CN115078504B

CN115078504B - Preparation method and detection device of multi-sensing integrated MIGFET biosensor

Info

Publication number: CN115078504B
Application number: CN202210801450.5A
Authority: CN
Inventors: 赵红; 孙伟斌; 秦鹏; 唐雪; 白成林; 许恒迎; 杨立山
Original assignee: Liaocheng University
Current assignee: Liaocheng University
Priority date: 2022-07-08
Filing date: 2022-07-08
Publication date: 2024-03-19
Anticipated expiration: 2042-07-08
Also published as: CN115078504A

Abstract

The invention discloses a preparation method and a detection device of a multi-sensor integrated MIGFET biosensor, belongs to the technical field of biosensors, and aims to provide a multi-sensor integrated graphene field effect transistor biosensor based on micro-flow control, which integrates graphene field effect transistor biosensing and fluorescence biosensing and combines magnetic nano-particle MPs to construct a multi-sensor integrated biochip based on micro-flow control to realize photo-electromagnetic multi-detection sensor detection of the same target molecules, thereby improving detection accuracy and reliability of the biosensor. The invention combines the field effect tube and the micro-flow control, the biosensor does not need to rely on large-scale instrument analysis, and is easy to integrate and automate, and meanwhile, the invention adopts a photo-electromagnetic multi-detection mode to detect the same target object, thereby improving the accuracy and reliability of detection.

Description

Preparation method and detection device of multi-sensing integrated MIGFET biosensor

Technical Field

The invention belongs to the technical field of biosensors, and particularly relates to a preparation method and a detection device of a multi-sensor integrated MIGFET biosensor.

Background

In order to detect biological or biomimetic signals, biosensors have been developed. The biosensor aims at qualitatively or quantitatively analyzing an object to be detected (including nucleic acid, protein, cell, etc.), and the detection principle is that after the object to be detected is combined with a biosensing material, one or more physical and chemical properties can be changed when the object to be detected passes through a specific physicochemical transducer, and the change of the properties can be detected as a detectable signal. Compared with the traditional detection means, the biosensor has the advantages of greatly improving sensitivity, economy, specificity, response time and the like, and has become a hot technological development direction in the current world. The DNA quantitative detection and the research on the binding kinetics thereof have important application values in the aspects of clinical diagnosis, microanalysis, drug research and development, food screening, industrial production, environmental detection and the like. Thus, research into DNA biosensors attracts more and more expert scholars.

In recent years, research and development of DNA biosensors based on different operating characteristics have become an important subject for widening scientific boundaries for domestic and foreign scholars, and have been made to obtain great achievements through continuous efforts, and particularly, biosensors having high sensitivity, wide detection range, good selectivity and high response speed are more favored by researchers. Of the many biosensors, optical and electronic biosensors are particularly excellent.

The working principle of the optical biosensor is to realize quantitative analysis of biomolecules to be detected by detecting the variation of optical signals, and the optical biosensor is widely applied to the fields of disease diagnosis, forensic identification, pollution environment monitoring, life science research and the like. In optical methods, label-free surface plasmon resonance (Surface plasmon resonance, SPR) is a standard tool commonly used for DNA hybridization kinetic process detection. However, the optical response of the SPR sensor is related to the molecular weight of the analyte, and weak signal changes generated by the binding between small molecules, such as oligopeptide and oligonucleotide binding, are difficult to detect. Therefore, SPR detection methods have limitations in terms of the sensitivity of quantitative analysis of DNA hybridization kinetics. In addition to SPR, fluorescence biosensors based on fluorescence resonance energy transfer (Fluorescence resonance energy transfer, FRET) are another commonly used optical technique for monitoring DNA hybridization kinetics. Fluorescent biosensors based on FRET have the advantages of high analysis sensitivity, strong selectivity, simple and convenient use and the like, and become an optical sensing technology most commonly applied at present. FRET is a non-radiative transition process that uses interactions between distant dipoles to transfer the donor energy of an excited state to a proximal ground state acceptor. Typically, the change in the spacing of the donor and acceptor molecules is on the order of nanometers (proportional to r 6), whereas FRET has an inherent sensitivity to this process. FRET is therefore very attractive for biological analysis, which has the potential to achieve highly sensitive detection of DNA. Based on this principle, a ultrasensitive biosensor based on FRET between carbon dots and AuNPs has been reported to be useful for detecting DNA sequences associated with HIV.

