A graphene nanofunctional gold electrical probe and its preparation method and application

Abstract

The present invention discloses a graphene nano-functional gold electrical probe and its preparation method and application, which belong to the field of biosensor technology. The graphene nano-functional gold electrical probe provided by the present invention includes: a tapered optical fiber needle; the cone top of the tapered optical fiber needle is a flat end face; a gold film, the gold film is formed on the tapered side surface and the cone top flat end face of the tapered optical fiber needle; an insulating layer, the insulating layer is formed on the outer surface of the tapered side surface of the gold film; a graphene layer, the graphene layer is arranged on the gold film surface of the cone top flat end face of the tapered optical fiber needle; a functional layer, the functional layer is modified on the graphene layer, and the functional layer includes L-polylysine and albumin antibody. The graphene nano-functional gold electrical probe provided by the present invention has a simple structure, good specificity, high sensitivity, and the detection limit of albumin in complex culture medium can still be as low as 0.1 pg / mL, and the detection method is simple to operate, highly efficient, and low cost.

Description

Technical Field

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

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Liver organoids are advanced three-dimensional cell culture systems that closely mimic the complex structure and physiological functions of the human liver in vitro, including cellular heterogeneity, metabolic activity, toxic reactions, and pathological changes. Therefore, they hold significant application value in liver disease research, drug screening, and toxicity assessment. During organoid growth and differentiation, hepatocytes continuously secrete a variety of biomarkers, such as albumin, urea, bilirubin, and liver enzymes. Albumin, a key indicator of liver function, directly reflects the health and metabolic capacity of hepatocytes. Therefore, accurate albumin detection is crucial for the early diagnosis of liver disease, drug toxicity assessment, and the development of personalized treatment strategies.

[0004] Currently, albumin detection relies primarily on traditional biochemical analysis methods, including enzyme-linked immunosorbent assay (ELISA), Western blot, and real-time quantitative PCR (qPCR). While these methods offer high specificity and quantitative capabilities, they generally suffer from complex procedures, long detection cycles, high sample consumption, high costs, and difficulty in achieving real-time dynamic monitoring. These limitations are particularly pronounced in research scenarios requiring high-throughput screening and continuous monitoring. Therefore, developing a highly sensitive, real-time, non-invasive, and high-throughput 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 nanofunctional gold electrical probe, a preparation method and application thereof. The graphene nanofunctional gold electrical probe provided by the present invention has the advantages of high sensitivity, low power consumption and miniaturization. It can be directly inserted into liver organoids or other biological samples to realize 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 nanofunctional gold electrical probe, comprising:

[0007] A tapered optical fiber needle; the top of the tapered optical fiber needle is a flat end surface;

[0008] A gold film formed on the tapered side surface and the flat end surface of the tapered optical fiber needle;

[0009] an insulating layer formed on an outer surface of the tapered side of the gold film;

[0010] A graphene layer, the graphene layer being provided on the gold film surface of the flat end face of the cone top of the tapered optical fiber needle;

[0011] The functional layer is modified on the graphene layer, and the functional layer includes L-polylysine and albumin antibody.

[0012] In a second aspect, the present invention provides a method for preparing the above-mentioned graphene nanofunctional gold electrical probe, comprising the following steps:

[0013] 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;

[0014] A layer of graphene is transferred onto the gold film on the flat end surface of the cone top of the tapered optical fiber needle to obtain a nanoelectric probe; the nanoelectric probe is then incubated in an L-polylysine solution and washed after the incubation is completed to obtain a nanoelectric probe modified with L-polylysine; the nanoelectric probe modified with L-polylysine is then incubated in an albumin antibody solution to obtain a graphene nanofunctional gold electric probe.

[0015] In a third aspect, the present invention provides a use of the graphene nanofunctional gold electrical probe or the graphene nanofunctional gold electrical probe prepared by the above preparation method, wherein the graphene nanofunctional gold electrical probe is used to detect albumin concentration in liver organoids;

[0016] The detection method comprises the following steps:

[0017] The graphene nanofunctional gold electroprobe was placed in a liver organoid for incubation. A three-electrode system was then formed using the incubated graphene nanofunctional gold electroprobe as a working electrode, a saturated Ag / AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode. Cyclic voltammetry was then performed in PBS buffer, and the peak current was recorded. The albumin concentration in the liver organoid was determined based on a standard curve between the peak current change value and the albumin concentration.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0019] (1) The graphene nanofunctional gold electrical probe provided by the present invention has a simple structure, wherein 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. The present invention modifies L-polylysine (PLL) and albumin antibodies on the graphene surface, so that the probe of the present invention can effectively identify 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.

