CN115616058A - Graphene sensor, manufacturing method thereof and real-time in-situ detection method for hepatocyte differentiation - Google Patents
Graphene sensor, manufacturing method thereof and real-time in-situ detection method for hepatocyte differentiation Download PDFInfo
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Abstract
The invention discloses a graphene sensor, a manufacturing method thereof and a real-time in-situ detection method for hepatocyte differentiation, wherein the graphene sensor comprises the following components: the graphene transistor comprises a substrate, wherein a source electrode, a drain electrode and a graphene conducting layer bridging the source electrode and the drain electrode are arranged on the substrate; the sample cell is provided with a first mounting cavity and a second mounting cavity, the sample cell is arranged above the substrate to be matched with the substrate to define a communicating cavity which is communicated with the first mounting cavity and the second mounting cavity, and the second mounting cavity is used for mounting a grid; the cell has the cultivation chamber that is used for holding the thing of waiting to detect and installs in first installation cavity, and at least partly of graphite alkene conducting layer is located the intercommunication chamber and sets up with the second installation cavity is relative, and the cell is equipped with the micropore of intercommunication cultivation chamber and first installation cavity. The graphene sensor provided by the embodiment of the invention can be used for culturing and detecting cells simultaneously, realizes real-time in-situ detection, does not need to be marked, does not change the property of detection molecules, is simple and convenient to operate, has short detection time, and has high sensitivity and stability.
Description
Technical Field
The invention relates to the technical field of biosensing, in particular to a graphene sensor, a manufacturing method of the graphene sensor and a real-time in-situ detection method of hepatocyte differentiation.
Background
The liver organoids can be induced to differentiate into parenchymal liver cells and express albumin, an important biomarker of the parenchymal liver cells. In the related art, albumin concentration monitoring is performed by methods such as fluorescent labeling, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA), which are expensive, time-consuming, low in sensitivity, complicated in steps, and incapable of performing real-time albumin concentration detection.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a graphene sensor for real-time in-situ detection, which can perform cell culture and detection, thereby realizing real-time in-situ detection.
Another object of the present invention is to provide a real-time in-situ detection system having the graphene sensor.
The invention further provides a manufacturing method of the graphene sensor.
The invention also provides a real-time in-situ detection method for hepatocyte differentiation.
The graphene sensor for real-time in-situ detection according to the embodiment of the invention comprises: the graphene transistor comprises a substrate, wherein a source electrode, a drain electrode and a graphene conducting layer bridging the source electrode and the drain electrode are arranged on the substrate; the sample cell is arranged above the substrate to be matched with the substrate to define a communication cavity for communicating the first mounting cavity with the second mounting cavity, and the second mounting cavity is used for mounting a grid; the small chamber is provided with a culture chamber for containing an object to be detected and is arranged in the first installation chamber, at least one part of the graphene conducting layer is located in the communicating chamber and is opposite to the second installation chamber, and the small chamber is provided with micropores communicated with the culture chamber and the first installation chamber.
According to the graphene sensor for real-time in-situ detection, the micropores communicated with the culture cavity and the first installation cavity are formed in the small chamber, so that cells can be cultured and detected at the same time, real-time in-situ detection is realized, the practicability is high, the popularization is easy, the graphene conducting layer is used as a detection probe, no mark is needed, the property of detection molecules is not changed, the operation is simple and convenient, the detection time is short, and the sensitivity and the stability are high.
In addition, the graphene sensor for real-time in-situ detection according to the above embodiment of the invention may further have the following additional technical features:
according to some embodiments of the invention, the graphene conductive layer is an albumin antibody functionalized graphene thin film.
According to some embodiments of the invention, the graphene thin film is single-layer graphene.
According to some embodiments of the invention, the bottom wall of the cell is a permeable membrane and the micropores are formed.
According to some embodiments of the invention, the permeable membrane is a polycarbonate membrane.
According to some embodiments of the invention, the bottom wall of the cell is spaced apart from the base by a predetermined gap.
According to some embodiments of the invention, the pore size of the micropores is between 1 μm and 3 μm.
According to some embodiments of the present invention, the substrate is a glass plate and has a first region and a second region, the first mounting cavity is opposite to the first region, the second mounting cavity is opposite to the second region, and the source electrode, the drain electrode, and the graphene conductive layer are disposed in the second region.
According to some embodiments of the invention, the bottom wall of the sample cell is provided with an upwardly concave groove, and the base covers a notch of the groove to define the communication cavity in cooperation with the sample cell.
According to some embodiments of the invention, the graphene sensor further comprises: the sample cover, sample cover detachable lid is located the sample cell, and be equipped with the second installation cavity is relative dodges the hole.
The real-time in-situ detection system comprises a probe station and a graphene sensor for real-time in-situ detection, wherein the graphene sensor is placed on the probe station.
