Nanoparticle, biosensor and preparation method and application thereof

Abstract

The present application discloses a nanoparticle, a biosensor, and their preparation methods and applications. The nanoparticle includes Fe3O4 nanoparticles, Au nanoparticles, and cysteine; the Au nanoparticles are wrapped on the surface of the Fe3O4 nanoparticles. The cysteine is connected to the Au nanoparticles. This biosensor not only has high sensitivity and selectivity, but also has good stability after 15 regeneration treatments. This biosensor can detect dopamine, with a concentration of 1 μmol L ‑1 ~120 μmol L ‑1 , and the detection limit is 3.3 nmol L ‑1 . Finally, this platform has been successfully applied to the analysis of dopamine in the fish brain under the global neurons of the cerebral cortex. This FET biosensor is the first biosensor that can remotely control the sensitivity and detection limit online through a permanent magnet. It opens the door to reusable, inexpensive, and large-scale production.

Description

Technical Field

[0001] The present application relates to a nanoparticle, a biosensor, and a preparation method and application thereof, and belongs to the field of sensors. Background Art

[0002] Magnetism is a property of matter itself, which is caused by the orbital motion of electrons around the nucleus and the spin motion of the electrons inside the matter. Among them, the most widely used is Fe3O4 nanoparticles. Since its discovery, Fe3O4 nanoparticles have been widely used in catalysis, magnetic fluid, magnetic resonance imaging and environmental science due to their stable chemical properties and good magnetic properties. 3 O 4 Nanoparticles have attracted extensive attention from researchers in the fields of medical materials, visualization and therapeutic agents in bioanalysis and biotechnology, and drug delivery systems due to their advantages such as convenient separation and delivery, large specific surface area, controllable synthesis and surface modification, good biocompatibility, and recyclability. There are many methods for preparing ferroferric oxide, mainly coprecipitation, pyrolysis, microemulsion, and hydrothermal methods. Among them, coprecipitation is a relatively simple and commonly used method for synthesizing nanoparticles with controllable morphology.

[0003] Dopamine is an important neurotransmitter biomolecule. When the dopamine content in the body is abnormal, it may cause Parkinson's disease, heart disease and schizophrenia. Therefore, it is crucial to have an accurate and rapid dopamine detection method in clinical diagnosis. The currently reported dopamine detection methods mainly include chromatography, fluorescence, high performance liquid chromatography, and chemiluminescence. However, these methods have defects such as cumbersome operation and low sensitivity. Electrochemical methods have attracted widespread attention due to their high sensitivity. For example, Cheng et al. developed an electrochemical sensing platform based on gold nanoparticles / multi-walled carbon nanotube hybrids to detect dopamine. The linear range of the sensor is 50nM to 2.7mM, the detection limit is 15nM, and the sensor can remove the interference of ascorbic acid and uric acid. Summary of the invention

[0004] Dopamine is a neurotransmitter in brain physiology, and its detection is crucial for studying brain activity and understanding brain function. However, regenerative biosensors for monitoring dopamine in physiological and pathological processes remain challenging due to the lack of platforms with repeatable online detection-regeneration cycles. To this end, we developed a regenerative magnetically controlled field-effect transistor (FET) sensor combined with an in vivo monitoring system.

[0005] Gold nanoparticles have good conductivity and bioaffinity and are often used in biosensors to improve the performance of biosensors. They can also be easily activated by thiol coupling with cysteine ​​to make them more biocompatible. 3 O 4 @Au@Cys) solution, based on the fact that tyrosinase can catalyze dopamine, 3 O 4 @Au@Cys) solution is added with tyrosinase and fixed on the prepared interdigital electrodes to detect dopamine. Since ferroferric oxide nanoparticles have good magnetic response, when the mixed solution of ferroferric oxide@gold@cysteine ​​and tyrosinase is fixed on the interdigital electrodes in a magnetic field, the distribution of the solution will be locally enriched, thus improving the sensitivity of the sensor.

[0006] According to a first aspect of the present application, a nanoparticle is provided.