[0020] (2) Compared with traditional methods such as enzyme-linked immunosorbent assay (ELISA), the graphene nanofunctional gold electroprobe of the present invention does not require the consumption of a large amount of reagents and samples when used for the detection of albumin, which can significantly reduce the experimental cost. At the same time, the present invention is easy to operate and greatly improves the experimental efficiency. Moreover, the detection method can monitor the albumin secreted by liver organoids in real time and continuously without destroying or removing samples. It is highly practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0022] Figure 1 1 is a flow chart of steps (1) to (4) of an embodiment of the present invention;

[0023] Figure 2 These are scanning electron microscope images of the probes obtained in step (2) and step (3) of the embodiment of the present invention and a physical image of the probe obtained in step (4), wherein a is a scanning electron microscope image of the probe after the gold film is deposited in step (2); b is a scanning electron microscope image of the probe after the insulating layer is wrapped in step (3); and c is a physical image of the probe after the graphene is transferred in step (4);

[0024] Figure 3 CV curves of the probe after the gold film was deposited in step (2) of the embodiment of the present invention, wherein a is the CV curve in 5 mM K3Fe(CN)6 / K4Fe(CN)6 and 1×PBS buffer, and b is the CV curve in 5 mM H2SO4 and 1×PBS buffer;

[0025] Figure 4 This is a Raman image of a graphene transfer test on a silicon wafer according to an embodiment of the present invention;

[0026] Figure 5 Schematic diagram of the process of steps (4) to (6) of the embodiment of the present invention;

[0027] Figure 6 The graphs are the corresponding CV curves and the linear relationship between the peak current and the scan rate obtained by scanning the graphene nanofunctional gold probe at different scan rates of 5 to 100 mV / s according to the embodiment of the present invention; wherein a is the corresponding CV curve obtained by scanning at different scan rates, and b is the linear relationship between the peak current and the scan rate;

[0028] Figure 7 are the CV curves of the probes in step (4), step (5) and step (6) of 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);

[0029] Figure 8 1 is a graph showing a CV curve of the graphene nano-functionalized gold probe used to detect rat albumin in 1×PBS buffer and a linear relationship between the current change value and the concentration; wherein a is a CV curve of the graphene nano-functionalized gold probe used to detect the concentration of rat albumin in 1×PBS buffer, and b is a linear relationship between the current change value and the concentration of rat albumin in 1×PBS buffer;

[0030] Figure 9 Figure 1 is a graphene nanofunctional gold probe used to detect mouse albumin in organoid culture medium using a CV curve and a linear relationship diagram between the current change value and the concentration. a is a graphene nanofunctional gold probe used to detect the concentration of mouse albumin in organoid culture medium using a CV curve, and b is a graph showing the linear relationship between the current change value and the concentration of mouse albumin in the organoid culture medium.

[0031] Figure 10 Graphs showing the specific detection of mouse albumin by the graphene nanofunctional gold electroprobe according to an embodiment of the present invention, wherein a is a CV curve showing the response of the electroprobe to different proteins and different mixtures, and b is a bar graph showing the current change of the electroprobe to different proteins and different mixtures;

[0032] Figure 11 This is a graph showing the current response changes of the graphene nano-functional gold electric probe according to an embodiment of the present invention when different substances are added step by step within 2000 s;

[0033] Figure 12Figure 1 shows the graphene nanofunctional gold electrical probe used in the present invention to detect albumin secreted by differentiated organoids in real time. (a) shows the CV curve of the electrical probe inserted into the organoid surface to detect albumin secretion during 0-6 days of organoid differentiation. (b) shows the current change in response to the electrical probe sensing albumin secretion during 0-6 days of organoid differentiation.

[0034] Figure 13 This is a representative photo of a graphene nanofunctional gold electrical probe inserted into the surface of an organoid according to an embodiment of the present invention. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0036] The present invention provides a graphene nano-functional gold electrical probe, comprising:

[0037] A tapered optical fiber needle; the top of the tapered optical fiber needle is a flat end surface;

[0038] A gold film formed on the tapered side surface and the flat end surface of the tapered optical fiber needle;

[0039] an insulating layer formed on an outer surface of the tapered side of the gold film;

[0040] A graphene layer, the graphene layer being provided on the gold film surface of the flat end face of the cone top of the tapered optical fiber needle;

[0041] The functional layer is modified on the graphene layer, and the functional layer includes L-polylysine and albumin antibody.