The manufacturing method of the graphene sensor comprises the following steps: obtaining the substrate, the source electrode, the drain electrode, the grid electrode, the graphene film, the sample cell and the small chamber; attaching the source electrode, the drain electrode and the graphene film on the substrate, so that the graphene film bridges the source electrode and the drain electrode; placing the sample cell on the base to define the communication cavity communicating the first mounting cavity and the second mounting cavity; and performing albumin antibody functionalization treatment on the graphene film to obtain a graphene conducting layer.
According to some embodiments of the invention, the albumin antibody functionalization treatment comprises the steps of: adding an intermediate into the communicating cavity, and incubating for a first predetermined time; sucking out the intermediate, and cleaning the communicating cavity through a buffer solution; adding albumin antibody into the communicating cavity, and incubating for a second preset time; sucking out the albumin antibody, and cleaning the communicating cavity through a buffer solution; adding ethanolamine into the communicating cavity for packaging for third preset time; and sucking out the ethanolamine, and washing the communicating cavity by using a buffer solution.
According to some embodiments of the invention, the concentration of the intermediate is 1mM to 5mM, the concentration of the albumin antibody is 1ug/ml to 2ug/ml, and the concentration of ethanolamine is 10mM to 100mM.
According to some embodiments of the invention, 1-pyrenebutanoic acid N-hydroxysuccinimide ester serves as an intermediate for linking the graphene thin film and albumin antibody in the albumin antibody functionalization treatment.
The real-time in-situ detection method for hepatocyte differentiation provided by the embodiment of the invention adopts the real-time in-situ detection system provided by the embodiment of the invention, and comprises the following steps: obtaining hepatocytes; the mixture of the hepatocytes and the substrate gel is spread on a permeable membrane of the chamber and stabilized by adding a culture medium; installing the small chamber into the first installation cavity, and adding a differentiation solution for culture; placing the graphene sensor on the probe station, accessing a detection circuit, and inserting the grid into the second mounting cavity; and recording an I-V curve of the graphene conducting layer.
According to some embodiments of the invention, the gate has a voltage ranging from-1V to 1V, the step size is 0.08V to 0.1V, and the constant voltage of the source and the drain is 0.1V to 0.5V.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a cross-sectional view of a graphene sensor according to an embodiment of the invention;
fig. 2 is a schematic view of a partial structure of a graphene sensor according to an embodiment of the invention;
FIG. 3 isbase:Sub>A cross-sectional view taken along line A-A of FIG. 2;
FIG. 4 is a schematic diagram of a sample cell according to an embodiment of the present invention;
FIG. 5 is a perspective view of a sample cell according to an embodiment of the present invention;
FIG. 6 is a cross-sectional view of a sample cell according to an embodiment of the invention;
FIG. 7 is a schematic diagram of a cell structure according to an embodiment of the invention;
FIG. 8 is a perspective view of a sample cover according to an embodiment of the present invention;
fig. 9 is a schematic diagram of an albumin antibody functionalization process of a graphene thin film;
FIG. 10 is a graph showing the transmission curves and line relationships of the graphene sensor for detecting albumin at different concentrations according to an embodiment of the present invention, in which albumin is dissolved in 0.1 × PBS;
FIG. 11 is a schematic diagram showing the transmission curves and linear relationship between different albumin concentrations in a cell culture solution and a graphene sensor according to an embodiment of the present invention;
FIG. 12 is a schematic view of a microscope of 3D liver organoid status under different step procedures;
fig. 13 is a schematic diagram of a transmission characteristic curve and a linear relationship of a graphene sensor for real-time in-situ detection of albumin according to an embodiment of the invention.
Reference numerals:
a graphene sensor 100;
a substrate 10;
a source electrode 21; a drain electrode 22; a graphene conductive layer 23; a gate electrode 24;
a sample cell 30; a first mounting cavity 31; a second mounting cavity 32; a recess 33; a communication chamber 34;
a chamber 40; a permeable membrane 41;
a sample cover 50; a relief hole 51;
a culture medium 60.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention and are not to be construed as limiting the present invention.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention.
In the description of the present invention, "a first feature" or "a second feature" may include one or more of the features, and "a plurality" means two or more, and the first feature may be "on" or "under" the second feature, and may include the first and second features being in direct contact, or may include the first and second features being not in direct contact but being in contact with another feature therebetween, and the first feature being "on", "above" and "above" the second feature may include the first feature being directly above and obliquely above the second feature, or merely indicating that the first feature is higher in level than the second feature.
The liver organoid is a hepatobiliary progenitor cell embedded in matrigel in a three-dimensional (3D) culture system with a fixed culture medium, can be self-assembled into a long-term expandable three-dimensional structure, and maintains the key characteristics and genetic stability of the original tissue in the self-renewal process. The liver organoids can be induced to differentiate into parenchymal liver cells and express albumin, an important biomarker of the parenchymal liver cells. The liver organoid and the differentiation technology thereof have wide research potential in the aspects of developmental biology, disease pathology, precise medicine, drug tests and the like. Albumin, a specific protein secreted by hepatocytes, can be used to assess the conversion of non-hepatocyte cells into hepatocyte cells, and is present in the extracellular space, which substantially represents the intracellular space. Therefore, the level of albumin can be measured to help determine the level of hepatic stem cells differentiating into hepatic parenchymal cells. Therefore, a method for detecting microalbumin in real time is urgently needed.
