Electrochemical sensor and method of manufacturing the same

Abstract

This invention relates to an electrochemical sensor and its fabrication method. The electrochemical sensor comprises a modification layer, an electrode layer, and an encapsulation layer sequentially formed on a cell culture chip via 3D printing. The electrochemical sensor and its fabrication method of this invention are manufactured directly on the cell culture chip using 3D printing technology, achieving integration with the cell culture chip and thus enabling real-time monitoring of changes in the cell culture medium environment. The fabrication can be achieved using only a 3D printer, making the process simple and low-cost.

Description

Technical Field

[0001] This invention relates to the field of biochemical sensors, and more specifically to an electrochemical sensor and its preparation method. Background Technology

[0002] In modern science, especially medical research, laboratory animals have traditionally been treated merely as experimental tools or methods. Humans have often viewed them simply as living reagents or teaching aids, and have generally been indifferent to their suffering and death. However, with societal progress and the rapid development of laboratory animal science in my country, this outdated attitude towards laboratory animals is gradually gaining attention, and more and more people are recognizing the importance of improving their welfare. Animal experiments in biomedical science involve the sacrifice of small animals to obtain medical principles and scientific knowledge; therefore, the lives of laboratory animals should not be treated as resources to be used at will. In the process of using laboratory animals, unnecessary harm, fright, and torture should be avoided, and alternative solutions should be actively sought to reduce their use and killing.

[0003] However, laboratory animals, as a crucial foundation and supporting condition for life and medical science research, currently play an irreplaceable role in the field of biomedical research. Almost all research, teaching, and safety evaluation activities in the pharmaceutical field rely on laboratory animals. Therefore, while minimizing the harm and use of laboratory animals, biomedical research will inevitably be significantly impacted. If pharmaceutical research is affected, it means that a large number of patients will not receive new drug support, will continue to suffer from illness, and may even lose their lives. Therefore, there is an urgent need to establish a new standardized drug testing platform that can highly simulate human organs and internal environment to partially replace related animal experiments. To promote the standardization, normalization, and modernization of drug testing, it is necessary to develop new drug testing technologies based on data mining and humanized model activity experiments.

[0004] Organoids are 3D cell cultures (organoid models) derived from stem cells in vitro. They possess histological characteristics and physiological functions highly similar to their source organs (tissues), making them excellent preclinical disease models. The emergence of organoid technology can reduce the use of laboratory animals, and its results also compensate for the shortcomings of species differences between laboratory animals and humans, providing ideas for building novel drug experimental platforms. Currently, the biochemical and physiological state measurements of organoid-on-a-chip systems in drug trials often rely on complex and bulky automated peripheral detection equipment. This limits the flexibility of organoid-on-a-chip systems and their true widespread adoption in the field of drug trials. The challenge for organoid-on-a-chip systems to truly be applied to practical drug research lies in achieving fully miniaturized integration of organoid-on-a-chip systems with real-time sensing systems such as biochemical analysis and biophysical monitoring, thus eliminating reliance on current bulky and complex large-scale scientific instruments. Summary of the Invention

[0005] The purpose of this invention is to provide an electrochemical sensor and its preparation method, which can be directly 3D printed on a cell culture chip, is easy to integrate with the cell culture chip, and the device is not only small in size but also flexible and variable in form, enabling metabolic monitoring in on-chip organoids and cell culture processes.

[0006] To achieve the above objectives, the present invention provides an electrochemical sensor comprising a modification layer, an electrode layer, and an encapsulation layer sequentially formed on a cell culture chip by 3D printing.

[0007] Optionally, the modification layer is made of polyaniline to enable pH detection.

[0008] Optionally, the electrode layer includes a working electrode, a reference electrode, and a counter electrode, wherein the working electrode, the reference electrode, and the counter electrode are disposed independently and spaced apart on the modification layer.

[0009] Optionally, the working electrode includes a working electrode body and a working electrode detection end, the reference electrode includes a reference electrode body and a reference electrode effective contact end, the counter electrode includes a counter electrode body and a counter electrode effective contact end, the encapsulation layer covers the working electrode body, the reference electrode body, and the counter electrode body, and the working electrode detection end, the reference electrode effective contact end, and the counter electrode effective contact end are exposed outside the encapsulation layer.