[0042] The graphene nanofunctionalized gold electrochemical probe described herein uses a tapered optical fiber needle as its substrate material, which features a smooth surface and excellent stability, making it suitable as a substrate material. The gold film, which exhibits excellent electrical conductivity, chemical stability, and electrochemical responsiveness, is coated on the surface of the tapered optical fiber needle, ensuring the probe's electrical conductivity. An insulating layer covers the outer surface of the tapered side of the gold film to prevent direct contact between the gold probe and cells, reducing signal instability or interference caused by biological reactions. The graphene layer, applied to the flat end of the tapered optical fiber needle, provides excellent electrical conductivity and biocompatibility. This layer enhances the electrochemical response of the gold film, enhancing current changes and thus improving detection sensitivity. Its excellent biocompatibility also makes it easy to functionalize the surface. L-Poly-lysine (PLL), a polymer material with excellent biocompatibility, is rich in cationic groups. This allows for a strong electrostatic bond with the graphene layer and a covalent chemical bond with the antibody, effectively improving the probe's stability. Albumin antibodies can specifically bind to albumin, thereby ensuring the specificity of the probe.

[0043] In the present invention, the diameter of the flat end surface of the cone top of the tapered optical fiber needle is 0.5~2μm. The tapered optical fiber needle can be inserted into cells, organoids, etc. for detection, and can more accurately capture albumin at a specific location, thereby improving the spatial resolution of the detection. At the same time, the tip of the tapered electrode causes the electric field to be highly concentrated in this area, forming an electric field enhancement effect.

[0044] In the present invention, the thickness of the gold film is 40-60 nm; the thickness of the insulating layer is 10-30 nm.

[0045] In the present invention, the material of the insulating layer is selected from SiO2, Al2O3, Si3N4 or polyimide.

[0046] In the present invention, the graphene in the graphene layer is a single-layer graphene, which has high carrier mobility, high sensitivity, and high specific surface area, and is conducive to enhancing molecular adsorption.

[0047] The albumin type described in the present invention is preferably mouse albumin, which is an albumin secreted by mouse liver cell-like cells, and the albumin antibody used is the corresponding mouse albumin antibody, thereby realizing real-time in situ detection of hepatocyte differentiation of liver organoids.

[0048] The present invention also provides a method for preparing the above-mentioned graphene nanofunctional gold electrical probe, comprising the following steps:

[0049] 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;

[0050] A layer of graphene is transferred onto the gold film on the flat end surface of the cone top of the tapered optical fiber needle to obtain a nanoelectric probe; the nanoelectric probe is then incubated in an L-polylysine solution and washed after the incubation is completed to obtain a nanoelectric probe modified with L-polylysine; the nanoelectric probe modified with L-polylysine is then incubated in an albumin antibody solution to obtain a graphene nanofunctional gold electric probe.

[0051] The optical fiber used in the optical fiber tapering process of the present invention is preferably a quartz optical fiber with an inner diameter of 40-60 μm and an outer diameter of 100-150 μm. Prior to tapering, the present invention also includes a solvent cleaning process for the optical fiber material to ensure that the surface is free of dust, dirt, and oxides. The present invention does not impose any particular restrictions on the specific optical fiber tapering process parameters; commonly used optical fiber tapering processes in the art can be used.

[0052] The present invention does not impose any special restrictions on the specific process of depositing the gold film using the evaporation coating technology. The gold film can be deposited using the evaporation coating technology commonly used in the art.

[0053] The present invention does not impose any 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 adhesion.

[0054] The present invention does not impose any particular restrictions on the graphene transfer process, and a method for transferring graphene commonly used in the art can be used. The present invention does not impose any particular restrictions on the method for synthesizing graphene, and the present invention preferably uses chemical vapor deposition to prepare the graphene. The present invention does not impose any particular restrictions on the specific process of preparing graphene by chemical vapor deposition, and a method for preparing graphene by chemical vapor deposition commonly used in the art can be used.

[0055] In the present invention, the concentration of the L-polylysine solution is 0.1 to 5 mg / mL, more preferably 1 to 3 mg / mL. The incubation time in the L-polylysine solution is 1 to 3 hours, and the incubation temperature is 10 to 40°C, preferably at room temperature. After incubation in the L-polylysine solution, the cells are washed with deionized water.

[0056] 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.

