer is uneven, the thickness of the most protruding part is relatively large, when the antibody is grafted, the thickness of the antibody at the distance from the channel is inconsistent, the protruding part is far away, and the biological signal is weak), so the ADDA molecule with a -COOH functional group is selected as the functional modification layer in the present invention;

[0101] (2) Optimizing deposition conditions: Depositing the ADDA layer at different deposition rates and substrate temperatures, and testing the roughness of the ADDA layer obtained under different deposition rates and substrate temperatures, such as Figure 7As shown in the figure, af represents different deposition rates or deposition temperatures. The results show that the deposition rate is The roughness is the smallest when the substrate temperature is 25℃ and 50℃, and the roughness difference is very small;

[0102] (3) Deposition thickness selection: The semiconductor layer surface of the device obtained in step 4 of Example 2 is ADDA molecules of different thicknesses (0nm, 3nm, 6nm, 12nm) were deposited at a deposition rate of 25°C and a deposition temperature (substrate temperature) of 25°C to obtain molecular crystal thin film OFETs with different thicknesses of ADDA. The electrical properties of the OFETs devices were characterized. The results are as follows: Figure 8 shown.

[0103] Figure 8 Transfer curves of molecular crystal thin film OFETs deposited with different thicknesses of ADDA. Figure 8 As can be seen from the figure, as the thickness of the ADDA layer increases, the mobility of the device slowly decreases, the threshold voltage moves to the left, and the switching ratio has almost no effect. Considering the above optimization results, the deposition rate is selected to be The vacuum deposition conditions of the ADDA functional modification layer were 25°C substrate temperature and 3 nm deposition thickness.

[0104] Example 4

[0105] Preparation of molecular crystal thin film OFETs biosensors based on ultrathin sensing layers (preparation process as shown in Fig. 9 The steps are as follows:

[0106] Step 1 to step 4 are the same as in Example 2;

[0107] Step 5: Deposition of ADDA functional modification layer: On the surface of the semiconductor layer of the device obtained in step 4 (the surface of the source and drain electrodes not covered by the C6-DPA molecular crystal film and the surface of the source and drain electrodes are deposited) ADDA molecules with a thickness of 3 nm were deposited at a deposition rate of 25°C and a deposition temperature (substrate temperature) of 25°C to obtain a molecular crystal film OFETs biosensor based on an ultra-thin sensing layer (C6-DPA molecular crystal film + ADDA functional modification layer as the sensing layer, the thickness of the C6-DPA molecular crystal film is 2.8 nm, the thickness of the functional modification layer is 3 nm, so the thickness of the sensing layer is 5.8 nm).

[0108] The electrical properties of the molecular crystal thin film OFETs biosensor based on the ultra-thin sensing layer prepared in this embodiment were characterized by a Keithley 4200SCS semiconductor parameter analyzer. The results are as follows: Fig.10 As shown, (a) is the transfer curve, (b) is the output curve, Fig.10 It can be seen that the molecular crystal thin film OFETs biosensor with a functional modification layer thickness of 3 nm still has typical P-type charge carrier transport characteristics, with a maximum mobility of 3.25 cm 2 V -1 s -1 , the threshold voltage is 4.59V. By measuring its output curve, V G The frequency was swept from 0 to -60 V in steps of -10 V, and the results showed a clear transition from the linear state to the saturation state.

[0109] Example 5

[0110] The highly efficient grafting and immobilization of the antibody of liver cancer marker AFP on the molecular crystal thin film OFETs biosensor based on ultrathin sensing layer (i.e., the preparation of the biosensor for detecting AFP, the process is as follows Fig. 9 The steps are as follows:

[0111] First, the carboxyl groups on the functional modification layer ADDA of the molecular crystal thin film OFETs biosensor based on the ultrathin sensing layer obtained in step 5 of Example 4 were activated using EDC and NHS for 1 h (the specific operation was to drop 20 μL of a mixed solution of EDC and NHS on the surface of the functional modification layer and let it stand at room temperature for 1 h. The concentration of EDC in the mixed solution was 20 mg / mL, and the concentration of NHS was 10 mg / mL). The activated surface was rinsed 3 times with a phosphate buffer solution (0.01×PBS, pH=7), and then 20 μL of a mouse monoclonal AFP antibody (antibody-AFP) with a concentration of 10 μg / mL was incubated (incubated at room temperature for 1 h) on the functional modification layer, and the surface was rinsed 3 times with a phosphate buffer solution (0.01×PBS, pH=7).

