Graphene nano functional gold electric probe as well as preparation method and application thereof

Abstract

The invention discloses a graphene nano functional gold electric probe as well as a preparation method and application thereof, and belongs to the technical field of biosensing. The graphene nano functional gold electric probe provided by the invention comprises a conical optical fiber probe head; the conical top of the conical optical fiber needle head is a flat end surface; the gold film is formed on the conical side surface and the conical top flat end surface of the conical optical fiber needle head; the insulating layer is formed on the outer surface of the conical side surface of the gold film; the graphene layer is arranged on the surface of the gold film on the flat end face of the conical top of the conical optical fiber needle head; the functional layer is modified on the graphene layer, and the functional layer comprises L-polylysine and an albumin antibody. The graphene nano functional gold electric probe provided by the invention is simple in structure, good in specificity and high in sensitivity, the detection limit of albumin in a complex culture medium can still be as low as 0.1 pg / mL, and the detection method is simple and convenient to operate, high in efficiency and low in cost.

Description

Technical Field

[0001] The present invention relates to the field of biosensing technology, and in particular, to a graphene nano-functional gold electrical probe, a preparation method thereof, and an application thereof. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to increase the understanding of the overall background of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Liver organoids are an advanced three-dimensional cell culture system that can highly simulate the complex structure and physiological functions of the human liver in vitro, including cell heterogeneity, metabolic activity, toxic reactions, and pathological changes. Therefore, they have important application values in the fields of liver disease research, drug screening, and toxicity assessment. During the growth and differentiation of organoids, hepatocytes continuously secrete various biomarkers, such as albumin, urea, bilirubin, and liver enzymes. Among them, albumin, as an important indicator of liver function, its secretion level directly reflects the health status and metabolic capacity of hepatocytes. Therefore, the accurate detection of albumin is crucial for the early diagnosis of liver diseases, drug toxicity assessment, and the formulation of personalized treatment strategies.

[0004] Currently, the detection of albumin mainly relies on traditional biochemical analysis methods, including enzyme-linked immunosorbent assay (ELISA), Western blot, and real-time quantitative PCR (qPCR). Although these methods have high specificity and quantitative ability, they generally have disadvantages such as complex operation steps, long detection cycles, large sample consumption, high costs, and difficulty in achieving real-time dynamic monitoring. Especially in research scenarios that require high-throughput screening and continuous monitoring, the limitations of these methods are more obvious. Therefore, developing a highly sensitive, real-time, non-invasive, and high-throughput applicable albumin detection method is crucial for the research and application of liver organoids. Summary of the Invention

[0005] In view of this, the present invention provides a graphene nano-functional gold electrical probe, a preparation method thereof, and an application thereof. The graphene nano-functional gold electrical probe provided by the present invention has advantages such as high sensitivity, low power consumption, and miniaturization. It can directly insert into liver organoids or other biological samples to achieve real-time monitoring of the physiological state of cells or tissues, and is particularly suitable for detecting albumin secreted by cells.

[0006] In a first aspect, the present invention provides a graphene nano-functional gold electrical probe, comprising: A tapered optical fiber needle tip; the tip of the tapered optical fiber needle tip is a flat end face; A gold film, which is formed on the conical side surface and the flat end surface of the cone top of the conical optical fiber needle tip; An insulating layer, which is formed on the outer surface of the conical side surface of the gold film; A graphene layer, which is arranged on the surface of the gold film on the flat end surface of the cone top of the conical optical fiber needle tip; A functional layer, which is modified on the graphene layer, and the functional layer includes L-polylysine and albumin antibody.