Traditional antibody-antigen based albumin concentration monitoring methods include fluorescence labeling, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA), which are expensive, time-consuming, low in sensitivity, complex in steps, and incapable of real-time detection.
Based on this, the present invention proposes a graphene sensor 100 for real-time in-situ detection, wherein the graphene sensor 100 utilizes high carrier and high mobility (10) of graphene nanomaterial 5 cm 2 V · s), ultra-high body surface ratio (2630 m) 2 Per gram), high transparency (97.7% monolayer) and biocompatibility, graphiteThe alkene sensor 100 has the advantages of high sensitivity and stability. The low dangling bond density zero band gap graphene surface reduces the level of electron scattering and scintillation noise compared to one-dimensional and other two-dimensional nanomaterials. The graphene sensor 100 of the embodiment of the invention has a wide application prospect in the aspects of real-time detection, accuracy and high-flux biomolecule detection.
A graphene sensor 100 for real-time in-situ detection according to an embodiment of the present invention is described below with reference to the accompanying drawings.
Referring to fig. 1 to 8, a graphene sensor 100 for real-time in-situ detection according to an embodiment of the present invention may include: a substrate 10, a sample cell 30 and a chamber 40.
Specifically, the substrate 10 is provided with a source electrode 21, a drain electrode 22, and a graphene conductive layer 23 bridging the source electrode 21 and the drain electrode 22. The sample cell 30 has a first mounting cavity 31 and a second mounting cavity 32, the sample cell 30 is disposed above the substrate 10 (e.g., the sample cell 30 is adhered to the substrate 10) to cooperate with the substrate 10 to define a communication cavity 34, and the communication cavity 34 communicates the first mounting cavity 31 and the second mounting cavity 32. The second mounting cavity 32 is used for mounting the grid 24 (such as an Ag/AgCl reference electrode), and at least a part of the graphene conductive layer 23 is located in the communication cavity 34 and is opposite to the second mounting cavity 32 so as to be matched with the grid 24 for detection.
The chamber 40 has a culture chamber for containing an object to be detected (e.g., a mixture of cells to be detected and the glue of the substrate 10), and the chamber 40 may be installed in the first installation chamber 31. The culture chamber can be used for culturing cells, for example, hepatic cells (all mammals including human and mouse) are differentiated in the culture chamber. The cell 40 is provided with the micropore of intercommunication culture cavity and first installation cavity 31, makes the secretion of cultivateing the intracavity cell can pass through micropore entering intercommunication chamber 34 and second installation cavity 32 to contact with graphite alkene conducting layer 23, the volume of secretion is different then the electric signal response of graphite alkene conducting layer 23 changes differently, thereby realizes the volume detection of secretion, realizes the real-time in situ detection of cell.
In addition, in the embodiment of the invention, the cells do not need to be marked, the performance of detection molecules is not changed, the operation is simple and convenient, the detection time is short, and the sensitivity is high.
In some embodiments, as shown in fig. 2, the substrate 10 may be a glass substrate 10, and the source electrode 21 and the drain electrode 22 may be both Indium Tin Oxide (ITO) conductive films. Two ITO conductive films are arranged on the glass substrate 10 at intervals, the ITO conductive film on one side is used as a source electrode 21, the ITO conductive film on the other side is used as a drain electrode 22, and a part without the ITO conductive film between the two ITO conductive films is used as a sensing channel. The graphene conductive layer 23 spans the sensing channel, i.e., one side of the graphene conductive layer 23 contacts the source electrode 21 and the other side contacts the drain electrode 22.
In some embodiments, as shown in fig. 1 to 3, the substrate 10 is a glass plate and has a first region and a second region, the first mounting cavity 31 is opposite to the first region, the second mounting cavity 32 is opposite to the second region, and the source electrode 21, the drain electrode 22 and the graphene conductive layer 23 are disposed in the second region. The small chamber 40 may be opposite to pure glass when installed in the first installation cavity 31; the gate electrode 24 may be opposite to the graphene conductive layer 23 when mounted into the second mounting cavity 32. Further, as shown in FIG. 1, the lower portion of the first installation chamber 31, the communication chamber 34, and the lower portion of the second installation chamber 32 may be filled with a culture medium 60 so that secretion of cells in the chamber 40 can flow and maintain activity among the first installation chamber 31, the communication chamber 34, and the second installation chamber 32.
In some embodiments, the substrate 10 may have dimensions of 45mm x 30mm x 2mm; the size of the ITO conductive film on two sides is 20mm multiplied by 13mm, the thickness is 185nm, and the resistance of the ITO conductive film is 1.0k omega. The dimensions of the sensing channel are 20mm x 4mm.