[0112] Firstly, the height and roughness of the molecular crystal thin film OFETs biosensor before and after grafting the biosensitive probe AFP antibody were characterized by atomic force microscopy (AFM). Fig.11 As shown, a and c are the height characteristics before and after incubation, and b and d are the roughness characteristics before and after incubation. It can be seen that after the antibody-AFP is fixed, the relative height of the sensor surface increases from 5.8nm to 10.53nm. At the same time, after the antibody-AFP is fixed, the roughness of the sensor surface increases from 1.25nm to 1.98nm, indicating that the antibody-AFP immobilization effect is good;

[0113] In order to more intuitively observe the fixation of antibody-AFP on the surface of the functional modification layer ADDA, the antibody-AFP with a fluorescent marker was fixed (grafted) on the surface of the film modified with ADDA molecules (the device prepared in Example 2), and the antibody-AFP before and after grafting was characterized using a confocal laser scanning microscope (CLSM). The results are shown in Figure 2. Fig.12 As shown, a is before antibody-AFP grafting (AFP in the figure represents antibody-AFP), and b is after antibody-AFP grafting. Fig.12 It can be seen that the film before antibody-AFP grafting shows dark fluorescence, while the film after antibody-AFP grafting shows green fluorescence due to the covalent binding of antibody-AFP and ADDA. Therefore, the modification of ADDA molecules is conducive to the construction of high-performance molecular crystal thin film OFETs biosensors.

[0114] XPS can also confirm the successful immobilization of antibody-AFP. XPS characterization of the biosensor surface before and after the immobilization of antibody-AFP is performed. The results are as follows Fig.13 As shown, a is the N element and b is the oxygen element; Fig.13 It can be seen that before antibody-AFP was fixed, no nitrogen signal was detected on the sensor surface modified with ADDA molecules; however, after antibody-AFP was fixed, nitrogen signal appeared and oxygen signal was significantly enhanced due to the presence of protein. The above characterization results fully prove that the sensor surface modified with ADDA molecules can successfully fix antibody-AFP.

[0115] Example 6

[0116] Sensing of liver cancer marker AFP antigen by molecular crystal thin film OFETs biosensor based on ultrathin sensing layer (process as shown in Fig. 9 ef in the figure), the steps are as follows:

[0117] Step 1: first, using EDC and NHS to activate the carboxyl groups on the functional modification layer ADDA of the molecular crystal thin film OFETs biosensor based on the ultra-thin sensing layer obtained in step 5 of Example 4 for 1 hour (the specific operation is to drop 20 μL of a mixed solution of EDC and NHS on the surface of the functional modification layer and let it stand at room temperature for 1 hour. The concentration of EDC in the mixed solution is 20 mg / mL, and the concentration of NHS is 10 mg / mL), and then rinse the activated surface 3 times with a phosphate buffer solution (0.01×PBS, pH=7), and then incubate 20 μL of mouse monoclonal AFP antibody (incubate at room temperature for 1 hour) on the functional modification layer to obtain a sensor for detecting AFP, and prepare multiple sensors for detecting AFP at the same time;

[0118] Step 2: Rinse the sensor surface for detecting AFP in step 1 three times with phosphate buffer solution (0.01×PBS, pH=7), dry it at room temperature for 5 seconds, test the transfer curve of the sensor and obtain the output threshold voltage value V0; add 20 μL of AFP antigen standard solution of different concentrations (concentrations are 10 -2 , 10 -1 , 10 0 , 10 1 , 10 2 , 10 3 ng mL -1 ) was dripped onto the surface of different sensors after the test (one sensor was used for one concentration of AFP antigen standard solution, and each sensor was fully rinsed before testing V0 before grafting AFP antigen to improve the accuracy and reproducibility of characterization), incubated at room temperature for 1 h, and dried at room temperature for 5 s, and the transfer curve of the biosensor exposed to different concentrations of AFP antigen was tested and the corresponding output threshold voltage value V was obtained;

[0119] Step 3: Calculate the △V corresponding to the AFP antigen concentration TH =V-V0 as the sensor output signal;

[0120] Step 4: Take the AFP antigen concentration as the horizontal axis and calculate the △V corresponding to the AFP antigen concentration. TH Draw a curve as the ordinate and calibrate to obtain a standard curve. The result is as follows Fig.14 As shown in the figure, the detection limit of the molecular crystal thin film OFETs biosensor based on the ultrathin sensing layer for the liver cancer marker AFP antigen is as low as 1.70 pg mL -1 , with high sensitivity, lower than the clinical detection value of AFP 10ng mL -1 .

