An electrochemical magnetic field detection device based on magnetic induction protein and its preparation method

Abstract

The present invention relates to an electrochemical magnetic field detection device based on magnetic induction protein and its preparation method. The method comprises: sputtering indium tin oxide on a substrate, cleaning; applying glue, pre-baking, exposing, then developing, and hard-baking; etching, de-gumming, and transferring graphene to the surface of a working electrode; applying a bovine serum albumin solution on the resulting substrate surface and curing to denature the protein into a film; applying glue, pre-baking, exposing, developing, and plasma etching; and dripping a mixed solution of EDC, NHS, and MagR / Cry onto the protein film on the working electrode area of ​​the electrochemical device after etching, incubating, rinsing, and sealing. The device has the advantages of digitization and miniaturization, and can achieve enhanced detection of signals excited by magnetic fields from biological magnetic induction proteins, as well as the detection of magnetic induction protein response signals to magnetic fields over a wide bandwidth.

Description

Technical Field

[0001] The present invention belongs to the field of sensors and their preparation, and in particular relates to an electrochemical magnetic field detection device based on magnetic induction protein and a preparation method thereof. Background Art

[0002] Currently, in the field of non-autonomous navigation, there are satellite navigation systems such as Beidou and GPS. However, in certain special circumstances, satellite positioning and navigation may experience signal obstruction and interruption due to natural disasters and other conditions. Therefore, autonomous navigation and positioning technology is particularly important. Existing autonomous navigation technologies include inertial navigation, polarization navigation, geomagnetic navigation, and others. Existing geomagnetic navigation systems generally use magnetic field detection devices such as fluxgates, optically pumped magnetometers, superconducting quantum interferometers, and magnetoelectric sensors. Each of these has high costs and requires a special operating environment, which are all limitations of magnetoelectric systems. Inspired by animal navigation and positioning, the development of biomimetic magnetic field detection devices based on the biomaterial-based magnetic induction protein MagR / Cry4 has become possible.

[0003] The recently proposed protein receptor MagR / Cry complex, a biological compass model, is widely present in the retinas of higher animals. Biophysical and physical experiments have confirmed that both MagR polymers and MagR / Cry protein complexes possess a significant intrinsic magnetic moment. Optical observations have also demonstrated that the MagR protein complex can sense the Earth's magnetic field (0.35-0.65 gs). Experiments have also demonstrated the protein's magnetic properties in aqueous solution, and its crystals also exhibit significant magnetism, capable of interacting with external magnetic fields. By combining this magnetic field-sensing biological protein with micro-nanofabrication techniques, miniaturized, integrated magnetic field detection devices compatible with biological systems can be developed.

[0004] Therefore, it is very meaningful to study new electrochemical magnetic field detection devices based on biomimetic magnetoresistors. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an electrochemical magnetic field detection device based on magnetic induction protein and a preparation method thereof, so as to fill the gap in the prior art.

[0006] The present invention provides a method for preparing an electrochemical magnetic field detection device based on magnetic induction protein, comprising the following steps:

[0007] (1) Using DC magnetron sputtering to deposit indium tin oxide on the substrate and cleaning;

[0008] (2) spin-coating a positive photoresist on the substrate with the indium tin oxide conductive layer cleaned in step (1), pre-baking, exposing, developing, and hard-baking;

[0009] (3) etching the indium tin oxide on the substrate after hard baking in step (2) with an etching solution to remove the glue, and transferring the graphene to the working electrode surface of the indium tin oxide after the glue is removed;

[0010] (4) spreading the bovine serum albumin solution on the surface of the substrate obtained in step (3), and heating it at 70-100° C. for 2-5 minutes to denature the protein into a film;

[0011] (5) spin coating the substrate formed in step (4) with a positive photoresist, pre-baking, exposing, developing, and plasma etching;

[0012] (6) EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution, NHS (N-hydroxysuccinimide) solution and MagR / Cry solution are mixed in a volume ratio of 0.95-2:0.95-2:0.95-2 to obtain a mixed solution, and the mixed solution is dropped onto the protein film on the working electrode area of ​​the electrochemical device etched in step (5), incubated, rinsed, and sealed to obtain an electrochemical magnetic field detection device based on magnetic induction protein.

[0013] Preferably, in step (1), the substrate is a glass sheet, and organic and inorganic impurities are removed from the substrate.

[0014] Preferably, the organic and inorganic impurities on the substrate are removed as follows: first, the substrate is cleaned in an acetone solution to remove impurities, then the residual acetone is washed away with isopropyl alcohol, and finally the isopropyl alcohol on the glass is dissolved with deionized water and blown dry with nitrogen.