In some embodiments, the bottom wall of the chamber 40 may be a permeable membrane 41, and the permeable membrane 41 is formed with micropores, so that the secretion of the cells in the culture chamber can permeate out of the chamber 40 through the permeable membrane 41 to reach the graphene conductive layer 23 and be specifically bonded to the graphene conductive layer 23. For example, the permeable membrane 41 may be a polycarbonate membrane or the like.
In some embodiments, the pores have a pore size of 1 μm to 3 μm. In the aperture range, albumin secreted by the hepatic cells can seep out of the small chamber 40 through the micropores, and then has a specific structure with the graphene conducting layer 23, so that the improvement of hepatic cell differentiation detection is facilitated, and the detection is more accurate.
In some embodiments, as shown in fig. 1, 6 and 7, the first mounting chamber 31 is provided with a support portion, and the cell 40 is provided with a support fitting portion, and the support fitting portion is supported on the support portion so that the bottom wall of the cell 40 is spaced apart from the substrate 10 by a predetermined gap, thereby preventing the substrate 10 from affecting the permeability of the bottom wall of the cell 40.
In some embodiments, as shown in fig. 1, 4-7, the sample cell 30 is made of solid acrylic material (PMMA), the total size is 32mm × 25mm × 25mm, the first installation cavity 31 is a stepped hole, and includes a small hole section with a diameter of 10mm and a height of 15mm and a large hole section with a diameter of 20mm and a height of 10mm, and the step structure at the connection position of the large hole section and the small hole section forms the support portion. The cell 40 has inclined side walls formed to support the mating portion and at the junction of the large and small pore sections to effect a spacing of the cell 40. By having the chamber 40 with sloped side walls, the chamber 40 can also be placed on a well plate to facilitate the previous incubation of the cells.
According to the graphene sensor 100 for real-time in-situ detection provided by the embodiment of the invention, the micropores communicating the culture cavity and the first installation cavity 31 are formed in the small chamber 40, so that cells can be cultured and detected simultaneously, real-time in-situ detection is realized, the practicability is high, the popularization is easy, the graphene conducting layer 23 is used as a detection probe, no mark is needed, the property of detection molecules is not changed, the operation is simple and convenient, the detection time is short, and the sensitivity and the stability are high.
The real-time in-situ detection system comprises a probe station and the graphene sensor 100 for real-time in-situ detection according to the embodiment of the invention, wherein the graphene sensor 100 is placed on the probe station so as to be connected to a detection circuit. Since the graphene sensor 100 for real-time in-situ detection according to the embodiment of the present invention has the above-mentioned beneficial technical effects, according to the real-time in-situ detection system of the embodiment of the present invention, the micropores communicating the culture chamber and the first installation chamber 31 are formed through the small chamber 40, so that the cells can be detected while culturing, and the real-time in-situ detection is realized.
According to some embodiments of the present invention, as shown in fig. 1-3 and 9, the graphene conductive layer 23 is an albumin antibody functionalized graphene thin film. The graphene film may be single-layer graphene. The graphene film is functionalized by the albumin antibody, and the albumin antibody is used as a probe and can be combined with albumin due to specific interaction of the albumin antibody, so that the biological reaction is converted into an electric signal through a detection circuit and transmitted.
According to some embodiments of the present invention, as shown in fig. 1, 5 and 6, the bottom wall of the sample cell 30 is provided with an upwardly concave groove 33, and the base 10 covers the notch of the groove 33 to define a communication chamber 34 in cooperation with the sample cell 30. In other words, the space inside the groove 33 is formed as the communication chamber 34 so that the communication chamber 34 can communicate the first mounting chamber 31 and the second mounting chamber 32 of the sample cell 30, facilitating the circulation of the solution.
According to some embodiments of the present invention, as shown in fig. 1 and 8, the graphene sensor 100 further includes a sample cover 50, wherein the sample cover 50 is detachably disposed on the sample cell 30, and is provided with an avoiding hole 51 opposite to the second mounting cavity 32. When the sample cover 50 is separated from the sample cell 30, the small chamber 40 can be taken and placed, when the sample cover 50 is covered on the sample cell 30, the small chamber 40 and cells in the small chamber 40 can be shielded, and meanwhile, the grid 24 can penetrate through the avoiding hole 51 to extend into the second mounting cavity 32.
A method of fabricating the graphene sensor 100 according to an embodiment of the present invention is described below. The method for manufacturing the graphene sensor 100 may include the steps of:
s01: a substrate 10, a source electrode 21, a drain electrode 22, a gate electrode 24, a graphene thin film, a sample cell 30, and a cell 40 are obtained.
In some embodiments, the substrate 10 may be a glass plate having dimensions of 45mm x 30mm x 2mm; the source electrode 21 and the drain electrode 22 are ITO conductive films having a size of 20mm × 13mm, a thickness of 185nm, and a resistance of 1.0k Ω. Grid 24 is an Ag/AgCl reference electrode. The sample cell 30 may be made of a customized solid acrylic material, the total size is 32mm × 25mm × 25mm, and the first mounting cavity 31 is a stepped hole for placing the small chamber 40, and includes a small hole section with a diameter of 10mm and a height of 15mm and a large hole section with a diameter of 20mm and a height of 10 mm. The side walls of the chamber 40 include an inclined extension to allow the chamber 40 to be placed in the well plate. The bottom wall of the chamber 40 is a permeable membrane 41, the permeable membrane 41 has a plurality of micropores, the pore diameter of the micropores is 1 μm to 3 μm, and the material of the permeable membrane 41 is generally a polycarbonate membrane.