[0007] A nanoparticle comprising Fe 3 O 4 Nanoparticles, Au nanoparticles, and cysteine;

[0008] The Au nanoparticles are encapsulated in Fe 3 O 4 The surface of nanoparticles.

[0009] The cysteine ​​is attached to the Au nanoparticles.

[0010] The Fe 3 O 4 The molar ratio of nanoparticles, Au nanoparticles and mercapto compounds is (1-2):(0.5-1):(0.1-0.2), preferably 1:0.5:0.1;

[0011] Optionally, the size of the nanoparticles is 80nm to 1000nm.

[0012] Optionally, the size of the nanoparticles is independently selected from any value among 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm or any range therebetween.

[0013] According to a second aspect of the present application, a biosensor is provided.

[0014] A biosensor comprising interdigital electrodes, nanoparticles, tyrosinase and a film-forming agent;

[0015] The nanoparticles and tyrosinase are covered on the surface of the interdigital electrodes by a film-forming agent;

[0016] The nanoparticles are arranged in a linear pattern; preferably in a straight line;

[0017] The nanoparticles are selected from the nanoparticles described above.

[0018] Optionally, the film former comprises chitosan.

[0019] The mass ratio of the nanoparticles to tyrosinase is 50 to 10:1, preferably 10:1;

[0020] The nanoparticles include Fe 3 O 4 Nanoparticles, Au nanoparticles, and thiol compounds;

[0021] The Fe 3 O 4 The molar ratio of the nanoparticles, Au nanoparticles and the mercapto compound is (1-2):(0.5-1):(0.1-0.2), preferably 1:0.5:0.1.

[0022] Optionally, the sensitivity and detection limit of the biosensor or in-situ detection chip are controlled by a permanent magnet.

[0023] Optionally, the regeneration of the biosensor or in-situ detection chip is achieved by adding and removing a permanent magnet.

[0024] According to a third aspect of the present application, a method for preparing a biosensor is provided.

[0025] A method for preparing a biosensor comprises the following steps:

[0026] (1) obtaining interdigital electrodes;

[0027] (2) placing a mixed solution containing nanoparticles, tyrosinase and a film-forming agent on the surface of the interdigital electrodes; placing the interdigital electrodes in a magnetic field, and drying the interdigital electrodes to obtain the biosensor. Preferably, a bar magnet is placed under the interdigital electrodes.

[0028] Optionally, placing the interdigitated electrodes in a magnetic field is to bring them close to a magnet;

[0029] Optionally, the distance between the interdigital electrodes and the magnet is 50 μm to 200 μm. The distance is independently selected from any value among 50 μm, 100 μm, 150 μm and 200 μm, or any range between the two.

[0030] According to a fourth aspect of the present application, an application of a biosensor is provided.

[0031] The above-mentioned biosensor and the biosensor prepared by the above-mentioned preparation method are used in brain science and detection of dopamine.

[0032] This has stimulated a strong demand for regenerative sensor devices to specifically detect, quantify, and monitor various biological and chemical species. Field effect transistors (FETs), as one of the most promising platforms, offer a tailor-made solution to this need.

[0033] The beneficial effects that can be produced by the present application include: In the biosensor of the present application, the gold-plated magnetic nanoparticles (Fe3O4@AuNPs) act as the regeneration recognition unit of dopamine. By simply removing the permanent magnet, tyrosinase can catalyze dopamine on the biosensor interface, thereby achieving the regeneration of the biosensor. The results show that the FET biosensor not only has high sensitivity and selectivity, but also has good stability after 15 regeneration treatments. The biosensor is able to detect dopamine at a concentration of 1μmol L -1 ~120 μmol L -1 The detection limit was 3.3 nmol L -1 . Finally, the platform has been successfully applied to dopamine analysis in fish brain under global neurons in the cerebral cortex. This FET biosensor is the first biosensor with online remote control of sensitivity and detection limit by permanent magnets. It opens the door to reusable, cheap and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 middle,

[0035] Figure A: Fe prepared in Example 1 3 O 4 TEM images of

[0036] Figure B: Fe prepared in Example 1 3 O 4 @TEM image of Au;

[0037] Figure C: Fe prepared in Example 1 3 O 4 @Magnified TEM image of Au;

[0038] Figure D: Fe prepared in Example 1 3 O 4 Elemental analysis spectrum of @Au@Cys nanoparticles.