[0010] Optionally, the working electrode body, the reference electrode body, and the counter electrode body are all made of silver, the working electrode detection end and the counter electrode effective contact end are both made of carbon, and the reference electrode effective contact end is made of silver and silver chloride.

[0011] Optionally, the encapsulation layer is made of an insulating material.

[0012] Another aspect of the present invention provides a method for preparing an electrochemical sensor, which includes the following steps:

[0013] S10: Provides a cell culture chip;

[0014] S20: Using a 3D printer, a modification layer, an electrode layer, and an insulating encapsulation layer are printed layer by layer on the cell culture chip.

[0015] Optionally, the electrode layer includes a working electrode, a reference electrode, and a counter electrode. The working electrode includes a working electrode body and a working electrode detection end. The reference electrode includes a reference electrode body and a reference electrode effective contact end. The counter electrode includes a counter electrode body and a counter electrode effective contact end. Step S20 specifically includes:

[0016] The 3D printer is used to print the modification layer on the cell culture chip;

[0017] The working electrode body, the reference electrode body, and the counter electrode body are printed on the modification layer using the 3D printer.

[0018] The working electrode detection end and the counter electrode effective contact end are printed on the modification layer using the 3D printer.

[0019] The effective contact end of the reference electrode is printed on the modification layer using the 3D printer described above;

[0020] The 3D printer is used to print the encapsulation layer on the modification layer, the encapsulation layer covering the working electrode body, the reference electrode body and the counter electrode body.

[0021] The electrochemical sensor and its preparation method of the present invention are manufactured directly on a cell culture chip using 3D printing technology, achieving integration with the cell culture chip, thereby enabling real-time monitoring of changes in the cell culture medium environment; only one 3D printer is required for preparation, the process is simple and low cost. Attached Figure Description

[0022] Figure 1 A cross-sectional view of an electrochemical sensor according to an embodiment of the present invention;

[0023] Figure 2 This is a schematic diagram showing the test results after performing a pH detection performance test on the electrochemical sensor of an exemplary embodiment of the present invention;

[0024] Figure 3A Bright-field micrographs of meningioma cells according to an exemplary embodiment of the present invention;

[0025] Figure 3B A diagram showing the live / dead staining results of meningioma cells according to an exemplary embodiment of the present invention;

[0026] Figure 4 This is a schematic diagram showing the change of detection voltage over time in the drug-treated group and the untreated group according to an exemplary embodiment of the present invention.

[0027] Figure 5 This is a flowchart of a method for preparing an electrochemical sensor according to an embodiment of the present invention;

[0028] Figure 6 This is a flowchart illustrating step S20 of the method for preparing an electrochemical sensor according to an embodiment of the present invention. Detailed Implementation

[0029] The preferred embodiments of the present invention are given below with reference to the accompanying drawings and described in detail.

[0030] like Figure 1 As shown, this embodiment of the invention provides an electrochemical sensor, which includes a modification layer 200, an electrode layer 300, and an encapsulation layer 400 sequentially formed on a cell culture chip 100 by 3D printing. The modification layer 200 can be selected with suitable modification materials as needed to achieve specific detection of specific indicators, including but not limited to one or more of pH value, ion concentration, metabolite concentration, and dissolved gas content. For example, the material of the modification layer 200 for pH value detection can be polyamide or other conductive polymers with pH-responsive properties, while the modification layer material for other indicator detection is a specific recognition / response material layer for the corresponding detection indicator. Since the electrochemical sensor is directly printed on the cell culture chip 100 using 3D printing technology, it is easy to integrate with the cell culture chip 100 to achieve metabolic monitoring during cell culture. Furthermore, it does not require a cleanroom; only a 3D printer is needed for fabrication, resulting in a simple process and low cost.