Electronic biosensors are generally used for detecting electrical signals accompanied in biochemical reaction processes to realize quantitative analysis of target objects. In electrical methods, one-dimensional nanomaterials (carbon nanotubes, nanowires, etc.) have proven useful for real-time detection of a variety of bioactive molecules. Based on this principle, a top-down silicon nanowire Field effect transistor (Field-effect transistor, FET) biosensor has been reported that can achieve highly sensitive, label-free detection of target DNA.

However, since the device fabrication process based on the silicon nanowire FET biosensor is relatively costly, it is not widely applicable to various application scenarios, and the high probe density in the silicon nanowire FET biosensor can reduce the efficiency of DNA hybridization and kinetics. Thus, manufacturing a reliable and cost-effective silicon nanowire FET biosensor remains difficult.

Graphene is a single-layer two-dimensional carbon material that has been used in large scale for decades. The two-dimensional Graphene field-effect transistor (GFET) biosensor has the advantages of detecting DNA hybridization dynamics compared with a one-dimensional nanomaterial FET biosensor due to the excellent characteristics of zero band gap structure, extremely high carrier mobility, high specific surface area, relatively sensitive charge change caused by reaction or adsorption of analyte surface and the like. In conventional GFET biosensors, DNA hybridization kinetics detection is analyzed based primarily on the theory of double-conductive layer capacitance model of GFET biosensors. When a gate voltage is applied to the graphene film through the dielectric, a structure with a double conductive layer is generated at the interface of the graphene and the electrolyte, the resistivity of the structure is correspondingly changed, and the structure can be quantitatively detected by the GFET biosensor. Therefore, by supplying a certain gate voltage to the gate and analyzing the change of the electrical signal acquired by the GFET biosensor, detection of target DNA (tDNA) to be detected can be achieved. Based on this principle, a pleated GFET biosensor has been reported for detecting the presence of SARS-CoV-2 virus in a sample of a patient's viral transport medium.

The magnetic tweezer technology is a single-molecule force spectrometry, magnetic nano particles (Magnetic nanoparticles, MPs) are coupled at the tail end of tDNA, and under the action of an externally applied magnetic field, the distance between graphene and tDNA coupled with the MPs can be mechanically controlled by utilizing the magnetic force between the MPs and the magnetic field, so that the double-conductive-layer structure of a magnetic-control graphene field effect transistor (Magnetic graphene field-effect transistor, MGFET) biosensor is modulated. Therefore, MGFET biosensors have attracted considerable attention because of their unique advantages of high signal-to-noise ratio, stable physical and chemical properties, biocompatibility, environmental protection, and the like. Based on this principle, an MGFET biosensor has been reported for high sensitivity detection of DNA.

However, although the above FRET-based fluorescent biosensors and GFET biosensors have been widely studied, they generally employ only one sensing method in one biosensor, and the sensing method is single, resulting in difficulty in ensuring accuracy; meanwhile, the biosensor relies on the analysis of the existing large-scale instrument, and the integrated detection device is not enough. The micro-fluidic chip analysis system is a rapid and low-consumption micro-analysis experimental device which integrates sample injection and detection. The microfluidic chip has the advantages of low cost, less pollution, convenient carrying, automation and easy integration, and can realize high-flux and high-sensitivity analysis. Therefore, a mode of integrating a plurality of sensing technologies in a microfluidic chip analysis system to detect a target object is widely paid attention to.

Disclosure of Invention

Aiming at the defects in the prior art, the preparation method and the detection device of the multi-sensing integrated MIGFET biosensor provided by the invention are used for improving the detection sensitivity and the reliability of the traditional single sensor.