[0015] Preferably, the DC magnetron sputtering of indium tin oxide in step (1) is performed by using an indium tin alloy target, sputtering at 600-800° C. with a DC power of 200-400 W and an argon gas flow rate of 100-200 sccm.

[0016] Preferably, the cleaning in step (1) is: using toluene, acetone, anhydrous ethanol, and deionized water in sequence to clean the surface of indium tin oxide.

[0017] Preferably, the spin coating of the positive photoresist in step (2) is as follows: placing the substrate with the indium tin oxide conductive layer on a coating machine, and spin coating the positive photoresist at a low speed of 500 revolutions per minute and a high speed of 2500 revolutions per minute.

[0018] Preferably, the pre-baking temperature in step (2) is 100-110° C. and the pre-baking time is 5-10 minutes. The pre-baking is performed by placing the coated substrate on a hot plate.

[0019] Preferably, the exposure in step (2) is to pattern the working electrode and the counter electrode.

[0020] Preferably, the development in step (2) is: cleaning the photoresist in the exposed area in a corresponding alkaline developer.

[0021] Preferably, the hard baking temperature in step (2) is 100-120° C. and the hard baking time is 20-30 minutes. The hard baking is performed by placing the developed substrate on a hot plate.

[0022] Preferably, the etching solution in step (3) is a mixed solution of HNO3, HCl and H2O in a volume ratio of 2-4:45-55:45-55.

[0023] Preferably, in step (3), the etching temperature is 80-100° C., and the etching time is 2-3 minutes.

[0024] Preferably, the de-resisting step in step (3) is to soak the etched wafer in an acetone solution to clean off the unexposed photoresist.

[0025] Preferably, in step (3), the graphene is transferred to the working electrode surface of the debonded indium tin oxide according to a standard graphene transfer technique, specifically:

[0026] Graphene transfer steps: Cut the purchased copper-based graphene into the required size and place it in a 0.2g / ml ammonium persulfate solution to corrode the copper substrate for 2-3 hours. Once the copper substrate is completely corroded, transfer it to two boxes of deionized water using a glass slide. Then, use an electrode with an indium tin oxide film to remove the graphene and place it on the surface of the working electrode. After naturally draining for 3 hours, bake it in an 80°C oven for 30 minutes to fully adhere the graphene to the indium oxide surface. At this point, the graphene surface still has a layer of photoresist to protect it from contamination. This protective layer needs to be removed using an organic solvent such as acetone. Prepare three boxes of clean acetone and soak the electrode with the transferred graphene in each box of acetone for 10 minutes to remove the glue. Then, use anhydrous ethanol to rinse off any acetone residue on the electrode. Finally, use deionized water to remove any remaining ethanol.

[0027] Preferably, in step (4), the bovine serum albumin solution is spread on the surface of the substrate obtained in step (3) by placing the substrate on a sizing machine, rotating it at a low speed, and then slowly pouring the bovine serum albumin solution, and removing the substrate after the protein solution is evenly spread on the surface of the substrate.

[0028] Preferably, the concentration of the bovine serum albumin solution in step (4) is 0.08-0.2 g / ml.

[0029] Preferably, the spin coating of the positive photoresist in step (5) is carried out according to the spin coating of the positive photoresist in step (2).

[0030] Preferably, the pre-baking temperature in step (5) is 80° C. to 105° C., and the pre-baking time is 5 to 8 minutes. The pre-baking is performed on a hot plate.

[0031] Preferably, the exposure in step (5) is as follows: patterning the channel under a photolithography machine, and shielding the channel area from light.

[0032] Preferably, the development in step (5) is to wash out the pattern of the channel in an alkaline developer.

[0033] Preferably, oxygen is used as the etching gas in the plasma etching in step (5).

[0034] Preferably, in step (6), the concentration of the EDC solution is 0.3-0.8 mol / L; the concentration of the NHS solution is 0.08-0.2 mol / L; and the concentration of the MagR / Cry solution is 0.25 mg / ml-0.5 mg / ml.

[0035] Preferably, the solvent of the EDC solution and the NHS solution in step (6) is MES (2-(N-morpholino)ethanesulfonic acid) solution.

[0036] Preferably, the concentration of the MES solution is 0.08-0.2 mol / L, and the solvent is deionized water.

[0037] Preferably, the incubation in step (6) is: incubating at 32-42° C. in a moist dark environment for 0.8-1.5 hours.