[0007] In a second aspect, the present invention provides a preparation method of the above graphene nano-functional gold electrical probe, comprising the following steps: Using the optical fiber taper technology to stretch the end of the optical fiber to obtain a conical optical fiber needle tip, depositing a gold film on the conical side surface of the conical optical fiber needle tip by using the evaporation coating technology, and then wrapping an insulating layer on the outer surface of the gold film; Transferring a layer of graphene on the gold film on the flat end surface of the cone top of the conical optical fiber needle tip to obtain a nano-electrical probe; then incubating the nano-electrical probe in an L-polylysine solution, washing it after incubation is completed to obtain a nano-electrical probe modified with L-polylysine; and then incubating the nano-electrical probe modified with L-polylysine in an albumin antibody solution to obtain the graphene nano-functional gold electrical probe.

[0008] In a third aspect, the present invention provides an application of the above graphene nano-functional gold electrical probe or the graphene nano-functional gold electrical probe prepared by the above preparation method, and uses the graphene nano-functional gold electrical probe for detecting the albumin concentration in liver organoids; The method for the detection comprises the following steps: Placing the graphene nano-functional gold electrical probe in liver organoids for incubation, then using the incubated graphene nano-functional gold electrical probe as a working electrode, using a saturated Ag / AgCl electrode as a reference electrode, using a platinum wire electrode as a counter electrode to form a three-electrode system, and then performing cyclic voltammetry scanning in a PBS buffer solution, recording the peak current, and determining the albumin concentration in the liver organoids according to the standard curve of the peak current change value and the albumin concentration.

[0009] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The graphene nano-functional gold electroprobe provided by the present invention has a simple structure. Among them, the gold film has good chemical stability and excellent electrochemical responsiveness. At the same time, the addition of graphene makes the electrochemical response of the gold film layer more sensitive, which can enhance the current change and thus improve the detection sensitivity. In the present invention, L-polylysine (PLL) and albumin antibody are modified on the surface of graphene, so that the probe of the present invention can effectively recognize and detect the albumin marker secreted by liver organoids, with good specificity and high sensitivity. The detection limit in PBS buffer is as low as 0.01 pg / mL, and the detection limit in organoid culture medium can still be as low as 0.1 pg / mL.

[0010] (2) Compared with traditional methods such as enzyme-linked immunosorbent assay (ELISA), when the graphene nano-functional gold electroprobe of the present invention is applied to the detection of albumin, it does not require a large amount of reagents and samples, which can significantly reduce the experimental cost. At the same time, the operation of the present invention is simple, which greatly improves the experimental efficiency. Moreover, this detection method can monitor the albumin secreted by liver organoids in real time and continuously, and does not require the destruction or removal of samples, with strong practicability and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0012] Figure 1 is a schematic flow chart of steps (1) to (4) of the embodiment of the present invention; Figure 2 is a scanning electron microscope image of the probe prepared in steps (2) and (3) and a physical image of the probe prepared in step (4) in the embodiment of the present invention. Among them, a is the scanning electron microscope image of the probe after depositing the gold film in step (2); b is the scanning electron microscope image of the probe after wrapping the insulating layer in step (3); c is the physical image of the probe after transferring graphene in step (4); Figure 3 is the CV curve of the probe after depositing the gold film in step (2) of the embodiment of the present invention. Among them, a is the CV curve in 5 mM K 3 Fe(CN) 6 / K 4 Fe(CN) 6 and 1×PBS buffer, and b is the CV curve in 5 mM H 2 SO 4 and 1×PBS buffer; Figure 4It is the Raman image of the graphene transfer test on the silicon wafer in the embodiment of the present invention; Figure 5 It is the process schematic diagram of steps (4) to (6) in the embodiment of the present invention; Figure 6 It is the corresponding CV curve obtained by scanning the graphene nano-functional gold electrical probe in the embodiment of the present invention at different scanning rates of 5 to 100 mV / s and the linear relationship diagram between the peak current and the scanning rate; wherein, a is the corresponding CV curve obtained by scanning at different scanning rates, and b is the linear relationship diagram between the peak current and the scanning rate; Figure 7 It is the CV curve of the probe in steps (4), (5) and (6) in the embodiment of the present invention; wherein, "gold + graphene" is the probe prepared in step (4), "gold + graphene + PLL" is the probe prepared in step (5), and "gold + graphene + PLL + antibody" is the probe prepared in step (6); Figure 8 It is the CV curve of the graphene nano-functional gold electrical probe in the embodiment of the present invention for detecting mouse albumin in 1×PBS buffer and the linear relationship diagram between the current change value and the concentration; wherein, a is the CV curve of the graphene nano-functional gold electrical probe for detecting the concentration of mouse albumin in 1×PBS buffer, and b is the linear relationship diagram between the current change value and the concentration of mouse albumin in 1×PBS buffer; Figure 9 It is the CV curve of the graphene nano-functional gold electrical probe in the embodiment of the present invention for detecting mouse albumin in organoid culture medium and the linear relationship diagram between the current change value and the concentration; a is the CV curve of the graphene nano-functional gold electrical probe for detecting the concentration of mouse albumin in organoid culture medium, and b is the linear relationship diagram between the current change value and the concentration of mouse albumin in organoid culture medium; Figure 10 It is the specific detection diagram of the graphene nano-functional gold electrical probe for mouse albumin in the embodiment of the present invention. Among them, a is the CV curve of the electrical probe's response changes to different proteins and different mixtures, and b is the bar chart of the current change of the electrical probe's response changes to different proteins and different mixtures; Figure 11 It is the current response change diagram of the graphene nano-functional gold electrical probe in the embodiment of the present invention when different substances are added step by step within 2000 s; Figure 12 It is the relevant picture of the real-time detection of albumin secreted after organoid differentiation by the graphene nano-functional gold electrical probe in the embodiment of the present invention. Among them, a is the CV curve of the electrical probe inserted on the surface of the organoid to detect the albumin secreted during the differentiation of the organoid from 0 to 6 days; b is the current change of the electrical probe's sensing response to the albumin secreted during the differentiation of the organoid within 0 to 6 days; Figure 13It is a representative photo of the graphene nano-functional gold electrical probe of the embodiment of the present invention inserted into the surface of an organoid. Detailed implementation manners