In order to achieve the aim of the invention, the invention adopts the following technical scheme: a preparation method of a multi-sensor integrated MIGFET biosensor comprises the following steps:

s1, spin-coating a layer of polymethyl methacrylate (PMMA) on graphene growing on the surface of a copper foil by using a spin coater;

s2, baking PMMA/graphene/copper foil on a constant-temperature heating table at 150 ℃ for 15 minutes to combine PMMA with the surface of graphene;

s3, etching the underlying copper substrate by using a ferric trichloride solution with the concentration of 1M to obtain a PMMA/graphene film;

s4, carrying out deionized washing treatment on the PMMA/graphene film, and transferring the washed PMMA/graphene film to an ITO electrode glass substrate with patterns;

s5, baking the PMMA/graphene/glass substrate on a constant-temperature heating table at 180 ℃ for 15 minutes to remove moisture;

s6, soaking the PMMA/graphene/glass substrate with the water removed in an acetone solution to remove the PMMA, and washing with absolute ethyl alcohol and deionized water solution in sequence to obtain an ITO glass substrate of graphene;

s7, bonding a reaction cavity with a microfluidic channel, which is made of polydimethylsiloxane materials, to an ITO glass substrate of graphene to construct a Y-shaped micro-channel;

s8, based on a Y-shaped micro-channel, an AgCl electrode is adopted as a grid input voltage to form a GFET biosensor as an electric channel, a narrow-band LED is adopted as an excitation light source, the narrow-band LED is conducted to a sensitive area through an optical fiber to form a fluorescent biosensor, the fluorescent biosensor is used as an optical channel of the MIGFET biosensor, a periodic magnetic field is applied above the MIGFET biosensor to form a magnetic channel of the MIGFET biosensor, and the preparation of the multi-sensing integrated MIGFET biosensor is completed.

The beneficial effects of the invention are as follows: the invention aims to provide a Multi-sensing integrated graphene field effect transistor (Multi-sensing integrated graphene field-effect transistor, MIGFET) biosensor based on micro-flow control, which integrates graphene field effect transistor biosensing and fluorescence biosensing and combines MPs to construct a Multi-sensing integrated biochip based on micro-flow control to realize photo-electromagnetic Multi-detection sensor detection of the same target molecules, so that the detection accuracy and reliability of the biosensor are improved. The invention combines the field effect tube and the micro-flow control, the biosensor does not need to rely on large-scale instrument analysis, and is easy to integrate and automate, and meanwhile, the invention adopts a photo-electromagnetic multi-detection mode to detect the same target object, thereby improving the accuracy and reliability of detection.

Further, the multi-sensing integrated MIGFET biosensor includes the following functionalization processes:

a1, injecting 10mM of N-hydroxysuccinimide ester of 1-pyrenebutyric acid (PBASE) solution dissolved in dimethyl sulfoxide (DMSO) into the prepared microfluidic reaction cavity, and incubating the solution at room temperature for 2 hours;

a2, washing the MIGFET biosensor by using DMSO and deionized water solutions respectively in sequence, removing redundant unbound PBASE molecules, injecting 2uM probe aptamer solution into a microfluidic reaction cavity, and incubating for 4 hours at room temperature;

and A3, washing the MIGFET biosensor by sequentially selecting PBS (phosphate buffer solution) containing 0.2% of dialkyl sodium sulfate SDS and PBS solution, removing redundant probe aptamer, and completing the functionalization treatment of the MIGFET biosensor.

The beneficial effects of the above-mentioned further scheme are: the invention makes the biosensor have specificity to the object to be detected by means of the specific recognition of the aptamer.

The invention provides a detection device of a multi-sensor integrated MIGFET biosensor, which comprises an optical path module, a circuit module connected with the optical path module and an upper computer connected with the circuit module.

Further, the optical path module comprises an optical fiber collimator and a photomultiplier;

focusing and coupling light emitted by the LED lamp by using an optical fiber collimator to form a first collimated beam; transmitting the first collimated beam to the MIGFET biosensor via an optical fiber to provide an excitation light source to excite the fluorophore 6' -carboxyfluorescein labeled on the target DNA; after the fluorescent group 6' -carboxyl fluorescein is excited, the fluorescent group is converted into a second collimated light beam through an optical fiber collimator, and the second collimated light beam is transmitted to a high-pass filter for filtering through the optical fiber collimator; and converging the filtered collimated light beams to an effective sensitive area of the photomultiplier, and displaying and storing the obtained optical signals by using an upper computer after photoelectric conversion, and reflecting the fluorescence intensity.