[0038] Preferably, the sealing time in step (6) is 0.8 to 1.5 hours.

[0039] Preferably, in step (6), the blocking is performed by using a bovine serum albumin solution to block excess reactive groups on the graphene surface, wherein the concentration of the bovine serum albumin solution is 0.5-1.2%.

[0040] The present invention also provides an electrochemical magnetic field detection device based on magnetic induction protein prepared by the above preparation method.

[0041] The present invention also provides an application of an electrochemical magnetic field detection device based on magnetic induction protein in a magnetoelectric system.

[0042] The present invention utilizes MEMS micro-nano processing technology to produce the main body of the electrochemical device and the indium tin oxide film, which has excellent electrical conductivity. Then, a micro electrolyte pool produced by 3D printing is bonded to it. Together with an exogenous commercial solid reference electrode, an electrochemical reaction system is formed. Graphene is used to enhance the conductivity of the system. The protein film coated on the graphene surface can not only protect the cleanliness of the graphene surface from being damaged by the external environment, but also its exposed chemical groups can fix the magnetic induction protein by covalent incubation rather than by physical adsorption. When the protein complex responds to an external magnetic field signal, there is a transfer of charge, and this partial charge can be detected and extracted by AC impedance technology. In addition, the presence of graphene enhances the conductivity of the indium tin oxide working electrode, improves the signal-to-noise ratio of the impedance signal, and is conducive to the mapping of impedance data and magnetic field intensity information.

[0043] Beneficial effects

[0044] The present invention uses microelectrodes made from indium tin oxide conductive thin films through micro-nanofabrication as the basic structure of the detection device, offering the advantages of miniaturization and digitization. It can enhance the detection of signals from biological magnetic induction proteins stimulated by magnetic fields, and can detect the response of magnetic induction proteins to magnetic fields over a wide bandwidth.

[0045] The magnetic field detection method used in the present invention has the characteristics of simple equipment and high upper limit of sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the structure of the electrochemical sensor device of the present invention, wherein 1 is a glass substrate, 2 is an indium tin oxide film, and 3 is a magnetic field transducer core covalently coupled to a magnetic induction protein (for specific protein fixation, see Figure 3 ), 4 is the electrolyte, and 5 is the electrolyte chamber.

[0047] Figure 2 The figure is a flow chart of the preparation process of the electrochemical magnetic field detection device based on magnetic induction protein of the present invention.

[0048] Figure 3 Schematic diagram of the covalent coupling of magnetic induction proteins in the present invention.

[0049] Figure 4 This is a schematic diagram of magnetic field detection by the electrochemical magnetic field detection device based on magnetic induction protein of the present invention.

[0050] Figure 5 This is the impedance spectrum test diagram of the indium tin oxide (ITO) electrode before and after graphene transfer in the present invention.

[0051] Figure 6This is the electrochemical impedance spectroscopy (EIS) test diagram after the magnetic induction protein is fixed in the present invention, where 0gs corresponds to the case of no magnetic field, which is equivalent to the test baseline. EIS tests are performed under increasing magnetic fields of 100gs (10mT) and 1000gs (0.1T). DETAILED DESCRIPTION

[0052] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0053] Reagent Source:

[0054]

[0055] Example 1

[0056] Step 1: Electrochemical sensor device fabrication

[0057] Substrate Preparation: Using a glass substrate, remove organic and inorganic impurities from the glass surface. First, clean the impurities in an acetone solution, then use isopropyl alcohol to remove any remaining acetone. Finally, dissolve the isopropyl alcohol on the glass with deionized water and blow dry with nitrogen.

[0058] DC magnetron sputtering: using an indium tin alloy target, sputtering was performed at 600°C with a DC power of 200 W and an argon gas flow rate of 100 sccm.

[0059] Indium tin oxide surface cleaning: Use toluene, acetone, anhydrous ethanol, and deionized water to clean it in sequence.

[0060] Glue coating: Place the glass sheet with the indium tin oxide conductive layer on a glue coater and spin-coat the positive photoresist at a low speed of 500 rpm and a high speed of 2500 rpm.

[0061] Pre-baking: Place the coated substrate on a hot plate and bake at 105°C for 8 minutes.

[0062] Exposure: Patterning the working electrode and the counter electrode.

[0063] Development: The photoresist in the exposed area is washed away in the corresponding alkaline developer.

[0064] Hard bake: Place the developed substrate on a hot plate and bake at 105°C for 20 minutes.

[0065] Wet etching: HNO3, HCl, and H2O were prepared as an etching solution in a volume ratio of 3:50:50, and indium tin oxide was etched in a water bath at 80°C for 2 minutes.