S02: the source electrode 21, the drain electrode 22, and the graphene thin film are attached on the substrate 10 such that the graphene thin film bridges the source electrode 21 and the drain electrode 22.
In some embodiments, the surface of the substrate 10 is divided into two regions, a first region and a second region, the first region is made of pure glass, the second region is provided with an Indium Tin Oxide (ITO) conductive film at two sides, one side is a source electrode 21, the other side is a drain electrode 22, and the portion without the ITO conductive film in the middle is used as a sensing channel.
The preparation of the graphene film is realized by utilizing a chemical vapor deposition method under the conditions of low pressure and high temperature, taking metal copper as a substrate, taking methane as a carbon source and taking hydrogen as etching gas for growth. And if the graphene film is required to be obtained, etching the metal copper substrate. Firstly, cutting graphene/copper into a size of 1.5cm multiplied by 1.5cm, and coating a layer of supporting adhesive on the surface; then placing the graphene/copper coated with the supporting glue on a heating plate for baking, so that the supporting glue is well attached to the surface of the graphene; and after cooling, putting the support glue/graphene/copper into the prepared etching solution to etch the metal copper, and cleaning the support glue/graphene film with deionized water. Then, the supporting glue/graphene is transferred to the cleaned substrate 10 with the ITO conductive film, so that it is erected on the sensing channel, and one side of the substrate is in contact with the source 21 and the other side of the substrate is in contact with the drain 22. After natural airing, the support adhesive/graphene/substrate 10 is placed on a heating plate for baking to ensure that moisture is completely removed and that the support adhesive/graphene is better cured on the surface of the substrate 10, and finally a process of removing the adhesive is performed.
Wherein the thickness of the metal copper is 200um, the supporting glue is polymethyl methacrylate (PMMA) solution, and the etching solution is 1M FeCl 3 The baking temperature of the solution is 140-150 DEG C20-30 min. The degumming solution is acetone solution. The cleaning agent selected for cleaning the substrate 10 is acetone, ethanol and deionized water, the cleaning time is 10-20 min each time, and impurities and organic matters on the surface are removed.
S03: the sample cell 30 is placed on the substrate 10 to define a communication chamber 34 communicating the first mounting chamber 31 and the second mounting chamber 32.
In some embodiments, the sample cell 30 is divided into two parts, one part having a first mounting cavity 31 for placing the chamber 40 for culturing the 3D liver organoids, and the other part having a second mounting cavity 32 for placing the grid 24. The sample cell 30 is adhered to the substrate 10, such that the bottom of the second mounting cavity 32 is the graphene film in step S2, and the bottom of the chamber 40 in the first mounting cavity 31 is a pure glass portion. And the base 10 covers the notch of the groove 33 of the sample cell 30, so that the base 10 and the sample cell 30 are matched to define a communicating cavity 34, and two ends of the communicating cavity 34 are respectively communicated with the lower parts of the first mounting cavity 31 and the second mounting cavity 32, so that the free flow of the solution is facilitated, and the purpose of detecting the albumin can be achieved.
S04: the graphene film is subjected to albumin antibody functionalization treatment to obtain the graphene conductive layer 23.
The graphene conducting layer 23 takes an albumin antibody as a probe and can be specifically combined with albumin secreted by hepatocytes, so that the secretion amount of the albumin is detected, the real-time in-situ detection of hepatocyte differentiation is realized, the graphene conducting layer 23 is not required to be marked, fluorescent dye is not required to be added, the property of the albumin is not changed, the graphene conducting layer 23 is suitable for real-time detection, and the albumin has high sensitivity, selectivity and specificity and good safety performance.
In some embodiments, the albumin antibody functionalization treatment in step S04 may include the following steps:
s041: the intermediate is added to the communicating chamber 34 and incubated for a first predetermined time.
Since graphene has high chemical stability, is not hydrophilic, has weak interaction with other media, and is difficult to dissolve in organic solvents, an intermediate is required to connect the graphene film and the albumin antibody so as to improve the connection stability of the graphene film and the albumin antibody. An I-V curve can be recorded during the incubation process so as to detect the binding degree of the graphene film and the intermediate.
In some embodiments, as shown in FIG. 9, the intermediate may be organic 1-pyrenebutanoic acid N-hydroxysuccinimide ester. In some embodiments, the concentration of the intermediate is 1mM to 5mM. In some embodiments, the first predetermined time may be 1.5 hours.
S042: the intermediate is aspirated and the communication chamber 34 is washed with buffer.
In some embodiments, the buffer is Phosphate Buffered Saline (PBS), pH = 7.2-7.4, and washes 2-3 times.