[0057] The present invention also provides the use of the graphene nanofunctional gold electrical probe or the graphene nanofunctional gold electrical probe prepared by the above preparation method, wherein the graphene nanofunctional gold electrical probe is used to detect the albumin concentration in liver organoids;

[0058] The detection method comprises the following steps:

[0059] The graphene nanofunctional gold electroprobe was placed in a liver organoid for incubation. A three-electrode system was then formed using the incubated graphene nanofunctional gold electroprobe as a working electrode, a saturated Ag / AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode. Cyclic voltammetry was then performed in PBS buffer, and the peak current was recorded. The albumin concentration in the liver organoid was determined based on a standard curve between the peak current change value and the albumin concentration.

[0060] In the present invention, the incubation time is 20-40 min, and the incubation temperature is 36-38°C.

[0061] The present invention does not impose any particular limitation on the specific parameters of the cyclic voltammetry scan. The present invention preferably performs the scan at a scan rate of 2 to 100 mV / s within a voltage range of -0.2 to 0.6 V.

[0062] 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 nanofunctional gold electric probe is placed in a blank PBS buffer and a series of organoid culture media with known albumin concentrations for incubation, and then the incubated graphene nanofunctional gold electric probe 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. Then, a cyclic voltammetry scan is performed in the PBS buffer, the peak current is recorded, and a standard curve is established according to 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 value detected after incubation at a specific albumin concentration relative to the peak current value detected after incubation in the blank PBS buffer.

[0063] The technical solution of the present invention is further described below with reference to specific examples. The present invention has no particular limitation on the sources of the reagents used in the following examples, and commercially available products known to those skilled in the art can be used.

[0064] The optical fiber material used in the following examples is quartz optical fiber with an inner diameter of 50 μm and an outer diameter of 125 μm. The single-layer graphene film is prepared by growing graphene on copper foil using chemical vapor deposition, followed by etching the copper substrate with a ferric chloride solution. Room temperature refers to 25 ± 3°C.

[0065] Example

[0066] This embodiment provides a method for preparing a graphene nanofunctional gold electrical probe.

[0067] (1) The selected optical fiber material is cleaned with acetone, anhydrous ethanol, and water in sequence, and then cut into optical fiber segments with a length of 2 cm. One end of the optical fiber segment is then fixed to the stretching machine through a clamping device to ensure the stability of the optical fiber during the entire stretching process. Next, the optical fiber taper technology is used to precisely control the stretching rate and temperature of the optical fiber, and the end of the optical fiber is tapered to form a micron-level probe head. The flat end face diameter of the probe head cone is about 1 μm.

[0068] (2) A gold film is deposited on the conical side and flat end surface of the conical probe head formed in step (1) by evaporation coating technology. The evaporation coating parameters are 0.2 nm / s and the vacuum degree is ≤10 -6 Torr, and the thickness of the formed gold film is 50 nm.

[0069] (3) The tapered side of the gold film formed in step (2) is coated with a SiO2 insulating layer with a thickness of about 20 nm by the sol-gel method.

[0070] (4) Place the single-layer graphene film in deionized water, then adsorb the top of the probe obtained in step (3), and then naturally dry to obtain the probe after transferring graphene.

[0071] (5) The graphene-transferred probe obtained in step (4) was immersed in a 2 mg / mL PLL solution and incubated at room temperature for 1.5 h. After the incubation, deionized water was used to remove excess unbound PLL.

[0072] (6) The probe obtained in step (5) was immersed in 1 μM mouse albumin antibody (A90-134A, purchased from Bethyl) in PBS buffer and incubated for 12 h. The unbound mouse albumin antibody was washed away with PBS buffer and dried naturally to obtain the graphene nanofunctional gold electrochemical probe.

[0073] Figure 1 Schematic diagram of the process of steps (1) to (4) of this embodiment. Figure 2 Figure a is a scanning electron microscope image of the probe after the gold film is deposited in step (2) of this embodiment. It can be seen that the gold film is evenly deposited on the surface of the optical fiber. Figure 2 Figure b is a scanning electron microscope image of the probe after being wrapped with 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 Figure c is a real picture of the probe after graphene is transferred in step (4) of this embodiment.

[0074] The probe after gold film deposition in step (2) was scanned at a rate of 50 mV / s in 5 mM K3Fe(CN)6 / K4Fe(CN)6 and 1×PBS buffer, and its cyclic voltammetry (CV) curve was tested, as shown in Figure 2. Figure 3 As shown in a, the curve is S-shaped, which is a typical curve for testing gold electrodes. The cyclic voltammetry (CV) curve was tested in 5 mM H2SO4 and 1×PBS buffer at a rate of 50 mV / s. Figure 3 As shown in b, this is also a typical curve of the gold electrode, indicating that the gold film is successfully deposited on the surface of the optical fiber.