[0066] Resin removal: Soak the etched wafer in acetone solution to clean off the unexposed photoresist.

[0067] Graphene Transfer: Graphene is transferred to the indium tin oxide working electrode using standard graphene transfer techniques. Specifically, purchase copper-based graphene is cut to the desired size and etched in a 0.2g / ml ammonium persulfate solution for 2-3 hours. Once the copper substrate is completely etched, the graphene is transferred sequentially to two boxes of deionized water using a glass slide. The graphene is then removed using an electrode coated with an indium tin oxide film and placed on the working electrode surface. After draining for 3 hours, the graphene is baked in an 80°C oven for 30 minutes to fully adhere to the indium tin oxide surface. A layer of photoresist is still present on the graphene surface to protect it from contamination. This protective layer needs to be removed using an organic solvent such as acetone. Prepare three boxes of clean acetone and soak the electrode with the transferred graphene in each box for 10 minutes to remove the adhesive. Residual acetone residue is then rinsed off with anhydrous ethanol. Finally, any residual ethanol is removed with deionized water.

[0068] Prepare a 0.1g / ml bovine serum albumin solution. Place the substrate on a spinner and spin at low speed. Then slowly pour 15ml (4-inch slide) of the bovine serum albumin solution. Once the protein solution is evenly spread on the substrate surface, remove the substrate. Place on an 80°C hot plate for 3 minutes to denature the protein and form a film.

[0069] Channel patterning

[0070] Glue coating: Spin-coat positive photoresist again according to the previous steps.

[0071] Pre-baking: Bake on a hot plate at 105°C for 8 minutes.

[0072] Exposure: The trench is patterned under a photolithography machine, and the trench area is shielded from light.

[0073] Development: Wash out the groove pattern in alkaline developer.

[0074] Plasma etching: Plasma etching was performed using oxygen as the etching gas for 5 minutes.

[0075] Step 2: Immobilization of MagR / Cry4 protein complex

[0076] Prepare the corresponding solutions: prepare 0.1mol / L MES (2-(N-morpholino)ethanesulfonic acid) solution, 0.5mol / L EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution, and 0.1mol / L NHS (N-hydroxysuccinimide) solution. The solvent for the MES solution is deionized water, and the solvent for the EDC and NHS solutions is MES solution. Prepare 0.25mg / ml of MagR / Cry protein in solution. The MagR / Cry magnetic induction protein is produced and purified by genetically modifying the CG8198 gene of Drosophila in Escherichia coli. The purified protein is generally freeze-dried, and PBS solvent can be added to obtain the corresponding solution-state protein.

[0077] A mixed solution of EDC solution, NHS solution, and MagR / Cry solution was prepared in a 1:1:1 volume ratio. Using a pipette, 20 μl of the solution was dripped onto the protein film at the working electrode area of ​​the electrochemical device. The transistor was then placed in an incubation chamber in a humidified dark environment at 37°C for 1 hour. After incubation, unbound MagR / Cry protein complexes were rinsed with phosphate buffer. Finally, excess reactive groups on the graphene surface were blocked with 1% bovine serum albumin solution for 1 hour.

[0078] Step 3: Magnetic field detection method based on electrochemical magnetic field detector of magnetic induction protein

[0079] The prepared indium tin oxide electrode is bonded to the micro-chamber printed by 3D printing, and a three-electrode electrochemical system is formed with a commercial reference electrode. The three electrodes are connected to the corresponding interfaces of the electrochemical workstation, and the AC impedance test module of the electrochemical workstation is used to perform impedance testing under a certain bandwidth, such as Figure 6 shown.

[0080] Figure 1 The device measures 20 mm x 20 mm, with 180 nm of sputtered indium tin oxide and a 1.1 cm thick glass substrate. The working electrode is circular, with a diameter between 1 mm and 4 mm, and is covered with graphene and a protein film.

[0081] Figure 5 It shows that after transferring graphene, the electrode impedance drops by two orders of magnitude, that is, the conductivity is enhanced by two orders of magnitude.

[0082] Figure 6The results show that under the influence of a magnetic field, the spatial conformation of the magnetoreceptor protein changes, its structure tending to be unified by the magnetic field direction, resulting in a decrease in impedance. The frequency band selected for the EIS analysis in the figure is precisely the frequency band where charge transfer occurs at the working electrode during the electrochemical reaction. The rate of mass transfer within this frequency range cannot keep up with the frequency change. Therefore, changes in this frequency band reflect the resistance during the charge transfer process. The figure shows that the magnetic field detection device achieves a detection limit of 100 gs.