[0039] Figure 2 Schematic diagram of tyrosinase catalyzing dopamine.

[0040] Figure 3 Magnetic control of magnetic nanoparticles from dispersed to linear shapes.

[0041] Figure A: Magnetic nanoparticles without magnetic field.

[0042] Panel B: Magnetic nanoparticles with added magnetic field.

[0043] Figure 4A :Fe 3 O 4 @Au+Tyr (magnetic) and Fe 3 O 4 @Au+Tyr (non-magnetic) detection Dopa time-current curve;

[0044] Figure 4B :Fe 3 O 4 @Au+Tyr (magnetic) and Fe 3 O 4 @Au+Tyr (non-magnetic) detection of Dopa steady-state current and Dopa concentration calibration curve;

[0045] Figure 4C :Fe 3 O 4 @Au+Tyr (magnetic) detects the response signal of Dopa and its interferents;

[0046] Figure 4D :Fe 3 O 4 @Au+Tyr (magnetic) stability test detects Dopa's electrical signal for 28 days.

[0047] Figure 5A :Fe 3 O 4 @Au+Tyr(magnetic),Fe 3 O 4 @Au+Tyr(non-magnetic),Fe 3 O 4 +Tyr (magnetic), Fe 3 O 4 +Tyr (non-magnetic), Tyr (magnetic), Tyr (non-magnetic) detection Dopa time-current curve;

[0048] Figure 5B Figure: Fe at different heights of 100 to 250μm magnets 3 O 4 @Au+Tyr (magnetic) detects Dopa;

[0049] Figure 5C :Fe 3 O 4 @Au+Tyr (with magnetism) repeated stability test. DETAILED DESCRIPTION

[0050] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0051] Unless otherwise specified, the raw materials and catalysts in the examples of the present application were purchased through commercial channels. Unless otherwise specified, the testing methods all adopted conventional methods, and the instrument settings all adopted the settings recommended by the manufacturer.

[0052] Ferric chloride hexahydrate (FeCl3.6H2O), ferrous chloride tetrahydrate (FeCl2.4H2O) (analytical grade) and dopamine (Dopa) were all from Sinopharm Group; ammonia (GR, 25-28%), sodium citrate, dihydrate (AR, 99%) and L-cysteine ​​(99%) were purchased from Shanghai McLean Co., Ltd.; 0.1 mol L-1 PBS solution, chitosan and chloroauric acid were purchased from Aladdin. The deionized water used in the experiment was water with a resistivity of 18.2 MΩ.

[0053] Table 2-1 Basic information of instruments required for the experiment

[0054]

[0055] Example 1 Synthesis of Nanoparticles

[0056] (1) Preparation of Fe 3 O 4

[0057] Using the alkaline coprecipitation method, 0.448 g of ferric chloride and 0.163 g of ferrous chloride were dissolved in 10 ml of deionized water and 10 ml of ethanol nitrogen for 30 minutes, and then ammonia water was added under nitrogen protection and rapidly shaken for 10 minutes. The mixture was centrifuged, ultrasonically cleaned with deionized water for 3 times to obtain ferrosoferric oxide, which was then dispersed in deionized water for further use.

[0058] (2) Preparation of Fe 3 O 4 @Au

[0059] FeCl 3 It is a yellow solution, FeCl 2 It is light green, and turns into a turbid black liquid after adding ammonia water to react and form ferroferric oxide. It is easily separated from the solution under the action of a magnet. Then, a layer of nano-gold particles is coated on the surface of the synthesized ferroferric oxide by reducing chloroauric acid with sodium citrate to form a core-shell structure. Gold-coated ferroferric oxide, tyrosinase and chitosan are mixed together in a ratio of 1:1:1 to obtain a mixed solution, which is later fixed to the surface of the interdigitated electrode.

[0060] Example 2 Fabrication of interdigitated electrodes and Fe 3 O 4 / Construction of interdigital electrode biosensor

[0061] To ensure the yield and performance of the interdigital electrode, all process steps are completed in a clean room. The electrode is made using the stripping process of large-scale integrated circuits. First, a 300nm silicon dioxide wafer is grown with thermal oxygen to ensure the insulation of the substrate. The silicon wafer is ultrasonically cleaned with acetone, ethanol, and ultrapure water, and dried with nitrogen to ensure the surface is clean. Then spin-coat the photoresist, bake it before, and then perform photolithography. After the electrode pattern is transferred to the silicon dioxide substrate by photolithography and development, the residual glue is removed by oxygen plasma etching. Then, a layer of 10nm / 70nm titanium / gold is deposited by electron beam evaporation, in which titanium is the adhesion layer of the gold electrode. Finally, ultrasound is carried out in acetone, ethanol, and ultrapure water in turn, and the interdigital electrode is obtained by slicing after stripping.

[0062] The interdigital electrodes were ultrasonically cleaned 3 times with ethanol and then dried naturally. 3 O 4 @Au, tyrosinase (tyr) and 1wt% chitosan solution (chitosan) were mixed evenly in a volume ratio of 1:1:1, and then 8μL was dropped on the interdigital area of ​​the interdigital electrode. In order to ensure the activity of tyrosinase, the interdigital electrode was placed in a refrigerator at minus 4 degrees to dry overnight to modify the solution. Chitosan solution has a film-forming effect, and it is used as a film-forming agent here. In previous experiments, it was found that when the ferroferric oxide powder was under the action of a bar magnet, it would move with the movement of the magnet, and when the ferroferric oxide powder was placed on an edge of the bar magnet, the straight line formed by the ferroferric oxide powder was the best. In order to verify that under the action of a magnetic field, magnetic ferroferric oxide nanoparticles would enhance the response of the biosensor, the simplest method was used to place the interdigital electrode under the bar magnet when modifying the solution.

[0063] Test Example 1 Characterization of FeO3 Nanoparticles

[0064] Characterization of Fe prepared in Example 1 (1) 3 O 4

[0065] In order to verify that the ferrosoferric oxide particles have been successfully synthesized, TEM was used to characterize the morphology of the ferrosoferric oxide prepared in Example 1 (1). Figure 1 As shown in Figure A, the TEM image of ferroferric oxide shows that it is a sphere with a diameter of about 5 nanometers, and the particle size is relatively uniform, but there is also a certain amount of agglomeration in some parts.

[0066] Characterization of Fe prepared in Example 1 (2) 3 O 4 @Au

[0067] When a layer of gold nanoparticles was wrapped around ferroferric oxide, gold clusters appeared on the surface of ferroferric oxide, and the particle size increased to about 20 nanometers. Figure 1 As shown in Figure B

[0068] Test Example 2 Principle of Dopamine Detection

[0069] Fe 3 O 4 / The reason why the interdigitated electrode biosensor can detect dopamine is mainly because tyrosinase (Tyr) has a catalytic effect on dopamine. As shown in Figure 5, dopamine can form dopaquinone under the catalytic action of tyrosinase. During the oxidation process, the hydroxyl group (-OH) of dopamine forms a carbon-oxygen double bond (C=O) to release hydrogen ions, which leads to an increase in hydrogen ions in the solution. When a voltage is applied to the electrodes, hydrogen ions will be transferred between the electrodes to form a current. In other words, when dopamine is added, Fe 3 O 4 / The interdigital electrode biosensor will have an electrical response.

[0070] Test Example 3 Fe 3 O 4 / Interdigital electrode biosensor for detection of dopamine (Dopa)

[0071] First, in order to observe the Fe 3 O 4 The morphology of Au+Tyr solution with and without magnetic field is different. 3 O 4 @Interdigital electrodes of Au+Tyr solution and Fe modification without magnetic field 3 O 4 @Au+Tyr solution interdigitated electrode for SEM comparison. Figure 3 As shown in the figure, when there is no magnetic field, the interdigital region of the electrode has nanoparticles, but the distribution is irregular. When modified under a magnetic field, the magnetic nanoparticles form a straight line under the action of the magnetic field. This shows that under the action of a magnetic field, a solution containing magnetic nanoparticles can be distributed regularly, which can not only increase the local concentration but also form a magnetic nanowire between the interdigital electrodes.

[0072] Figure 3 The SEM image of Fe 3 O 4 @Au+Tyr solution will form a dense straight line on the interdigitated electrode. Now, based on tyrosinase, dopamine can be catalyzed to oxidize to detect Fe under the action of magnetic field. 3 O 4 @Au+Tyr biosensor electrochemical response changes. Figure 4 shows the electrochemical response of chitosan / Fe3 O 4 @Au+Tyr(non-magnetic) and chitosan / Fe 3 O 4 @Au+Tyr (magnetic) control was used to detect dopamine using the chronoamperometry. Figure 4 is the time-current curve for detecting dopamine (10μmol L-1). During the test, 10μL of dopamine solution was added to 5ml (50mmol L-1) PBS solution every 50s. Each group of sensors had an obvious electrical response to dopamine, and the response time was about 5s, which indicates that tyrosinase can quickly catalyze the oxidation of dopamine. 3 O 4 @Au+Tyr(non-magnetic) and chitosan / Fe 3 O 4 @Au+Tyr (with magnetism) time-current curve shows that the step change of current in each group increases significantly after the magnetic field is added. Due to the differences between the electrodes themselves, the initial current of each time-current curve is different. The step current value of each group of time-current curves is fitted linearly, and the slope of the fitted line is the signal sensitivity of the biosensor. This shows that the signal sensitivity of the sensor can be improved under the action of the magnetic field. This is because the ferroferric oxide magnetic nanoparticles not only increase the local concentration of tyrosinase under the action of the magnetic field, but also form a dense curve between the interdigitated electrode channels, which improves the transmission capacity of electrons and the conductivity. Therefore, the signal sensitivity of the biosensor can be improved under the action of the magnetic field.

[0073] This shows that the addition of magnetic field can indeed improve signal sensitivity. This is because under the action of magnetic field, the magnetic field force on ferroferric oxide will form a straight line along the direction of the magnet. This straight line is equivalent to forming a channel for electrons between the interdigitated electrodes and will increase the concentration of the modified solution on this line. 3 O 4 @Au+Tyr signal sensitivity. Adding Fe 3 O 4 @Au solution with the same volume of deionized water to ensure the same concentration of tyrosinase. Similarly, when testing dopamine at the same concentration, compared with pure tyrosinase, the addition of Fe 3 O 4 @Au particles, and the introduction of a very easy-to-implement magnetic field, the sensor's signal sensitivity has increased by 2 orders of magnitude, which is a very simple and easy measure compared to other methods of improving the sensitivity of biosensors. It is now known that the sensitivity of biosensors can be significantly improved after adding a magnetic field, and the position of the magnetic field will be optimized in the next work.

[0074] Example 4 Optimization of magnetic field height

[0075] The present invention has found through experiments that the straight line formed by magnetic nanoparticles on the edge of a bar magnet is the best. The present invention has also found that different distances from the edge will also affect the formed straight line, so in the process of fixing the magnet, it is preferred to select the edge of the bar magnet to place the interdigital electrode. In the electrical test, in order to make the height change the same as much as possible each time, ordinary A4 paper is used, and the distance from the magnet is controlled by increasing the number of layers of paper each time. The thickness of the paper measured with a vernier caliper is 50μm. Then time-current measurements are performed at four distances of 100μm, 150μm, 200μm, and 250μm. And it can be seen from the figure that the current has a good linear relationship with the dopamine concentration between 10μM-50μM. In order to more intuitively see the relationship between the signal sensitivity and the height of the interdigital electrode from the magnet, the current change curve with the dopamine concentration at different interdigital electrode heights from the magnet under the same voltage is made, as shown in Figure 5, after 5, 10, and 15 cycles, the number of recovery times of the EN-FETs biosensor is controlled by a magnet.