[0075] Figure 4 This is a Raman image of graphene grown by chemical vapor deposition and transferred to a silicon wafer for testing. The image shows that the 2D peak is twice the G peak and there are no defect peaks, proving that the graphene is of good quality.

[0076] Figure 5 It is a flowchart of steps (4) to (6).

[0077] The graphene nanofunctional gold probe prepared in this example was scanned in PBS buffer at different scan rates from 5 to 100 mV / s to obtain the corresponding CV curves, as shown in FIG. Figure 6 As shown in a in the figure, the relationship between peak current and scan rate is shown in Figure 6 As shown in b, it can be seen that the peak current has a good correlation with the scan rate, indicating that the graphene nanofunctional gold probe of this embodiment has good electrochemical response characteristics and is suitable for high-sensitivity analysis of biomarkers such as albumin. The CV curves of the probes in step (4) (gold + graphene), step (5) (gold + graphene + PLL) and step (6) (gold + graphene + PLL + antibody) in PBS buffer were tested by cyclic voltammetry. Figure 7 As shown, it can be seen that the CV curve is constantly changing, indicating that the probe surface is successfully modified.

[0078] Test example

[0079] 1. Electrochemical response in PBS buffer:

[0080] Commercial standard rat albumin (purchased from Abcam) was prepared into 1×PBS buffer at different concentrations (0.01 pg / mL-100 ng / mL). The graphene nanofunctional gold probe prepared in the example was placed in 1×PBS buffer and different concentrations of rat albumin solution, incubated at 37°C for 30 minutes, and then removed. The incubated graphene nanofunctional gold probe 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 terminal, reference electrode terminal, and counter electrode terminal of the single cell analysis instrument, respectively. The other ends of the three electrodes were inserted into the 1×PBS buffer solution and then scanned in the voltage range of -0.2~0.6V at a scan rate of 50 mV / s. Figure 8 As shown in a, the peak current values in different concentrations of mouse albumin solution were recorded, and then the peak current change value-concentration standard curve was drawn. 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. Figure 8 As shown in Figure b, it can be seen that with the increase of target concentration, 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 and albumin antibody. The current changes induced by different concentrations of protein show a good linear correlation with the albumin concentration, with a correlation coefficient of 0.9869.

[0081] 2. Electrochemical response in complex culture medium:

[0082] In order to evaluate the electrochemical response characteristics of the graphene nanofunctional gold probe prepared in the example in complex culture medium, rat albumin was diluted in organoid culture medium (concentration ranged from 0.1 pg / mL to 100 ng / mL) and the electrochemical response of the graphene nanofunctional gold probe in the example was tested using a test method similar to the above-mentioned "Electrochemical Response in PBS Buffer". The test results are shown in Figure 2. Figure 9 As shown in a and b in Figure 1, it can be seen that the current gradually increases with increasing albumin concentration, with a detection limit as low as 0.1 pg / mL. The current change at each concentration is linearly correlated with the albumin concentration, with a correlation coefficient of 0.9771. This shows that the electrical properties of the graphene nanofunctional gold probe prepared in this example are sensitive, whether in pure PBS buffer or in complex culture medium, thus demonstrating the feasibility of the probe in detecting biomolecules and its application in detecting albumin in organoids.

[0083] 3. Specificity detection

[0084] In order to test the specificity of the graphene nanofunctional gold electroprobe to mouse albumin, human serum albumin (HSA), immunoglobulin (IgG), and bovine serum albumin (BSA) were selected to interfere with it. Figure 10 As shown in a and b in the figure, "albumin" refers to mouse albumin. It can be seen that the graphene nanofunctional gold electrical probe of the embodiment has a strong sensing signal only for mouse albumin, and the induced current change is significantly higher than that for other proteins, indicating that the sensor is not interfered by other substances in a complex system and has high specificity for mouse albumin.