S043: albumin antibody is added to the communicating lumen 34 and incubated for a second predetermined time.
The albumin antibody can be combined with the intermediate, so that the albumin antibody is combined with the graphene film, and the albumin antibody functionalization of the graphene film is realized. In some embodiments, the second predetermined time may be 12h. An I-V curve can be recorded during incubation to facilitate detection of the extent of binding of the albumin antibody to the intermediate. In some embodiments, the concentration of albumin antibody is between 1ug/ml and 2ug/ml.
S044: the albumin antibody is aspirated and the communication chamber 34 is washed with buffer.
S045: ethanolamine is added to the communicating chamber 34 for encapsulation for a third predetermined time.
The ethanolamine can prevent nonspecific adsorption of albumin antibody, and is beneficial to improving the accuracy of the graphene sensor 100. In some embodiments, the ethanolamine concentration is from 10mM to 100mM. In some embodiments, the third predetermined time may be 1h. The I-V curve can be recorded during the encapsulation process to facilitate detection of the extent of nonspecific adsorption of albumin antibody.
S046: the ethanolamine is aspirated and the communicating chamber 34 is washed with buffer.
Thus, the graphene sensor 100 is completely manufactured and can be stored for later use.
In some embodiments, as shown in fig. 9, 1-pyrenebutanoic acid N-hydroxysuccinimide ester (PBASE) serves as an intermediate for linking the graphene film and the albumin antibody in the albumin antibody functionalization process. Graphene has high chemical stability, is not hydrophilic, has weak interaction with other media, and is difficult to dissolve in organic solvents, so an intermediate is needed for connecting the graphene film and the albumin antibody. Compared with other organic matters, the PBASE can improve the albumin antibody functionalization degree of the graphene film, increase the number of albumin antibody probes on the graphene conducting layer 23, enable the graphene film and the albumin antibody to be combined more stably, and facilitate improvement of the detection stability and the service life of the graphene sensor 100.
The real-time in situ detection method of hepatocyte differentiation according to an embodiment of the present invention is described below. The real-time in-situ detection method is carried out by adopting the real-time in-situ detection system according to the embodiment of the invention, and comprises the following steps:
s11: obtaining the liver cells.
In some embodiments, obtaining hepatocytes comprises the steps of:
s111: adding digestive juice into the liver just extracted for digestion, and adding culture solution for culture after digestion and filtration.
Wherein the digestive juice can be mixture of Collagenase (Collagenase, 0.125 mg/ml) and dispase II (dispaseII, 0.125 mg/ml).
S112: and observing the growth density of the cells, carrying out passage and passing through 2-3 generations.
(1) Preparing crushed ice in advance and opening a centrifugal machine for cooling;
(2) Sucking the culture solution in the culture dish and discarding;
(3) 500ul of substrate medium (Basal medium) was added to each well and rinsed;
(4) Washing and putting into a 15ml centrifuge tube;
(5) Centrifuging: 100-200g,8 ℃,5min;
(6) Taking the supernatant, and then adding a matrix culture medium to 10ml;
(7) And (3) centrifuging again: 200-250g,8 ℃,5min;
(8) Taking clean supernatant, adding substrate 10 glue (Matrigel glue);
(9) The hepatocyte and basal 10 gel mixture was added to a 24-well plate, 50ul per well;
(10) 500ml of culture medium was added to each well.
S12: the mixture of hepatocytes and the glue of the substrate 10 is plated on the permeable membrane 41 of the chamber 40 and then stabilized by adding culture medium.
Specifically, the cell 40 can be stabilized in a 24-well plate for 3 to 4 days. Under the condition of room temperature, the substrate 10 is polymerized to form a three-dimensional matrix with biological activity, the structure, the composition, the physical characteristics and the functions of the in-vivo cell substrate 10 membrane are simulated, the culture and the differentiation of in-vitro cells are facilitated, and the method can be used for researching the cell morphology, the biochemical function, the migration, the infection, the gene expression and the like.
S13: the chamber 40 is mounted in the first mounting chamber 31, and a differentiation medium is added for culture.
S14: the graphene sensor 100 is placed on a probe station, the detection circuit is connected, and the grid 24 is inserted into the second mounting cavity 32.
S15: the I-V curve of the graphene conductive layer 23 was recorded.
The time for recording the I-V curve can be determined according to actual conditions. For example, the 3D liver organoid records an I-V curve once every day of culture, and the I-V curve is recorded every time without operations such as marking, and the like, and the detection can be directly carried out through a detection circuit, so that the detection while the culture is carried out, and the operation is simple. As the amount of albumin secreted increases, the response of the graphene sensor 100 to the electric signal changes differently. In addition, the differentiation solution can be changed every day of culture to ensure normal differentiation.
In some embodiments, the gate 24 voltage ranges from-1V to 1V, the step size is 0.08V to 0.1V, and the constant voltage of the source 21 and drain 22 is 0.1V to 0.5V.