Claims

1. A method for preparing an electrochemical magnetic field detection device based on magnetic induction protein, comprising the following steps: (1) Using DC magnetron sputtering to deposit indium tin oxide on the substrate and cleaning; (2) spin coating the substrate with the indium tin oxide conductive layer cleaned in step (1) with a positive photoresist, pre-baking, exposing, developing, and hard baking; (3) using an etching solution to etch the indium tin oxide on the substrate after hard baking in step (2), remove the glue, and transfer the graphene to the working electrode surface of the indium tin oxide after the glue is removed; wherein, the graphene transfer step: cutting the purchased copper-based graphene into the required size, placing it in a 0.2g / ml ammonium persulfate solution to corrode the copper substrate for 2 to 3 hours; after the copper substrate is completely corroded, using a glass slide to transfer it to two boxes of deionized water in turn; then using an electrode with an indium tin oxide film to fish out the graphene to the surface of the working electrode; after naturally draining the water for 3 hours, placing it in an 80°C oven and baking it for 30 minutes to make the graphene and the indium tin oxide surface completely adhere; at this time, there is still a layer of photoresist on the graphene surface to protect the graphene surface from contamination, and acetone is needed to remove this protective glue; prepare three boxes of clean acetone, and soak the electrode with the transferred graphene in each box of acetone for 10 minutes in turn to remove the glue; then use anhydrous ethanol to clean off the acetone remaining on the electrode; finally, use deionized water to remove the residual ethanol; (4) spreading the bovine serum albumin solution on the surface of the substrate obtained in step (3), and heating it at 70-100° C. for 2-5 minutes to denature the protein into a film; (5) spin coating the substrate formed in step (4) with a positive photoresist, pre-baking, exposing, developing, and plasma etching; (6) The EDC solution, the NHS solution, and the MagR / Cry solution are mixed in a volume ratio of 0.95-2:0.95-2:0.95-2 to obtain a mixed solution, and the mixed solution is dropped onto the protein film on the working electrode area of ​​the electrochemical device etched in step (5), incubated, rinsed, and sealed to obtain an electrochemical magnetic field detection device based on magnetic induction protein.

2. The preparation method according to claim 1, characterized in that In the step (1), the substrate is a glass sheet, and organic and inorganic impurities are removed from the substrate; DC magnetron sputtering of indium tin oxide is performed by using an indium tin alloy target, sputtering at 600-800°C with a DC power of 200-400W and an argon gas flow rate of 100-200sccm.

3. The preparation method according to claim 1, characterized in that In the step (2), the pre-baking temperature is 100-110° C., and the pre-baking time is 5-10 minutes; the exposure is: patterning the working electrode and the counter electrode; the hard-baking temperature is 100-120° C., and the hard-baking time is 20-30 minutes.

4. The preparation method according to claim 1, characterized in that The etching solution in step (3) is a mixed solution of HNO3, HCl and H2O in a volume ratio of 2-4:45-55:45-55; the etching temperature is 80-100°C, and the etching time is 2 minutes to 3 minutes.

5. The preparation method according to claim 1, characterized in that The concentration of the bovine serum albumin solution in step (4) is 0.08-0.2 g / ml.

6. The preparation method according to claim 1, characterized in that In the step (5), the pre-baking temperature is 80° C. to 105° C., and the pre-baking time is 5 to 8 minutes; and oxygen is used as the etching gas in the plasma etching.

7. The preparation method according to claim 1, characterized in that The concentration of EDC solution in step (6) is 0.3~ 0.8mol / L; NHS solution concentration is 0.08~0.2mol / L; MagR / Cry solution concentration is 0.25mg / ml~0.5mg / ml; the solvent of EDC solution and NHS solution is MES solution; the concentration of MES solution is 0.08~0.2mol / L, and the solvent is deionized water.

8. The preparation method according to claim 1, characterized in that In step (6), the incubation is as follows: incubating at 32-42° C. in a moist dark environment for 0.8-1.5 hours; the blocking time is 0.8-1.5 hours; and blocking is performed by using a bovine serum albumin solution to block excess reactive groups on the graphene surface, wherein the concentration of the bovine serum albumin solution is 0.5-1.2%.

9. An electrochemical magnetic field detection device based on magnetic induction protein prepared by the preparation method according to claim 1.

10. Use of the electrochemical magnetic field detection device based on magnetic induction protein as claimed in claim 9 in a magnetoelectric system.

Images