[0031] The electrode layer 300 includes a working electrode, a reference electrode, and a counter electrode, which are independently and spaced apart on the modification layer 200. The working electrode is the core detection electrode of the sensor, serving as the central site for the conversion between biochemical indicators and electrical signals. During cell culture monitoring, the detection end of the working electrode is in direct contact with the culture medium. The modification layer undergoes a specific response / electrochemical reaction with the target indicator, causing an electrical signal change on the working electrode surface related to the concentration / content of the target indicator, thus realizing the conversion of biochemical signals into quantifiable electrical signals. The reference electrode is the potential calibration electrode of the sensor. Its core function is to provide a stable, known, and unchanging reference potential, providing a precise calibration benchmark for the detection potential of the working electrode. In chemical detection, the electrical signal potential generated by the working electrode is affected by environmental factors such as culture medium temperature, ionic strength, and stirring state. Reading the working electrode potential alone cannot achieve quantitative detection. The reference electrode can serve as a potential reference. By detecting the potential difference between the working electrode and the reference electrode, the interference of environmental factors on the detection results can be eliminated, and the accurate quantification of the electrical signal of the target indicator can be achieved. The counter electrode (also known as the auxiliary electrode) is an auxiliary electrode for the electrochemical reaction of the sensor. Its core function is to form a current loop with the working electrode, providing an electron conduction channel for the electrochemical reaction on the surface of the working electrode, ensuring the smooth and full progress of the electrochemical reaction, and preventing polarization of the working electrode to avoid detection signal drift and distortion caused by polarization.

[0032] The working electrode includes a working electrode body and a working electrode detection end; the reference electrode includes a reference electrode body and a reference electrode effective contact end; and the counter electrode includes a counter electrode body and a counter electrode effective contact end. The working electrode body, the reference electrode body, and the counter electrode body are all made of silver. The working electrode detection end and the counter electrode effective contact end are both made of carbon, and the reference electrode effective contact end is made of silver and silver chloride.

[0033] The encapsulation layer 400 is made of insulating material and only covers the working electrode body, the reference electrode body, and the counter electrode body. The working electrode detection end, the effective contact end of the reference electrode, and the effective contact end of the counter electrode are exposed outside the encapsulation layer 400.

[0034] When the modification layer 200 is polyaniline, the electrochemical sensor exhibits excellent pH detection performance and is resistant to alcohol disinfection, making it ideal for pH monitoring in cell culture media. By monitoring changes in pH, cellular metabolic activities can be monitored. For example, the electrochemical sensor can be used to test related anticancer drugs. If the anticancer drug is effective, it can kill some or all cancer cells, slowing down or even stopping cell metabolism. This results in the culture environment no longer producing acidic substances due to cancer cell metabolism, and the pH change will be slower. Conversely, if the anticancer drug is ineffective, the cancer cells continue to metabolize vigorously, producing a large amount of acidic substances, and the pH in the culture environment will continue to decrease, without slowing down the rate of change. In other words, the effectiveness of anticancer drugs can be determined by detecting changes in pH.

[0035] This invention also tested the pH detection performance of the electrochemical sensor. During the test, seven standard pH test solutions with pH values ​​of 10, 9, 8, 7, 6, 5, and 4 were sequentially added to the electrochemical sensor. The test results are as follows: Figure 2 As shown, from Figure 2 It can be seen that during the process of continuously changing the test solution from pH 10 to 4 and then from 4 to 10, the voltage change of the electrochemical sensor is very uniform for each pH change of 1, that is, the sensor has linearity. After the sensor gradually measures from a solution with pH 10 to a solution with pH 4, and then gradually measures from a solution with pH 4 to 10, the curves on both sides are symmetrical, indicating that the device drift is very small. Throughout the entire test process, when retesting solutions with the same pH value, the signal is the same, which shows repeatability, proving that the sensor is stable enough during use.

[0036] like Figure 3A Bright-field micrographs of meningioma cells as an exemplary embodiment. Figure 3B This is an image showing the live / dead staining results of meningioma cells. Figure 3B It can be seen that the cells survived well during the culture process. Figure 3A and Figure 3B The meningioma cells shown were treated with a drug, and the pH of the cell culture medium was measured after treatment. A control group without drug treatment was also provided. Compared with the control group, the drug-treated group was treated with 10 μM cycloacetilimide. The pH of the culture medium was measured at 0, 2, 8, 20, and 24 hours after the addition of cycloacetilimide. The curves of the detection voltage change over time for the drug-treated and untreated groups are shown in the figure. Figure 4 As shown; from Figure 4It can be seen that the voltage drop rate of the drug-treated group is significantly lower than that of the untreated group, that is, the pH drop rate of the drug-treated group is significantly lower than that of the untreated group. This indicates that the drug has the ability to inhibit and kill meningioma cells, and that the electrochemical sensor of the present invention can observe the pH change of the culture medium in real time.