The graphene sensor 100 according to one embodiment of the present invention is described in detail below with reference to the accompanying drawings, and it is to be understood that the following description is only exemplary and should not be construed as limiting the invention.
As shown in fig. 1 to 9, the graphene sensor 100 includes a glass substrate 10, a source electrode 21, a drain electrode 22, a gate electrode 24, a sample cell 30, a graphene conductive layer 23 obtained by albumin antibody functionalization, and a sample cover 50.
Fig. 10 is a schematic diagram showing the transmission curves and linear relationship of the graphene sensor 100 for detecting albumin with different concentrations, in which albumin is dissolved in 0.1 × PBS buffer. As shown in fig. 10 (a), the purchased albumin was diluted in PBS (pH = 7.4) to different concentrations (0-100 ng/ml), and the transfer characteristic curve of the graphene sensor 100 was gradually shifted to the right with the injection of albumin at different concentrations, because the albumin antibody undergoes a conformational change after being exposed to the albumin solution, resulting in a change in the carrier concentration of graphene per unit area of monoatomic thickness. Since 0.1 × PBS solution is electrically neutral, albumin has an isoelectric point of 5.8, less than 7.4, and is considered to be negatively charged, and thus, the graphene surface holes increase, and the curve shifts to the right. FIG. 10 (b) shows Δ V of the transfer curve cnp (i.e., minimum point voltage V for albumin antibody) cnp Relative to V after albumin is added at different concentrations cnp Offset of) and albumin concentration, linear correlation coefficient R 2 ＝0.988，ΔV cnp There is a good linear relationship with the logarithmic concentration of albumin.
Fig. 11 is a schematic diagram showing a transmission curve and a linear relationship of the graphene sensor 100 for detecting albumin with different concentrations according to the embodiment of the present invention when albumin is dissolved in a cell culture solution. As shown in fig. 11 (a), the real environment is simulated, the purchased albumin is dissolved in the cell culture fluid and diluted to different concentrations (0-100 ng/ml), and the transmission characteristic curve of the graphene sensor 100 is gradually shifted to the right along with the injection of the albumin with different concentrations, which is consistent with the result of fig. 10 (a). The cell culture fluid contains a plurality of cytokines suitable for 3D liver organoid growth, and the components are more complex than that of a pure PBS solution, which shows that the selectivity and the specificity of the graphene sensor 100 in the invention are higher, and the minimum detection limit in PBS or the cell culture fluid is 1pg/ml, which is improved by several orders of magnitude than that in the related art, so that the cell culture fluid is suitable for the growth of 3D liver organoidsAnd shows that the sensitivity of the graphene sensor 100 is high. And as shown in FIG. 11 (b), Δ V of the transfer curve in the complex composition of the culture solution cnp Has a better linear relation with the logarithmic concentration of the albumin, and the linear correlation coefficient is R 2 ＝0.980。
FIG. 12 is a schematic view of a microscope showing the status of 3D liver organoids at different steps. As shown in fig. 12 (a), the liver is just extracted and digested, and bile duct cells are in the middle and surrounded by cell debris; as shown in FIG. 12 (b), the cells were cultured for 5 days, the number of cells increased, the volume increased, the cell density was observed, and when the cells proliferated to some extent, they were passaged 2 to 3 times, and then differentiation was performed by adding a differentiation medium; as shown in fig. 12 (c), the cells were differentiated for 7 days, which is different from the cells before differentiation.
Fig. 13 is a schematic diagram showing a transmission characteristic curve and a linear relationship of real-time in-situ albumin detection when the graphene sensor 100 according to the embodiment of the invention is applied to practical applications. As shown in FIG. 13 (a, b), the transfer curve and the linear relationship chart obtained by culturing 3D liver organoids on a 24-well plate, collecting differentiation medium once a day from the start of addition of differentiation medium for 0 to 6 days in total, and detecting with the graphene sensor 100, show that the amount of albumin gradually increases from day 2, and the linear relationship is good, and the linear coefficient is R 2 1 =0.99262. As shown in FIG. 13 (c, D), a chamber 40 is directly placed in a sample cell 30 for culturing a 3D liver organoid, albumin secreted by the organoid directly permeates into the bottom through the micropores of a permeable membrane 41 at the bottom of the chamber 40, and the albumin antibody reaching the surface of the graphene conductive layer 23 is specifically bound with the graphene conductive layer 23 along with the circulation of the solution, the detection while culturing is improved by several orders of magnitude compared with the non-in-situ detection, as can be seen from FIG. 13 (c, D), the result detected by the graphene sensor 100 is that the albumin secretion amount is increased from day 1 until day 6 reaches a stationary phase, and the linear correlation coefficient is R 2 2 =0.99215, indicating that the organoid has matured and is almost no longer secreting albumin. Thus, the graphene sensor 100 can be used for real-time in-situ detection and transmissionThe sensitive area can be incubated in the solution for at least 10 days, which proves that the graphene sensor 100 has good stability and can be popularized and applied.