[0013] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0014] The present invention provides a graphene nano-functional gold electrical probe, including: A tapered optical fiber needle tip; the apex of the tapered optical fiber needle tip is a flat end face; A gold film, which is formed on the tapered side surface and the flat end face of the apex of the tapered optical fiber needle tip; An insulating layer, which is formed on the outer surface of the tapered side surface of the gold film; A graphene layer, which is disposed on the surface of the gold film on the flat end face of the apex of the tapered optical fiber needle tip; A functional layer, which is modified on the graphene layer, and the functional layer includes L-polylysine and albumin antibody.

[0015] In the above graphene nano-functional gold electrical probe of the present invention, the tapered optical fiber needle tip is used as the base material, which has a smooth surface and good stability and is suitable as the base material. The gold film has excellent electrical conductivity, chemical stability and electrochemical responsiveness, and its coating on the surface of the tapered optical fiber needle tip ensures the electrical conductivity of the probe. The insulating layer coats the outer surface of the tapered side surface of the gold film to avoid direct contact between the gold electrical probe and cells, reducing signal instability or interference caused by biological reactions. The graphene layer is disposed on the surface of the gold film on the flat end face of the apex of the tapered optical fiber needle tip, which provides excellent electrical conductivity and biocompatibility, enables the electrochemical response of the gold film layer to be more sensitive, can enhance current changes, and thus improves the detection sensitivity; at the same time, its good biocompatibility makes it easy to perform surface functionalization. L-polylysine (PLL) is a polymer material with excellent biocompatibility, and its structure is rich in cationic groups, which can form a strong bond with the graphene layer through electrostatic interaction and form a chemical bond with the antibody through a covalent reaction, effectively improving the stability of the probe. The albumin antibody can specifically bind to albumin, thus ensuring the specificity of the probe.