The beneficial effects of the above-mentioned further scheme are: the invention can effectively remove stray light by utilizing the light path module.

Still further, the circuit module includes:

the power supply sub-module is used for supplying power to the detection device;

the LED lamp driving submodule is used for providing an excitation light source;

a PMT gain control sub-module for providing a gain voltage for the photomultiplier tube;

the signal conditioning sub-module is used for amplifying the electric signal subjected to photoelectric conversion and output by the photomultiplier, and a four-order low-pass filter is arranged as a filter circuit by combining the double operational amplifiers, and the electric signal is conditioned by the filter circuit and is transmitted to the AD signal acquisition sub-module;

the grid voltage output submodule is used for realizing grid voltage output in a positive and negative voltage range in an SPI communication mode;

the field effect tube equivalent resistance measuring submodule is used for collecting the voltage between the source electrode and the drain electrode of the MIGFET biosensor;

the stepping motor driving submodule is used for driving the stepping motor to apply a periodic magnetic field to the MIGFET biosensor in a pulse width modulation mode, and the mechanical movement of MPs/tDNA causes the fluctuation of the resistance intensity of the MIGFET biosensor so as to change the conductivity of graphene;

and the AD signal acquisition sub-module is used for converting the electric signal into a digital signal and communicating with the upper computer.

The beneficial effects of the above-mentioned further scheme are: the invention can synchronously collect three detection signals by arranging the detection device of the multi-sensor integrated MIGFET biosensor.

Drawings

FIG. 1 is a flow chart of the preparation method of the invention.

Fig. 2 is a schematic diagram of the MIGFET biosensor manufacturing process in this example.

FIG. 3 is a schematic diagram of a detecting device according to the present invention.

Fig. 4 is a schematic diagram of the MIGFET biosensor functionalization process in this embodiment.

FIG. 5 is a schematic diagram showing the MIGFET biosensor modification process in this embodiment.

FIG. 6 is a comparative schematic diagram of the detection result of sensitivity of MIGFET biosensor to DNA in this example.

FIG. 7 is a comparative schematic diagram showing the result of DNA specific detection by the MIGFET biosensor in this example.

FIG. 8 is a graph showing the comparison of the detection results of the MIGFET biosensor in this example.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

Examples

As shown in fig. 1, the invention provides a preparation method of a multi-sensing integrated MIGFET biosensor, comprising the following steps:

In this embodiment, the multi-sensing integrated MIGFET biosensor includes the following functionalization process:

As shown in fig. 3, the invention provides a detection device of a multi-sensing integrated MIGFET biosensor, which comprises an optical path module, a circuit module connected with the optical path module and an upper computer connected with the circuit module;

the optical path module comprises an optical fiber collimator and a photomultiplier;

The circuit module includes:

The invention is described in detail below with reference to the attached drawing figures:

(1) Process for coupling tDNA to be tested with MPs

The principle that MPs and tDNA to be tested can be coupled is that one end of tDNA is modified with amino, so that dehydration condensation reaction can be carried out between the MPs and carboxylated MPs (4 mg.mL-1), and then MPs are modified at the tail end of tDNA to be tested. Unbound tDNA was washed under enrichment of strong magnetic field and then dispersed in phosphate-buffered saline (PBS) for use.

Firstly, MPs (4 mg.mL-1) are modified by carboxyl groups, 20 mu L of MPs are taken and subjected to ultrasonic treatment by an ultrasonic instrument, and after 20 minutes, a uniformly dispersed MPs solution can be obtained. Next, 200. Mu. L N-hydroxysuccinimide (2 mg. ML-1) solution was mixed with 200. Mu.L of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (2 mg. ML-1) solution and the uniformly dispersed MPs solution was added and mixed for 15 minutes to obtain activated MPs. Then, 20. Mu.L of tDNA to be tested was added to the above mixed solution, which was slightly shaken and incubated at room temperature for 2 hours. Next, a strong magnetic field is used to enrich the MPs coupled tDNA under test (Magnetic nanoparticles/tDNA, MPs/tDNA). Finally, the excess unbound tDNA was washed with PBS solution and dispersed in PBS solution and stored at 4 ℃ for later use.