[0121] Example 7

[0122] V of OFETs biosensors with different numbers of molecular crystal films for AFP biomarkers TH Exploration of the sensing behavior of electrical signals

[0123] In this example, single-layer, double-layer and twelve-layer molecular crystal thin film OFETs with organic molecular crystal film thicknesses of 2.8 nm, 5.7 nm and 39.6 nm were prepared (the preparation steps were the same as in Example 2, only the thickness of the transferred C6-DPA molecular crystal film was different), and OFETs biosensors with different numbers of molecular crystal thin films were prepared after ADDA modification. The method of Example 6 (steps 1-3) was used to modify the concentration of 1 μg mL -1 The AFP antigen solution was tested.

[0124] In the biosensing process of the present invention, the binding of the charged target protein to the recognition molecule induces the accumulation or depletion of the carrier in the organic channel and produces a threshold voltage change, thereby quantitatively detecting target proteins of different concentrations. TH Obvious changes occurred before and after the target protein was fixed, indicating that the specific recognition reaction between the AFP antigen and the AFP antibody led to changes in the electrical signal of the biosensor. Based on the above working principle, the thinner the sensing layer, the stronger the electrostatic interaction and the larger the electrical signal. In order to verify the charge-induced biodetection principle, this embodiment prepared single-layer, double-layer and twelve-layer molecular crystal film OFETs with molecular crystal film thicknesses of 2.8nm, 5.7nm and 39.6nm, respectively, and prepared OFETs biosensors with different numbers of molecular crystal film layers after ADDA modification. And at three thicknesses, V TH The output signal detection concentration was 1 μg mL -1 The AFP antigen, the results are as follows Fig.15 As shown in the figure, as the thickness increases, the threshold voltage change value becomes smaller. The change values ​​of the single layer and double layer are very weak. It is speculated that under the condition of high concentration of the target analyte, the electrostatic interaction of the single layer and double layer thickness is strong enough to ensure the perfect conversion of biological signals to electrical signals. However, when the number of layers is twelve, it can be found that the electrical signal value is greatly reduced compared with fewer layers. This result is consistent with the charge-induced sensing mechanism.

[0125] Example 8

[0126] Selective testing of molecular crystal thin film OFETs biosensors for sensing AFP cancer markers

[0127] 1 μg mL -1 AFP antigen was used as the experimental group, 1 μg mL -1CEA, PSA, BSA and HSA were used as negative control groups, and 1×PBS (pH=7) phosphate buffer solution was used as blank control group to conduct a selectivity test of molecular crystal thin film OFETs biosensor for AFP cancer marker sensing. The test steps refer to steps 1-3 of Example 6. The test results are as follows: Fig.16 As shown. Fig.16 It can be seen that under the same conditions, after 1 μg mL -1 After the immune reaction between AFP antigen and AFP antibody, V TH The positive shift was 4.083±0.048 V. In the negative control experiment, at 1 μg mL -1 In the case of CEA, PSA, BSA and HSA, V TH Electrical signal (△V TH ) is almost negligible compared with the sensing signal of AFP, and the corresponding signal shown in the blank control experiment of 1×PBS phosphate buffer solution is weak, indicating that the sensor of the present invention has excellent selectivity for AFP.

[0128] Example 9

[0129] Anti-interference test of molecular crystal thin film OFETs biosensor for AFP cancer marker sensing

[0130] 1 μg mL -1 The AFP was mixed into the same concentration of PSA, CEA, CEA & PSA, CEA & PSA & HSA & BSA and human serum (Serum) samples to obtain 5 samples containing 1 μg mL -1 The AFP test solution sample was used, and human serum alone was used as a blank control to conduct an anti-interference test of the molecular crystal thin film OFETs biosensor for AFP cancer marker sensing. The test steps refer to steps 1-3 of Example 6. The test results are as follows: Fig.17 As shown. Fig.17 It can be seen that V TH Electrical signal (△V TH The above results fully demonstrate that the biosensor has good anti-interference ability for AFP sensing and is not easily affected by other interfering substances.