[0037] The electrochemical sensor of this invention is manufactured directly on a cell culture chip using 3D printing technology, achieving integration with the cell culture chip and thus enabling real-time monitoring of changes in the cell culture medium environment; it can be fabricated using only one 3D printer, with a simple process and low cost.

[0038] like Figure 5 As shown in the figure, this embodiment of the invention also provides a method for preparing an electrochemical sensor, which includes the following steps:

[0039] S10: Provides 100 cell culture chips;

[0040] S20: Using a 3D printer 500, a modification layer 200, an electrode layer 300, and an insulating encapsulation layer 400 are printed layer by layer on the cell culture chip 100.

[0041] like Figure 6 As shown, step S20 specifically includes the following steps:

[0042] S21: Use 3D printer 500 to print a modification layer 200 on cell culture chip 100;

[0043] S22: Using a 3D printer 500, the working electrode body 311, the reference electrode body 331, and the counter electrode body 321 are printed on the modification layer 200;

[0044] S23: Using a 3D printer 500, print the working electrode detection end 312 and the counter electrode effective contact end 322 on the modification layer 200;

[0045] S24: Using a 3D printer 500, print the effective contact end 332 of the reference electrode on the modification layer 200;

[0046] S25: Using a 3D printer 500, an encapsulation layer 400 is printed on the modification layer 200. The encapsulation layer covers the working electrode body 311, the reference electrode body 331, and the counter electrode body 321.

[0047] 3D printers can be controlled by control devices to achieve automated printing, thereby enabling the automated fabrication of electrochemical sensors.

[0048] The method for fabricating the electrochemical sensor in this invention involves directly manufacturing it on a cell culture chip using 3D printing technology, thereby achieving integration with the cell culture chip and enabling real-time monitoring of changes in the cell culture medium environment. The fabrication can be accomplished with just one 3D printer, making the process simple and low-cost.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. That is, all simple and equivalent changes and modifications made based on the claims and description of this invention fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. An electrochemical sensor, characterized in that, This includes a modification layer, an electrode layer, and an encapsulation layer, which are sequentially formed on a cell culture chip via 3D printing.

2. The electrochemical sensor according to claim 1, characterized in that, The modified layer is made of polyaniline to enable pH detection.

3. The electrochemical sensor according to claim 1, characterized in that, The electrode layer includes a working electrode, a reference electrode, and a counter electrode, which are independently disposed on the modification layer at intervals.

4. The electrochemical sensor according to claim 3, characterized in that, The working electrode includes a working electrode body and a working electrode detection end; the reference electrode includes a reference electrode body and a reference electrode effective contact end; the counter electrode includes a counter electrode body and a counter electrode effective contact end; the encapsulation layer covers the working electrode body, the reference electrode body, and the counter electrode body; the working electrode detection end, the reference electrode effective contact end, and the counter electrode effective contact end are exposed outside the encapsulation layer.

5. The electrochemical sensor according to claim 4, characterized in that, The working electrode body, the reference electrode body, and the counter electrode body are all made of silver. The detection end of the working electrode and the effective contact end of the counter electrode are both made of carbon. The effective contact end of the reference electrode is made of silver and silver chloride.

6. The electrochemical sensor according to claim 1, characterized in that, The encapsulation layer is made of insulating material.

7. A method for preparing an electrochemical sensor, characterized in that, Includes the following steps: S10: Provides a cell culture chip; S20: Using a 3D printer, a modification layer, an electrode layer, and an insulating encapsulation layer are printed layer by layer on the cell culture chip.

8. The method for preparing the electrochemical sensor according to claim 7, characterized in that, The electrode layer includes a working electrode, a reference electrode, and a counter electrode. The working electrode includes a working electrode body and a working electrode detection end. The reference electrode includes a reference electrode body and a reference electrode effective contact end. The counter electrode includes a counter electrode body and a counter electrode effective contact end. Step S20 specifically includes: The 3D printer is used to print the modification layer on the cell culture chip; The working electrode body, the reference electrode body, and the counter electrode body are printed on the modification layer using the 3D printer. The working electrode detection end and the counter electrode effective contact end are printed on the modification layer using the 3D printer. The effective contact end of the reference electrode is printed on the modification layer using the 3D printer described above; The 3D printer is used to print the encapsulation layer on the modification layer, the encapsulation layer covering the working electrode body, the reference electrode body and the counter electrode body.

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