(2) Preparation of MIGFET biosensor

Because the prepared MIGFET biosensor needs to be transferred out of high-quality graphene, the graphene film manufactured by a typical chemical vapor deposition method is transferred to a glass substrate with an Indium Tin Oxide (ITO) electrode by a wet transfer method, and the glass substrate is used as a source electrode and a drain electrode of the MIGFET biosensor. The MIGFET biosensor is fabricated as shown in fig. 2.

First, graphene grown on the surface of the copper foil was spin-coated with a thin layer of polymethyl methacrylate (PolymethylMethacrylate, PMMA) using a spin coater (fig. 2 a). After PMMA spin coating is completed, PMMA/graphene/copper foil is baked on a constant temperature heating table at 150 ℃ for 15 minutes, so that PMMA and the graphene surface are tightly combined. Then, the underlying copper substrate was etched with 1M ferric trichloride solution to obtain a PMMA/graphene thin film (fig. 2 b). After deionized water washing, clean PMMA/graphene was transferred onto a patterned ITO electrode glass substrate (fig. 2 c). Next, the clean PMMA/graphene/glass substrate was baked on a constant temperature heating stage at 180 ℃ for 15 minutes to remove moisture. Finally, PMMA/graphene/glass substrates were immersed in an acetone solution to remove PMMA. And sequentially washing the ITO glass substrate with the graphene transferred by absolute ethyl alcohol and deionized water solution. In order to prepare the GFET biosensor device, a reaction chamber with microfluidic channels made of polydimethylsiloxane material was bonded to an ITO glass substrate to construct a Y-shaped microchannel (fig. 2 d). An AgCl electrode is used as a grid input voltage to form a GFET biosensor as an electric channel; the narrow-band LED is used as an excitation light source and is conducted to a sensitive area through an optical fiber to form a fluorescent biosensor, and the fluorescent biosensor is used as an optical channel of the MIGFET biosensor; a periodic magnetic field is applied over the sensor, forming a magnetic channel of the MIGFET biosensor. Thus, a physical structure diagram of the MIGFET biosensor is obtained (see fig. 2 e).

(3) Photo-electromagnetic multi-sensing integrated biosensing detection system

As shown in FIG. 3, the integrated biological sensing detection system (Multi-sensing integrated biosensing detection system, MIBDS) based on the micro-fluidic photo-electromagnetic Multi-sensing mode mainly comprises an optical path part, a circuit part and an upper computer part written in LabView.

The light path part utilizes an optical fiber collimator to focus, couple and form a first collimated light beam emitted by the LED lamp, the first collimated light beam is transmitted to the MIGFET biosensor through an optical fiber, and an excitation light source is provided to excite a fluorescent group 6 '-carboxyfluorescein (6' -FAM) marked on target DNA. After the fluorescent group 6'-FAM is excited, the fluorescent group 6' -FAM is firstly converted into a second collimated light beam through an optical fiber collimator, then the second collimated light beam is transmitted to a high-pass filter of 520nm through an optical fiber for filtering, the light is converged into an effective sensitive area of a photomultiplier (Photoelectric multiplier tube, PMT) after being filtered, finally the obtained electric signal is displayed and stored in real time by an upper computer written in LabView after being subjected to photoelectric conversion of the PMT, and the current fluorescence intensity (The fluorescence intensity, IF) is reflected.