[0016] In the present invention, the diameter of the flat end face of the apex of the tapered optical fiber needle tip is 0.5 - 2 μm. The tapered optical fiber needle tip can be inserted into cells, organoids, etc. for detection, which can more accurately capture albumin at a specific position, improve the spatial resolution of detection, and at the same time, the tip of the tapered electrode will cause the electric field to be highly concentrated in this area, forming an electric field enhancement effect.

[0017] In the present invention, the thickness of the gold film is 40 - 60 nm; the thickness of the insulating layer is 10 - 30 nm.

[0018] In the present invention, the material of the insulating layer is selected from SiO 2 , Al 2 O 3 , Si 3 N 4 or polyimide.

[0019] In the present invention, the graphene in the graphene layer is single-layer graphene, which has a high carrier mobility, high sensitivity, and high specific surface area, and is beneficial to enhancing molecular adsorption.

[0020] The type of albumin in the present invention is preferably mouse albumin, which is the albumin secreted by mouse liver cells, and the albumin antibody used is the corresponding mouse albumin antibody, so as to realize the real-time in-situ detection of hepatocyte differentiation in liver organoids.

[0021] The present invention also provides a preparation method of the above graphene nano-functional gold electroprobe, including the following steps: Using the optical fiber taper technology to stretch the end of the optical fiber to obtain a tapered optical fiber needle tip, depositing a gold film on the tapered side surface of the tapered optical fiber needle tip by evaporation coating technology, and then wrapping an insulating layer on the outer surface of the gold film; Transferring a layer of graphene on the gold film of the flat end surface at the cone top of the tapered optical fiber needle tip to obtain a nano electroprobe; then incubating the nano electroprobe in an L-polylysine solution, washing it after incubation to obtain a nano electroprobe modified with L-polylysine; and then incubating the nano electroprobe modified with L-polylysine in an albumin antibody solution to obtain the graphene nano-functional gold electroprobe.

[0022] The optical fiber used in the optical fiber taper of the present invention is preferably a quartz optical fiber, with an inner diameter size of 40 - 60 μm and an outer diameter size of 100 - 150 μm. Before the optical fiber taper of the present invention, it also includes a process of cleaning the optical fiber material with a solvent to ensure that the surface is free of dust, dirt, and oxides. The present invention does not make special restrictions on the specific optical fiber taper process parameters, and the common optical fiber taper process in the art can be used.

[0023] The present invention also does not make special restrictions on the specific process of depositing the gold film by evaporation coating technology, and the common evaporation coating technology in the art can be used to deposit the gold film.

[0024] The present invention also does not make special restrictions on the specific process of wrapping the insulating layer. For example, the insulating layer can be wrapped by chemical vapor deposition, atomic layer deposition, electroplating, surface reaction, coating, or gluing.

[0025] The present invention does not impose special restrictions on the transfer process of graphene, and the commonly used methods for transferring graphene in the art can be adopted. The present invention also does not impose special restrictions on the synthesis method of graphene. Preferably, the present invention prepares graphene by chemical vapor deposition, and the present invention does not impose special restrictions on the specific process of preparing graphene by chemical vapor deposition, and the commonly used methods for preparing graphene by chemical vapor deposition in the art can be adopted.

[0026] In the present invention, the concentration of the L-polylysine solution is 0.1-5 mg / mL, more preferably 1-3 mg / mL; the incubation time in the L-polylysine solution is 1-3 h, and the incubation temperature is 10-40 °C, preferably at room temperature. After incubation in the L-polylysine solution, the present invention is washed with deionized water.

[0027] In the present invention, the concentration of the albumin antibody solution is 0.5-2 μM; the incubation time in the albumin antibody solution is 8-20 h, and the incubation temperature is 10-40 °C, preferably at room temperature.