[0085] In order to further evaluate the specificity of the electrical probe for mouse albumin, the current curve method in the electrical detection software was selected for testing, which was run for 2000s to detect the response current generated by the electrical probe in real time when detecting different substances. Figure 11 As shown in the figure, "albumin" refers to rat albumin. After the detection baseline stabilized, 2 μL PBS buffer, 2 μL BSA, 2 μL HSA, and 2 μL IgG (negative control) were added at 300s, 500s, 600s, and 1000s, respectively. No electrical signal was detected, indicating that PBS, BSA, HSA, and IgG did not interfere with the electrical signal. It was not until 1300s that a current response was generated after the addition of 2 μL of the target rat albumin. Subsequently, 5 μL, 10 μL, and 20 μL of rat albumin were added at 1600s, 1800s, and 1900s, respectively. As the concentration of rat albumin increased, the current response gradually increased, demonstrating that the probe can specifically detect the presence of rat albumin and that the response is positively correlated with the concentration of rat albumin.

[0086] 4. Real-time detection of albumin secreted by organoids after differentiation:

[0087] Ductal cell pellets collected from digested mouse liver tissue were embedded in Matrigel and seeded into 35 mm dishes. Following seeding, expansion medium containing penicillin, streptomycin, GlutaMAX-I, N2, B27, N-acetylcysteine, R-spondin 1, EGF (epidermal growth factor), FGF10 (fibroblast growth factor), HGF (hepatocyte growth factor), and gastrin was added, and culture and expansion continued. During the differentiation process, the expansion medium was replaced with differentiation medium (containing penicillin, streptomycin, GlutaMAX-I, N2, B27, N-acetylcysteine, R-spondin 1, EGF, FGF10, HGF, gastrin, A83-01, and DAPT). At this point, the cultured organoids were placed on a single-cell analysis stage for observation and monitoring. Next, the graphene nanofunctionalized gold electroprobes from the examples were inserted into the organoid culture matrix, ensuring precise contact between the probes and the organoid surface for real-time signal detection.

[0088] Figure 12 Figure (a) shows the electrical response of the probe inserted into the organoid surface to detect albumin secretion during organoid differentiation. Over six consecutive days of monitoring, the current response gradually increased, indicating that the organoids were continuously differentiating and that the secretion of the marker albumin increased as differentiation progressed. Figure 12 Figure b shows the relationship between current change and time. The current response increases linearly with time, with a correlation coefficient of 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 reveals the dynamic changes in albumin secretion during organoid differentiation, providing key information about the functional status of organoids. Figure 13 It shows a photo of the electric probe inserted into the surface of the organoid, intuitively demonstrating the contact between the electric probe and the surface of the organoid, providing visual support for subsequent experiments.

[0089] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A graphene nanofunctional gold electrical probe, characterized in that: include: A tapered optical fiber needle; the top of the tapered optical fiber needle is a flat end surface; the diameter of the flat end surface is 0.5 to 2 μm; A gold film formed on the tapered side surface and the flat end surface of the tapered optical fiber needle; an insulating layer formed on an outer surface of the tapered side of the gold film; A graphene layer, the graphene layer being disposed on the gold film surface of the flat end face of the tapered optical fiber needle; the graphene layer being prepared by chemical vapor deposition; 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, wherein: The thickness of the gold film is 40-60 nm; the thickness of the insulating layer is 10-30 nm.

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

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

5. The method for preparing a graphene nano-functional gold electrical probe according to any one of claims 1 to 4, wherein: 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 surface of the cone top of the tapered optical fiber needle to obtain a nanoelectric probe; the nanoelectric probe is then incubated in an L-polylysine solution and washed after the incubation is completed to obtain a nanoelectric probe modified with L-polylysine; the nanoelectric probe modified with L-polylysine is then incubated in an albumin antibody solution to obtain a graphene nanofunctional gold electric probe.

6. The preparation method according to claim 5, wherein 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.

7. The preparation method according to claim 5, wherein 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.

8. Use of the graphene nano-functional gold electrical probe according to any one of claims 1 to 4 or the graphene nano-functional gold electrical probe prepared by the preparation method according to any one of claims 5 to 7, characterized in that: The graphene nanofunctional gold electrical probe is used to detect the concentration of albumin in liver organoids; The detection method comprises the following steps: The graphene nanofunctional gold electroprobe was placed in a liver organoid for incubation. A three-electrode system was then formed using the incubated graphene nanofunctional gold electroprobe as a working electrode, a saturated Ag / AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode. Cyclic voltammetry was then performed in PBS buffer, and the peak current was recorded. The albumin concentration in the liver organoid was determined based on a standard curve between the peak current change value and the albumin concentration.

9. The use according to claim 8, characterized in that The incubation time is 20-40 min, and the incubation temperature is 36-38°C.

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