The circuit system design part mainly comprises a power supply module, an LED lamp driving module, a PMT gain control module, a signal conditioning module, a grid voltage output module, a field effect tube equivalent resistance measuring module, a stepping motor driving module and an AD signal acquisition module. The power supply module is connected with 220V alternating current input, and is rectified and reduced to +/-12V direct current and +/-5V direct current, and the power supply module supplies power for the whole system after voltage stabilization. The LED lamp driving module utilizes a constant current chip MAX1916 to provide an excitation light source with adjustable brightness and stable luminous intensity for the system. The PMT gain control module provides a gain control voltage for the PMT to achieve PMT gain control. The signal conditioning module amplifies the electric signal output after the PMT photoelectric conversion by adopting an AD623 instrument amplifier, and a four-order 'Butterworth response' low-pass filter is designed by combining a double operational amplifier LM358 and used as a filter circuit to condition the electric signal and then transmit the electric signal to the AD data acquisition module. The grid voltage output module adopts an STM32F103 microcontroller and combines with the DAC8501 module to realize the grid voltage output with adjustable positive and negative voltage ranges in an SPI communication mode. The field effect tube equivalent resistance measuring module designs the field effect tube equivalent resistance measuring module to collect the voltage (Voltage between the source and drain, vds) between the source electrode and the drain electrode of the MIGFET by utilizing the Wheatstone balance bridge detection principle, wherein the current intensity (The current intensity, IC) of the MIGFET can be calculated through the Vds. When the stepping motor driving module drives the stepping motor to apply a periodic magnetic field to the MIGFET biosensor in a pulse width modulation mode, the mechanical motion of MPs/tDNA can be caused, so that the resistance intensity (The resistance intensity, IR) of the sensor is caused to move, and the conductivity of graphene is changed. The electrical parameters in the MIGFET biosensor are amplified by the field effect tube equivalent resistance measuring module and transmitted to the AD data acquisition module. The AD data acquisition module capable of realizing analog-to-digital conversion can convert a voltage signal into a binary digital signal, and the voltage signal is communicated with an upper computer written by LabView through a USB data interface in a serial port manner. And displaying, processing and storing the data by using an upper computer written in LabView.

(4) Functionalization of MIGFET biosensor

The functionalization scheme of MIGFET biosensor is shown in fig. 4, first, 10mM solution of N-hydroxysuccinimide ester (1-pyrenebutyric acid N-hydroxysuccinimide ester, PBASE) dissolved in dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) 1-pyrenebutyric acid is injected into the prepared microfluidic reaction chamber, and incubated at room temperature for 2 hours. And pyrenyl groups capable of reacting with a six-membered ring of the graphene exist on the PBASE molecules, and the pyrenyl groups can be attached to the graphene film through pi-pi stacking so as to keep the inherent high conductivity of the graphene. The biosensor was then rinsed with DMSO, deionized water solution, respectively, in order to remove excess unbound PBASE molecules. 2uM probe Aptamer (Aptamer) solution was injected into the reaction chamber and incubated at room temperature for 4 hours, and since there is an amide bond in the molecule of PBASE that can react with the amino group on the Aptamer, the two can be tightly combined under the action of the chemical bond. Next, to remove excess Aptamer from the sensor, MIGFET biosensors were washed with PBS containing 0.2% sodium dodecyl sulfate (Sodium dodecyl sulfate, SDS) followed by PBS solution. Finally, respectively adding the coupling substance solutions of MPs/tDNA with different concentrations into the reaction cavity, and sequentially developing a series of detection experiments on the tDNA to be detected.

(5) tDNA detection to be tested

the tDNA detection step is specifically as follows, and MPs/tDNA to be detected is added into the MIGFET biosensor for 1 hour, so as to ensure that the MPs/tDNA to be detected and the Aptamer biosensor are fully hybridized and combined. The sensor reaction chamber was then washed with 0.2% SDS in PBS, after which it was washed again with PBS and repeated three times in order to wash away excess unbound fluorophore-6' -FAM labeled MPs/tDNA and excess MPs. The change of the electrical signal and the change of the fluorescence intensity of the MIGFET biosensor are recorded by adopting a self-made photoelectromagnetic MIBDS in a laboratory so as to realize the detection of tDNA. After each measurement, the apparatus was rinsed with 10mM NaOH solution (25 ℃) for 60s to regenerate the Aptamer, and the recombination experiment was carried out.

(6) MIGFET biosensor modification result detection

Since the graphene needs to be modified before the tDNA to be detected is detected, that is, whether the graphene can be successfully functionalized or not is achieved, the detection result of the tDNA to be detected is affected, and therefore, the invention respectively explores the modification results of the electric channel, the optical channel and the magnetic channel of the MIGFET biosensor.