Other configurations and operations of the graphene sensor 100 according to embodiments of the present invention are known to those of ordinary skill in the art and will not be described in detail herein.
In the description herein, references to the description of "an embodiment," "a specific embodiment," "an example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (17)
1. A graphene sensor for real-time in-situ detection, comprising:
the graphene transistor comprises a substrate, wherein a source electrode, a drain electrode and a graphene conducting layer bridging the source electrode and the drain electrode are arranged on the substrate;
the sample cell is provided with a first mounting cavity and a second mounting cavity, the sample cell is arranged above the substrate to be matched with the substrate to define a communicating cavity communicating the first mounting cavity with the second mounting cavity, and the second mounting cavity is used for mounting a grid;
a chamber having a culture chamber for containing an object to be detected and mounted to the first mounting chamber, wherein,
at least one part of the graphene conducting layer is located in the communicating cavity and is arranged opposite to the second installation cavity, and the small chamber is provided with a micropore for communicating the culture cavity with the first installation cavity.
2. The graphene sensor for real-time in-situ detection according to claim 1, wherein the graphene conducting layer is an albumin antibody functionalized graphene thin film.
3. The graphene sensor for real-time in-situ detection according to claim 2, wherein the graphene thin film is single-layer graphene.
4. The graphene sensor for real-time in-situ detection according to claim 1, wherein the bottom wall of the small chamber is a permeable membrane and the micropores are formed.
5. The graphene sensor for real-time in-situ detection according to claim 4, wherein the permeable membrane is a polycarbonate membrane.
6. The graphene sensor for real-time in-situ detection according to claim 4, wherein a bottom wall of the cell is spaced apart from the substrate by a predetermined gap.
7. The graphene sensor for real-time in-situ detection according to claim 1, wherein the pore size of the micropores is 1 μm to 3 μm.
8. The graphene sensor according to claim 1, wherein the substrate is a glass plate and has a first region and a second region, the first mounting cavity is opposite to the first region, the second mounting cavity is opposite to the second region, and the source electrode, the drain electrode and the graphene conductive layer are disposed in the second region.
9. The graphene sensor for real-time in-situ detection according to claim 1, wherein the bottom wall of the sample cell is provided with an upward concave groove, and the base covers a notch of the groove to define the communication cavity in cooperation with the sample cell.
10. The graphene sensor for real-time in-situ detection according to any one of claims 1-9, further comprising:
the sample cover, sample cover detachable lid is located the sample cell, and be equipped with the second installation cavity is relative dodges the hole.
11. A real-time in-situ detection system, comprising a probe station and a graphene sensor for real-time in-situ detection according to any one of claims 1 to 10, wherein the graphene sensor is placed on the probe station.
12. A method for fabricating a graphene sensor according to any one of claims 1-10, comprising the steps of:
obtaining the substrate, the source electrode, the drain electrode, the grid electrode, the graphene film, the sample cell and the small chamber;
attaching the source electrode, the drain electrode and the graphene film on the substrate so that the graphene film bridges the source electrode and the drain electrode;
placing the sample cell on the base to define the communication cavity communicating the first mounting cavity and the second mounting cavity;
and performing albumin antibody functionalization treatment on the graphene film to obtain a graphene conducting layer.
13. The method for manufacturing the graphene sensor according to claim 12, wherein the albumin antibody functionalization treatment includes the steps of:
adding an intermediate into the communicating cavity, and incubating for a first predetermined time;
sucking out the intermediate, and cleaning the communicating cavity by buffer solution;
adding albumin antibody into the communicating cavity, and incubating for a second preset time;
sucking out the albumin antibody, and cleaning the communicating cavity through a buffer solution;
adding ethanolamine into the communicating cavity for packaging for third preset time;
and sucking out the ethanolamine, and washing the communicating cavity by using a buffer solution.
14. The method of manufacturing a graphene sensor according to claim 13, wherein the concentration of the intermediate is 1mM to 5mM, the concentration of the albumin antibody is 1ug/ml to 2ug/ml, and the concentration of ethanolamine is 10mM to 100mM.
15. The method of manufacturing a graphene sensor according to claim 12, wherein 1-pyrenebutanoic acid N-hydroxysuccinimide ester is used as an intermediate for connecting the graphene thin film and the albumin antibody in the albumin antibody functionalization treatment.
16. A real-time in-situ detection method for hepatocyte differentiation, characterized in that the real-time in-situ detection system according to claim 11 is adopted and comprises the following steps:
obtaining hepatocytes;
the mixture of the hepatocytes and the substrate gel is spread on a permeable membrane of the chamber and stabilized by adding a culture medium;
mounting the small chamber into the first mounting cavity, and adding a differentiation solution for culture;
placing the graphene sensor on the probe station, accessing a detection circuit, and inserting the grid into the second mounting cavity;
and recording an I-V curve of the graphene conducting layer.
17. The method of claim 16, wherein the gate voltage ranges from-1V to 1V, the step size ranges from 0.08V to 0.1V, and the constant voltage of the source and the drain is 0.1V to 0.5V.
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