[0131] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A molecular crystal thin film organic field effect transistor biosensor based on an ultra-thin sensing layer, characterized in that: The invention comprises a substrate, an organic molecular crystal film, a source-drain electrode and a functional modification layer which are arranged in sequence; the organic molecular crystal film is 2,6-bis(4-hexylphenyl)anthracene, 2-decyl-7-phenyl[1]benzothieno[3,2-b[1]benzothiophene, perylene, dioctylbenzothiophene or 6,13-bis(triisopropylsilylethynyl)pentacene; the functional modification layer is 4,4'-(anthracene-9,10-diyl)dibenzoic acid; the thickness of the organic molecular crystal film is 2.8-39.6 nm, and the thickness of the functional modification layer is 3 nm; The method for preparing the molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer comprises the following steps: The interface of the substrate is modified; an organic molecular crystal film is grown on the surface of the liquid substrate; the organic molecular crystal film grown on the surface of the liquid substrate is transferred to the surface of the substrate after the interface modification, a source-drain electrode is prepared on the surface of the organic molecular crystal film, and a functional modification layer is deposited on the surface of the organic molecular crystal film and the surface of the source-drain electrode to obtain the molecular crystal film organic field-effect transistor biosensor based on the ultra-thin sensing layer.

2. The method for preparing a molecular crystal thin film organic field effect transistor biosensor based on an ultra-thin sensing layer according to claim 1, characterized in that: The following steps are involved: The interface of the substrate is modified; an organic molecular crystal film is grown on the surface of the liquid substrate; the organic molecular crystal film grown on the surface of the liquid substrate is transferred to the surface of the substrate after the interface modification, a source-drain electrode is prepared on the surface of the organic molecular crystal film, and a functional modification layer is deposited on the surface of the organic molecular crystal film and the surface of the source-drain electrode to obtain the molecular crystal film organic field-effect transistor biosensor based on the ultra-thin sensing layer.

3. The preparation method according to claim 2, characterized in that: The interface modification of the substrate comprises: firstly treating the substrate with plasma in an oxygen environment, and then modifying the substrate with octadecyltrichlorosilane.

4. The preparation method according to claim 3, characterized in that: The time for treating the substrate with plasma is 5-25 min, and the power is 80 W; the specific operation of modifying the substrate with octadecyltrichlorosilane is: placing the substrate in a modification solution composed of octadecyltrichlorosilane and n-heptane, and leaving it to stand at room temperature for 2-5 h for modification.

5. The preparation method according to claim 2, characterized in that: The method for growing an organic molecular crystal film on the surface of a liquid substrate comprises: dripping a mixed solution of organic molecules and a surfactant onto the surface of the liquid substrate, placing the solution under constant temperature and humidity conditions to completely evaporate the solvent, and obtaining an organic molecular crystal film floating on the surface of the liquid substrate; the organic molecules are 2,6-bis(4-hexylphenyl)anthracene, 2-decyl-7-phenyl[1]benzothieno[3,2-b[1]benzothiophene, perylene, dioctylbenzothiophene or 6,13-bis(triisopropylsilylethynyl)pentacene.

6. The preparation method according to claim 5, characterized in that: The surfactant is sodium perfluorooctanoate; the solvent of the mixed solution is toluene; the concentration of organic molecules in the mixed solution is 0.2 mg mL -1 , the concentration of surfactant was 0.001 mg mL -1 ; The liquid matrix is ​​glycerol.

7. The preparation method according to claim 2, characterized in that: The deposition rate of the functional modification layer was 0.3 Å s -1 .

8. A method for detecting a disease marker for non-diagnostic purposes, characterized in that: A molecular crystal thin film organic field effect transistor biosensor based on an ultra-thin sensing layer as described in claim 1.

9. The method according to claim 8, characterized in that The following steps are involved: Grafting disease marker antibodies as biosensitive probes on the surface of the functional modification layer of the molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer; Grafting disease marker standard solutions of different concentrations on the surface of the disease marker antibody, and detecting the threshold voltage of the transfer curve of the biosensor before and after the grafting of the disease marker standard solution, and taking the change value of the threshold voltage of the transfer curve of the biosensor before and after the grafting of the disease marker standard solution as the sensor output signal; The standard curve is drawn with the concentration of the disease marker standard solution as the horizontal axis and the change value of the threshold voltage at the corresponding concentration as the vertical axis; The test solution is grafted on the surface of the disease marker antibody, and the threshold voltage of the transfer curve of the biosensor before and after grafting the test solution is detected. The change value of the threshold voltage corresponding to the test solution is substituted into the standard curve to obtain the concentration of the disease marker in the test solution.

10. Use of the molecular crystal thin film organic field effect transistor biosensor based on the ultra-thin sensing layer as claimed in claim 1 in the preparation of a biosensor for detecting AFP.

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