The photo-electromagnetic MIGFET modification process is shown in fig. 5, fig. 5 (a) is an electrical channel detection result, fig. 5 (b) is an Aptamer regeneration after the sensor is cleaned, and fig. 5 (c) is an optical channel detection result; fig. 5 (d) shows the magnetic channel detection result. In experiments, the invention utilizes a grid voltage output module to set grid voltage (Vg) which is output at equal intervals of 50mV in the range of [ -1V,1.5V ], and detects and records the changes of IC, IF and IR generated by the MIGFET biosensor in the modification process under the condition of providing an excitation light source and applying a periodic magnetic field. When PBS, PBASE, 2 μm Aptamer solution is added, on the one hand, MIGFET biosensor electrical channels exhibit typical bipolar characteristics centered on Dirac (Dirac) points (fig. 5 (a)) due to the zero band gap structure of graphene and extremely high carrier mobility, and Dirac shifts to the right in turn, IC in MIGFET decreases in turn due to the opening of the band gap to fermi energy when foreign molecules adsorb onto graphene, resulting in a decrease in conductivity of the graphene absorption system. On the other hand, MIGFET has no excited fluorescent group, and thus IF remains substantially unchanged (fig. 5 (c)). Meanwhile, MIGFET has substantially unchanged IR since no MPs affect the graphene double conductive layer structure (fig. 5 (d)). Next, MPs/tDNA solution was added, the IF and IR of MIGFET increased significantly, the Dirac of MIGFET shifted to the left and the current continued to decrease. A significant increase in IF of MIGFET indicates that hybridization of MPs/tDNA labeled with 6' -FAM to Aptamer causes an IF change; the significant increase in IR of MIGFET indicates that MPs/tDNA are able to bind to the Aptamer and enrich; the Dirac of MIGFET shifts to the left and the current drops due to the non-electrostatic accumulation phenomenon of DNA molecules in contact with the graphene film, which in turn causes free electron transfer. From this, it was found that the graphene functionalization was successfully achieved by injecting the PBASE, aptamer solution, and then tDNA detection was achieved. In addition, 10mM NaOH solution was rapidly injected after each test, washed for 60 seconds, and then washed with PBS solution. As shown in fig. 5 (b), the Dirac point of the graphene recovered after washing, consistent with the Aptamer, indicating that the Aptamer was regenerated, and a recombination experiment was realized. Therefore, the MIGFET designed by the invention can realize the detection of DNA through three sensing modes of light and electromagnetic.

(7) MIGFET to DNA sensitivity detection

To examine the utility of MIGFET biosensors, the present invention investigated the binding kinetics of real-time DNA hybridization between tDNA and Aptamer at different concentrations. As shown in fig. 6, fig. 6 (a) is an optical channel; fig. 6 (b) is an electrical path; FIG. 6 (c) is a magnetic channel; FIG. 6 (d) is a time domain plot of the fluctuation in resistance for different complementary tDNA concentrations. First, MPs/tDNA with different concentration gradients are added to the modified graphene, and the content is 2uM, 100nM, 10nM, 1nM and 10pM from high to low. Then, based on the photo-electromagnetic multi-detection sensing characteristics of the MIGFET biosensor and the MIBDS monitoring device, the IF, IC, and IR changes can be obtained. For the electrical channels, the IC of MIGFET biosensor gradually decreased with increasing concentration of MPs/tDNA to be measured, and the Dirac point gradually shifted left (fig. 6 (a)). For the photo-magnetic channel, IF and IR in MIGFET biosensors gradually increase with increasing concentration of MPs/DNA to be measured under excitation light excitation and application of a periodic magnetic field (fig. 6 (b), (c), (d)). The above results all indicate that MPs/tDNA bind successfully to Aptamer. The photoelectromagnetic three channels of MIBDS showed a consistent trend in detecting tDNA sensitivity, and the sensitivity was 10pM with a low detection limit.