[0076] Any slight changes or modifications made using the technical contents disclosed above are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A biosensor or in situ detection chip, It is characterized in that The biosensor comprises interdigital electrodes, nanoparticles, tyrosinase and a film-forming agent; The nanoparticles and tyrosinase are covered on the surface of the interdigital electrodes by a film-forming agent; The nanoparticles are arranged in a linear pattern; The mass ratio of the nanoparticles to tyrosinase is 50-10:1; The nanoparticles include Fe 3 O 4 Nanoparticles, Au nanoparticles, and thiol compounds; The Fe 3 O 4 The molar ratio of the nanoparticles, Au nanoparticles and the thiol compound is (1-2):(0.5-1):(0.1-0.2); The Au nanoparticles are encapsulated in Fe 3 O 4 The surface of the nanoparticles; The thiol compound is connected to the Au nanoparticles; The size of the nanoparticles is 80nm~1000nm; The thiol compound includes cysteine.

2. The biosensor or in situ detection chip according to claim 1, It is characterized in that The mass ratio of the nanoparticles to tyrosinase is 10:

1.

3. The biosensor or in situ detection chip according to claim 1, It is characterized in that The Fe 3 O 4 The molar ratio of the nanoparticles, Au nanoparticles and the thiol compound is 1:0.5:0.

1.

4. The biosensor or in situ detection chip according to claim 1, It is characterized in that The film former includes chitosan.

5. The biosensor or in situ detection chip according to claim 1, It is characterized in that The sensitivity range of the biosensor or in-situ detection chip controlled by the permanent magnet is 1umol / L to 120umol / L, and the detection limit is 3.3nmol / L.

6. The biosensor or in situ detection chip according to claim 1, It is characterized in that The regeneration of the biosensor or in-situ detection chip is achieved by adding and removing the permanent magnet.

7. A method for preparing the biosensor or in-situ detection chip according to any one of claims 1 to 6, It is characterized in that The following steps are involved: Obtaining interdigitated electrodes; A mixed solution of nanoparticles, tyrosinase and a film-forming agent is placed on the surface of the interdigital electrode; and the interdigital electrode is placed in a magnetic field, and after drying, the biosensor or in-situ detection chip is obtained.

8. The method for preparing the biosensor or in-situ detection chip according to claim 7, It is characterized in that Placing the electrode in the magnetic field is to place the interdigitated electrodes close to a magnet; The distance between the interdigital electrodes and the magnet is 50 μm to 200 μm.

9. Application of the biosensor or in-situ detection chip according to any one of claims 1 to 6, or the biosensor or in-situ detection chip prepared by the preparation method according to claim 7 or 8 in brain science and detection of dopamine.

10. A nanoparticle, It is characterized in that The nanoparticles include Fe 3 O 4 Nanoparticles, Au nanoparticles, and thiol compounds; The Fe 3 O 4 The molar ratio of the nanoparticles, Au nanoparticles and the thiol compound is (1-2):(0.5-1):(0.1-0.2); The Au nanoparticles are encapsulated in Fe 3 O 4 The surface of the nanoparticles; The thiol compound is connected to the Au nanoparticles; The size of the nanoparticles is 80nm~1000nm; The thiol compound includes cysteine.

11. The nanoparticle according to claim 10, It is characterized in that The mass ratio of the nanoparticles to tyrosinase is 10:

1.

12. The nanoparticle according to claim 10, It is characterized in that The Fe 3 O 4 The molar ratio of the nanoparticles, Au nanoparticles and the thiol compound is 1:0.5:0.1.

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