[0028] The present invention also provides the application of the above graphene nano-functional gold electroprobe or the graphene nano-functional gold electroprobe prepared by the above preparation method, and uses the graphene nano-functional gold electroprobe to detect the albumin concentration in liver organoids; The method for the detection includes the following steps: Place the graphene nano-functional gold electroprobe in liver organoids for incubation, then use the incubated graphene nano-functional gold electroprobe as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode to form a three-electrode system, and then perform cyclic voltammetry scanning in a PBS buffer solution, record the peak current, and determine the albumin concentration in the liver organoids according to the standard curve of the change value of the peak current and the albumin concentration.

[0029] In the present invention, the incubation time is 20-40 min, and the incubation temperature is 36-38 °C.

[0030] The present invention does not impose special restrictions on the specific parameters of the cyclic voltammetry scanning. Preferably, the scanning is performed at a scanning rate of 2-100 mV / s in a voltage range of -0.2-0.6 V.

[0031] In the present invention, the process for determining the standard curve of the peak current change value and the albumin concentration is as follows: The graphene nano-functional gold electrical probe is respectively incubated in a blank PBS buffer solution and a series of organoid culture media with known albumin concentrations. Then, using the incubated graphene nano-functional gold electrical probe as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode, a three-electrode system is formed. Then, cyclic voltammetry scanning is performed in the PBS buffer solution, the peak current is recorded, and a standard curve is established based on the relationship between the peak current change value and the albumin concentration; the peak current change value refers to the change value of the peak current detected after incubation with a specific albumin concentration relative to the peak current detected after incubation in the blank PBS buffer solution.

[0032] The technical solution of the present invention will be further elaborated below in conjunction with specific embodiments. The present invention places no special restrictions on the sources of the reagents used in the following embodiments, and commercially available products well-known to those skilled in the art can be used.

[0033] In the following embodiments, the optical fiber material used is a quartz optical fiber with an inner diameter of 50 μm and an outer diameter of 125 μm; the preparation method of the single-layer graphene used is: using chemical vapor deposition to grow graphene on a copper foil, and then etching the copper substrate with a ferric chloride solution to obtain a single-layer graphene film. Room temperature refers to 25 ± 3 °C.

[0034] Example This embodiment provides a preparation method for a graphene nano-functional gold electrical probe.

[0035] (1) The selected optical fiber material is cleaned successively with acetone, absolute ethanol, and water, and then cut into optical fiber segments with a length of 2 cm. Then, one end of the optical fiber segment is fixed on a stretching machine through a clamping device to ensure the stability of the optical fiber during the entire stretching process. Next, using the optical fiber tapering technology, the stretching rate and temperature of the optical fiber are precisely controlled, and the end of the optical fiber is tapered to form a probe head at the micron level, and the flat end face diameter of the probe head apex is about 1 μm.

[0036] (2) A gold film is deposited on the conical side and the flat end face of the conical probe head formed in step (1) by evaporation coating technology. The parameters of evaporation coating are 0.2 nm / s, and the vacuum degree is ≤ 10 -6 Torr, and the thickness of the formed gold film is 50 nm.

[0037] (3) A layer of SiO 2 insulating layer with a thickness of about 20 nm is wrapped on the conical side of the gold film formed in step (2) by the sol-gel method.

[0038] (4) Place the single-layer graphene film in deionized water, then adsorb it on the top of the probe obtained in step (3), and then dry it naturally to obtain the probe after transferring graphene.

[0039] (5) Immerse the probe after transferring graphene obtained in step (4) into a 2 mg / mL PLL solution and incubate it at room temperature for 1.5 h; after incubation, use deionized water to remove the excessive unbound PLL.

[0040] (6) Immerse the probe obtained in step (5) into a PBS buffer solution containing 1 μM mouse albumin antibody (A90 - 134A, purchased from Bethyl Company), incubate for 12 h, wash off the unbound mouse albumin antibody with PBS buffer solution, and dry it naturally to obtain the graphene nano-functional gold electrode probe.

[0041] Figure 1 It is a schematic flow chart of steps (1) - (4) of this embodiment. Figure 2 In [reference], a is the scanning electron microscope image of the probe after depositing the gold film in step (2) of this embodiment. It can be seen that the gold film is uniformly deposited on the surface of the optical fiber. Figure 2 In [reference], b is the scanning electron microscope image of the probe after wrapping the insulating layer in step (3) of this embodiment. It can be seen that the surface roughness of the probe increases, indicating that the insulating layer is successfully coated on the surface of the gold film. Figure 2 In [reference], c is a physical picture of the probe after transferring graphene in step (4) of this embodiment.

[0042] The probe after depositing the gold film in step (2) was scanned at a rate of 50 mV / s in 5 mM K 3 Fe(CN) 6 / K 4 Fe(CN) 6 and 1×PBS buffer solution, and its cyclic voltammetry (CV) curve was tested. As shown in Figure 3 a in [reference], it can be seen from the figure that the curve is S-shaped, which is a typical curve for testing a gold electrode. In 5 mM H 2 SO 4 and 1×PBS buffer solution, it was scanned at a rate of 50 mV / s, and its cyclic voltammetry (CV) curve was tested. As shown in Figure 3 b in [reference], this is also a typical curve of a gold electrode, indicating that the gold film is successfully deposited on the surface of the optical fiber.

[0043] Figure 4 It is a Raman image of graphene grown by chemical vapor deposition method transferred on a silicon wafer for testing. It can be seen from the image that the 2D peak is twice the G peak and there is no defect peak, proving that the quality of this graphene is good.

[0044] Figure 5It is a schematic flow diagram of steps (4) to (6).

[0045] The corresponding CV curves obtained by scanning the graphene nano-functional gold electroprobe prepared in this example in PBS buffer at different scanning rates of 5 to 100 mV / s are as shown in Figure 6 a in, and the relationship curve between the peak current and the scanning rate is as shown in Figure 6 b in. It can be seen that there is a good correlation between the peak current and the scanning rate, indicating that the graphene nano-functional gold electroprobe of this example has good electrochemical response characteristics and is suitable for highly sensitive analysis of biomarkers such as albumin. The CV curves of the probes in steps (4) (gold + graphene), step (5) (gold + graphene + PLL), and step (6) (gold + graphene + PLL + antibody) in PBS buffer were tested by cyclic voltammetry, as shown in Figure 7 shown. It can be seen that the CV curves are constantly changing, indicating that the surface of the probe has been successfully modified.

[0046] Test Example 1. Electrochemical response in PBS buffer: Commercial standard mouse albumin (purchased from Abcam) was prepared into 1×PBS buffer with different concentrations (0.01 pg / mL - 100 ng / mL). The graphene nano-functional gold electroprobe prepared in the example was placed in 1×PBS buffer and mouse albumin solutions with different concentrations respectively, incubated at 37°C for 30 min, and then taken out. The incubated graphene nano-functional gold electroprobe was used as the working electrode, the saturated Ag / AgCl electrode was used as the reference electrode, and the platinum wire electrode was used as the counter electrode to form a three-electrode system. One end of the three electrodes was fixed to the working electrode end, reference electrode end, and counter electrode end of the single-cell analysis instrument respectively, and the other end of the three electrodes was inserted into the 1×PBS buffer solution. Then, it was scanned in the voltage range of -0.2 to 0.6 V at a scanning rate of 50 mV / s, as shown in Figure 8 a in, and the peak current values in mouse albumin solutions with different concentrations were recorded. Then, a peak current change value - concentration standard curve was plotted. The peak current change value refers to the change value of the peak current value detected after incubation with a specific albumin concentration relative to the peak current value detected after incubation with 1×PBS buffer. As shown in Figure 8 b in. It can be seen that as the target concentration increases, the current of the CV curve gradually increases, and the detection limit is as low as 0.01 pg / mL. This phenomenon is attributed to the specific binding of albumin to albumin antibody, and the current changes induced by different concentrations of protein have a good linear correlation with the albumin concentration, and the correlation coefficient is 0.9869.

[0047] 2. Electrochemical response in complex medium: To evaluate the electrochemical response characteristics of the graphene nano-functional gold electro-probe prepared in the examples in a complex culture medium, mouse albumin was diluted with an organoid culture medium (concentration: 0.1 pg / mL to 100 ng / mL), and the electrochemical response of the graphene nano-functional gold electro-probe of the examples was tested using a test method similar to the above-mentioned "electrochemical response in PBS buffer". The test results are as Figure 9 shown in a and b of Figure 9 . It can be seen that as the albumin concentration increases, the current also gradually increases. The detection limit is as low as 0.1 pg / mL, and the current change at each concentration shows a linear correlation with the albumin concentration, with a correlation coefficient of 0.9771. Thus, it can be seen that whether in pure PBS buffer or in a complex culture medium, the electrical characteristics of the graphene nano-functional gold electro-probe prepared in the examples respond sensitively, which proves the feasibility of using this probe to detect biomolecules and can be used for the detection of albumin in organoids.

[0048] 3. Specific detection To test the specificity of the graphene nano-functional gold electro-probe of the examples for mouse albumin, human serum albumin (HSA), immunoglobulin (IgG), and bovine serum albumin (BSA) were selected to interfere with it. As Figure 10 shown in a and b of Figure 10 , where "albumin" in the figure refers to mouse albumin. It can be seen that the graphene nano-functional gold electro-probe of the examples only has a strong sensing signal for mouse albumin, and the induced current change is significantly higher than that of other proteins, indicating that this sensor is not interfered by other substances in a complex system and has high specificity for mouse albumin.

[0049] To further evaluate the specificity of the electro-probe for mouse albumin, the current curve method (Amperomentric i-t curve) in the electrical detection software was selected for testing. It was run for 2000 s, and the response current generated by the electro-probe when detecting different substances was detected in real time. As Figure 11 shown in Figure 11 , where "albumin" in the figure refers to mouse albumin. After the detection baseline was stable, 2 μL of PBS buffer, 2 μL of BSA, 2 μL of HSA, and 2 μL of IgG (negative control) were added at 300 s, 500 s, 600 s, and 1000 s respectively, and no electrical signal was detected, indicating that PBS, BSA, HSA, and IgG do not produce electrical signal interference. Until 2 μL of the target mouse albumin was added at 1300 s, a current response began to be generated. Subsequently, 5 μL, 10 μL, and 20 μL of mouse albumin were added at 1600 s, 1800 s, and 1900 s respectively. As the concentration of mouse albumin increased, the current response gradually increased, proving that this probe can specifically detect the presence of mouse albumin, and this response shows a positive correlation with the concentration of mouse albumin.

[0050] 4. Real-time detection of albumin secreted after organoid differentiation: The duct cell pellet collected from digested mouse liver tissue was embedded in Matrigel and seeded in a 35 mm dish. After seeding, expansion medium containing penicillin, streptomycin, GlutaMAX-I, N2, B27, N-acetylcysteine, R-spondin1, EGF (epidermal growth factor), FGF10 (fibroblast growth factor), HGF (hepatocyte growth factor), and gastrin was added, and the culture was continued for expansion. During differentiation, the expansion medium was replaced with differentiation medium (containing penicillin, streptomycin, GlutaMAX-I, N2, B27, N-acetylcysteine, R-spondin1, EGF, FGF10, HGF, gastrin, A83-01, and DAPT). At this time, the cultured organoids were placed on a single-cell analysis stage to ensure observation and monitoring. On this basis, the graphene nano-functional gold electrical probe of the example was inserted into the culture matrix of the organoids to ensure that the probe could precisely contact the surface of the organoids and perform real-time signal detection.

[0051] Figure 12 a in [reference] shows the electrical response of detecting albumin secreted by organoids during differentiation after the probe is inserted into the surface of the organoids. Through continuous monitoring for 6 days, over time, the current response gradually increases, indicating that the organoids are continuously differentiating and the secreted marker albumin increases as the differentiation progresses. Figure 12 b in [reference] presents a graph of the relationship between current change and time, which shows that the current response increases linearly with time, and the correlation coefficient is 0.9959, indicating a high linear correlation between the current response and the organoid differentiation process. Analyzing the relationship between the current response and albumin concentration can reveal the dynamic changes of albumin secreted by organoids during differentiation and provide key information about the functional state of the organoids. Figure 13 [reference] then shows a photo of the electrical probe inserted into the surface of the organoids, visually demonstrating the contact between the electrical probe and the surface of the organoids, providing visual support for subsequent experiments.

[0052] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A graphene nanofunctional gold electrical probe, characterized in that: include: A conical optical fiber needle; the top of the conical optical fiber needle is a flat end surface; A gold film, wherein the gold film is formed on the tapered side surface and the flat end surface of the tapered top of the tapered optical fiber needle; An insulating layer, the insulating layer being formed on the outer surface of the tapered side of the gold film; A graphene layer, wherein the graphene layer is disposed on a gold film surface of a flat end surface of a cone top of the tapered optical fiber needle; The functional layer is modified on the graphene layer, and the functional layer includes L-polylysine and albumin antibody.

2. The graphene nanofunctional gold electrical probe according to claim 1, characterized in that: The diameter of the flat end surface of the cone top of the tapered optical fiber needle is 0.5-2 μm.

3. The graphene nanofunctional gold electrical probe according to claim 1, characterized in that: The thickness of the gold film is 40-60 nm; the thickness of the insulating layer is 10-30 nm.

4. The graphene nanofunctional gold electrical probe according to claim 1, characterized in that: The material of the insulating layer is selected from SiO2, Al2O3, Si3N4 or polyimide.

5. The graphene nanofunctional gold electrical probe according to claim 1, characterized in that: The graphene in the graphene layer is a single-layer graphene.

6. The method for preparing the graphene nano-functional gold electrical probe according to any one of claims 1 to 5, characterized in that: The steps include: The end of the optical fiber is stretched by using the optical fiber taper technology to obtain a tapered optical fiber needle, a gold film is deposited on the tapered side of the tapered optical fiber needle by using the evaporation coating technology, and then an insulating layer is wrapped on the outer surface of the gold film; A layer of graphene is transferred onto the gold film on the flat end face of the cone top of the tapered optical fiber needle to obtain a nano-electric probe; the nano-electric probe is then incubated in an L-polylysine solution, and after the incubation is completed, it is washed to obtain a nano-electric probe modified with L-polylysine; the nano-electric probe modified with L-polylysine is then incubated in an albumin antibody solution to obtain a graphene nano-functional gold electric probe.

7. The preparation method according to claim 6, characterized in that: The concentration of the L-polylysine solution is 0.1-5 mg / mL, the incubation time in the L-polylysine solution is 1-3 hours, and the incubation temperature is 10-40° C.

8. The preparation method according to claim 6, characterized in that: The concentration of the albumin antibody solution is 0.5-2 μM; the incubation time in the albumin antibody solution is 8-20 h, and the incubation temperature is 10-40° C.

9. Use of the graphene nano-functional gold electric probe according to any one of claims 1 to 5 or the graphene nano-functional gold electric probe prepared by the preparation method according to any one of claims 6 to 8, characterized in that: Using the graphene nanofunctional gold electrical probe to detect albumin concentration in liver organoids; The detection method comprises the following steps: The graphene nanofunctional gold electroprobe is placed in a liver organoid for incubation, and then the incubated graphene nanofunctional gold electroprobe is used as a working electrode, a saturated Ag / AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode to form a three-electrode system. Cyclic voltammetry scanning is then performed in a PBS buffer solution to record the peak current, and the albumin concentration in the liver organoid is determined based on a standard curve of the peak current change value and the albumin concentration.

10. The use according to claim 9, characterized in that The incubation time is 20-40 min, and the incubation temperature is 36-38°C.

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