(8) MIGFET to DNA specific detection

To verify the specificity of MIGFET biosensors, the present invention uses perfect mismatch MPs/tDNA for verification as shown in fig. 7, fig. 7 (a) is an electrical pathway; FIG. 7 (b) is an optical channel; fig. 7 (c) shows a magnetic channel. First, 2. Mu.M of a completely mismatched MPs/tDNA solution was prepared and added to the reaction cell of the MIGFET biosensor for 60 minutes. The MIGFET biosensor was then washed with 0.2% SDS and PBS to remove unreacted perfect mismatch MPs/DNA. Finally, as a control experiment, 2. Mu.M of perfectly matched MPs/tDNA was added to the reaction cell, washed again after 60 minutes, and the MIBDS was used to detect changes in IF, IC and IR throughout the reaction. For the electrical channels, there was no significant change in the IC and Dirac points for the perfectly mismatched MPs/tDNA, the IC for the perfectly matched MPs/tDNA was significantly reduced, and the Dirac points were shifted left (FIG. 7 (a)), indicating that the perfectly matched MPs/tDNA was perfectly bound to the Aptamer and the perfectly mismatched MPs/tDNA was not bound to the Aptamer. For the photo-magnetic channel, the IF and IR of the perfectly matched MPs/tDNA increased significantly, and the IF and IR of the perfectly mismatched MPs/tDNA were essentially unchanged (FIG. 7 (b), (c)), which also indicated that the Aptamer bound only to the perfectly matched MPs/tDNA. The invention proves that the MIGFET biosensor has excellent specificity.

(9) Comparison of photo-electromagnetic three-channel detection results

In order to verify the consistency and reliability of the detection results, the photo-electromagnetic three-channel detection results are compared. Fig. 8 shows that MIGFET biosensors after graphene functionalization, immobilized Vg at 500mV, separately detect tDNA at different concentrations, collect and record IC between source and drain, current IF, and IR generated under periodic magnetic fields by MIBDS. As shown in FIGS. 8 (b), (c), since one end of tDNA is labeled with a fluorescent group 6' -FAM and MPs are coupled, the IF and IR of the MIGFET biosensor gradually increase with increasing concentration of MPs/tDNA, indicating that MPs/tDNA has hybridized with Aptamer. Meanwhile, IC continuously decreases with increasing MPs/tDNA concentration (fig. 8 (a)), due to the increasing external negative charge in the electrolyte, which not only can dope the graphene but also enlarges the energy gap of the graphene, thus decreasing the conductivity of the graphene. In conclusion, the MIGFET biosensor designed by the invention can realize quantitative analysis of tDNA to be detected by combining the modified graphene with the electric channel and the fluorescent channel, and compared with a biosensor in a single sensing detection mode, the MIGFET biosensor has the advantages that the detection process is more efficient by adopting a multi-sensing mode, the detection result has consistency on the order of 10pM, mutual verification can be realized, and the detection result is more accurate.

Claims

1. The preparation method of the multi-sensing integrated MIGFET biosensor is characterized by comprising the following steps of:

s8, forming a GFET biosensor as an electric channel based on a Y-type micro-channel by adopting an AgCl electrode as a grid input voltage, adopting a narrow-band LED as an excitation light source, conducting to a sensitive area through an optical fiber to form a fluorescent biosensor, forming a light channel of the MIGFET biosensor, and applying a periodic magnetic field above the MIGFET biosensor to form a magnetic channel of the MIGFET biosensor to complete the preparation of the multi-sensing integrated MIGFET biosensor;

the detection device of the multi-sensing integrated MIGFET biosensor comprises an optical path module, a circuit module connected with the optical path module and an upper computer connected with the circuit module;

focusing and coupling light emitted by the LED lamp by using an optical fiber collimator to form a first collimated beam; transmitting the first collimated beam to the MIGFET biosensor via an optical fiber to provide an excitation light source to excite the fluorophore 6' -carboxyfluorescein labeled on the target DNA; after the fluorescent group 6' -carboxyl fluorescein is excited, the fluorescent group is converted into a second collimated light beam through an optical fiber collimator, and the second collimated light beam is transmitted to a high-pass filter for filtering through the optical fiber collimator; converging the filtered collimated light beams to an effective sensitive area of a photomultiplier, and displaying and storing the obtained optical signals by using an upper computer after photoelectric conversion, and reflecting the fluorescence intensity;

the circuit module includes:

2. The method of manufacturing a multi-sensor integrated MIGFET biosensor of claim 1, wherein the multi-sensor integrated MIGFET biosensor includes the following functionalization process: