Micro-fluidic chip system and chip manufacturing method thereof

Abstract

The invention provides a micro-fluidic chip system and a chip manufacturing method thereof. The micro-fluidic chip system comprises a cell culture chip, a DNA logic gate calculation chip and an electrochemical sensing chip. The cell culture chip is used for culturing cells, the DNA logic gate calculation chip is used for calculating a DNA molecular signal, the electrochemical sensing chip is used for converting the DNA molecular signal into a corresponding current signal, and visual output of a logic relation is carried out. According to the application, the intercellular interaction and communication mode which is difficult to directly observe originally can be converted into quantifiable and measurable current signals through DNA logic calculation; the logic relation between the cells is dynamically monitored in real time; and high sensitivity and high specificity can accurately detect the specific cell signal molecule logic relationship.

Description

Technical Field

[0001] The present application relates to the field of microfluidic chip technology, and in particular to a microfluidic chip system and a chip manufacturing method thereof. Background Art

[0002] Cancer is one of the greatest threats to human health and the disease with the highest mortality rate. Currently, most cancer treatments and therapies rely on single targets, resulting in unsatisfactory clinical outcomes. This is due to the intercellular interactions between tumor cells and surrounding cells, as well as signal transduction processes involving logical relationships between multiple biomolecules such as DNA, RNA, and proteins, during tumor development and progression. Therefore, deciphering the complex relationships between tumor cells and surrounding stromal and immune cells is crucial. DNA logic gates, a novel bioinformatics tool, are biocomputing systems that take DNA, RNA, nucleases, or aptamers as input and, after a series of biochemical operations such as DNA strand displacement reactions, produce single-stranded nucleic acid molecules as output. Through DNA sequence design, logic gates can perform information processing tasks involving Boolean logic such as "AND" and "OR." Therefore, compared to traditional signal processing methods, DNA logic gates demonstrate advantages in logic analysis for physiological and pathological applications such as miRNA detection, cancer diagnosis, and tumor typing, including more accurate analysis results. They also hold promise for deciphering the logical relationships underlying gene regulation during signal transduction and intercellular interactions. However, current signal readout methods for DNA logic gates still have limitations such as high cost, low throughput, and difficulty in integration with external sensors.

[0003] Electrochemical biosensors have attracted widespread attention in clinical diagnosis and disease monitoring applications due to their advantages such as high throughput, low cost, high detection sensitivity, and ease of integration. On the one hand, electrochemical sensors can be designed with electrode structures containing multiple parallel channels, which is conducive to multi-channel detection to improve detection efficiency. On the other hand, on specially modified electrodes, the signals generated by the pre-modified capture probes recognizing the target analyte can be amplified through enzymatic reactions, thereby greatly improving detection sensitivity. DNA logic computing based on electrochemical sensors integrates the output signals of DNA computing with electrochemical signal recognition modules, which facilitates the direct conversion of DNA computing results into visual signals such as current and voltage and in situ signal amplification. However, how to use cell-derived nucleic acid molecules for DNA logic computing and integrate them into multi-channel sensors remains a difficult problem in current research.

[0004] Microfluidic chips manipulate fluids in micron-scale channels through preprogrammed fluid control units, allowing multi-step liquid handling processes to be integrated into a single chip for automated, integrated sample processing and analysis. Microfluidic chips allow for customized input and output ports, making it easy to integrate electrochemical signal reading units. Currently, a variety of systems combining electrochemical biosensors with microfluidic chips have been developed and are widely used to detect disease-related biomarkers such as DNA and miRNA. Due to the excellent sealing and connectivity of microfluidic chips, microfluidic-based DNA logic computing achieves the integration and automation of signal reading and calculation processes, reducing errors caused by manual operation and sample exposure to the external environment. Furthermore, microfluidic chips have the advantages of being able to construct extracellular matrices (ECM) in vitro and provide continuous nutrient perfusion, thereby creating a suitable environment for cell growth. By integrating microfluidic chips and electrochemical sensors, cell-derived biomarkers can be directly collected, detected, and analyzed. However, current research still relies on detecting discrete single or multiple biological targets, and there is no microfluidic chip system that integrates on-chip cell culture, DNA logic computing, and electrochemical sensors for interpreting logical relationships between cells. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of this application is to provide a microfluidic chip system and a chip manufacturing method thereof, so as to solve the technical problem that there is currently no system for visualizing the logical relationship between cells.

[0006] To achieve the above-mentioned objectives and other related objectives, the first aspect of the present application provides a microfluidic chip system, comprising: a first cell culture chip, a second cell culture chip, a DNA logic computing chip and an electrochemical sensor chip; the first cell culture chip is connected to the second cell culture chip via a liquid circulation tube; the second cell culture chip is connected to the DNA logic computing chip via a liquid circulation tube, and the DNA logic computing chip is connected to the first cell culture chip and the electrochemical sensor chip via a liquid circulation tube; wherein, two types of cells grown for different days on the first cell culture chip and the second cell culture chip are stained for live and dead, and the target cell signal molecules secreted by the cells are amplified and transmitted to the DNA logic computing chip; the DNA logic computing chip calculates DNA molecular signals; and the electrochemical sensor chip converts the calculated DNA molecular signals into current signals and performs a visual output of the logical relationship.

[0007] In some embodiments of the first aspect of the present application, the two cells grown on the first cell culture chip and the second cell culture chip for different days are stained live and dead, which includes: using calcein acetoxymethyl ester and iodide to perform live and dead staining on the two cells grown on the first cell culture chip and the second cell culture chip for 1 day and 3 days.

[0008] In some embodiments of the first aspect of the present application, the target cell signaling molecule secreted by the cell is amplified, including: using an amplification reagent to amplify the target miRNA signaling molecule secreted by the cell.

[0009] In some embodiments of the first aspect of the present application, the DNA logic computing chip calculates DNA molecular signals; the electrochemical sensor chip converts the calculated DNA molecular signals into current signals and performs a visual output of the logical relationship, including any one of the following: using an OR gate DNA logic gate and an electrochemical sensor chip to detect target miRNA signal molecules of different concentrations; using an AND gate DNA logic gate and an electrochemical sensor chip to detect target protein signal molecules of different concentrations.

[0010] In some embodiments of the first aspect of the present application, the use of an OR gate DNA logic gate and an electrochemical sensor chip to detect target cell signaling molecules of different concentrations includes: preparing an OR gate DNA logic gate, adding target miRNA signaling molecules of different concentrations and other DNA chains that undergo DNA reactions, and after sufficient mixing, using an electrochemical sensor chip to detect the output results. The detection result shows that the concentration value of the target miRNA signaling molecule is linearly related to the generated current value.

[0011] In some embodiments of the first aspect of the present application, the use of an AND gate DNA logic gate and an electrochemical sensor chip to detect target protein signal molecules of different concentrations includes: preparing an AND gate DNA logic gate, adding target protein signal molecules, aptamers and other reacting DNA chains of different concentrations, mixing them thoroughly, and then using an electrochemical sensor chip to detect the output results. The detection result shows that the target protein concentration is linearly related to the current signal.

[0012] To achieve the above-mentioned objectives and other related objectives, the second aspect of the present application provides a method for manufacturing an electrochemical sensor chip, wherein the manufactured chip is used as the electrochemical sensor chip in the microfluidic chip system; the manufacturing method comprises: adding a tetrachloroauric acid solution to a screen-printed carbon electrode, reducing the tetrachloroauric acid solution to gold on the electrode by electrodeposition to form a nano-gold layer; utilizing gold-sulfur bonds to modify a DNA tetrahedron structure on the screen-printed carbon electrode to form a capture probe, so that the DNA tetrahedron is fixed on the electrode; placing a polydimethylsiloxane cover sheet having an S-shaped channel structure and the DNA tetrahedron-modified electrode together in a plasma machine for treatment; covering the electrode portion with the DNA tetrahedron with a cap, and laminating the treated polydimethylsiloxane cover sheet and the electrode to irreversibly encapsulate the two to form a chip comprising a microfluidic channel and a DNA tetrahedron-modified electrode; injecting the DNA tetrahedron into the microfluidic channel and incubating to fix the DNA tetrahedron on the chip, and cleaning and drying the chip.

[0013] To achieve the above-mentioned objectives and other related objectives, the third aspect of the present application provides a method for manufacturing a microfluidic lung cancer cell culture chip, wherein the manufactured chip is used as the first cell culture chip in the microfluidic chip system; the manufacturing method comprises: manufacturing a chip mold, mixing a polydimethylsiloxane prepolymer and a curing agent in a preset proportion and then casting the mixture onto the chip mold, curing the dimethylsiloxane at a certain temperature, and peeling the upper and lower polydimethylsiloxane chips from the chip mold after curing to obtain upper and lower polydimethylsiloxane chips; preparing a polycarbonate porous membrane; plasma cleaning the upper and lower polydimethylsiloxane chips and the polycarbonate porous membrane respectively; aligning the upper and lower polydimethylsiloxane chips and the polycarbonate porous membrane after plasma cleaning, and using a bonding method to form a chemical bond between the upper and lower polydimethylsiloxane chips and the polycarbonate porous membrane; performing chip post-processing after bonding, inoculating lung cancer cells on the chip, and placing the chip in a cell culture incubator for culture.

[0014] To achieve the above-mentioned objectives and other related objectives, the fourth aspect of the present application provides a method for manufacturing a microfluidic macrophage culture chip, wherein the manufactured chip is used as the second cell culture chip in the microfluidic chip system; the manufacturing method includes: making a chip mold, mixing a polydimethylsiloxane prepolymer and a curing agent in a preset proportion and then casting the mixture onto the chip mold, curing the polydimethylsiloxane at a certain temperature, and peeling the upper and lower polydimethylsiloxane chips from the chip mold after curing to obtain upper and lower polydimethylsiloxane chips; plasma cleaning the upper and lower polydimethylsiloxane chips; after plasma cleaning, aligning the upper and lower polydimethylsiloxane chips, and using a bonding method to form a chemical bond between the upper and lower polydimethylsiloxane chips; after bonding is completed, performing chip post-processing and inoculating macrophages on the chip, and placing the chip in a cell culture incubator for culture.

[0015] To achieve the above-mentioned purpose and other related purposes, the fifth aspect of the present application provides a method for manufacturing a DNA logic computing chip, and the chip manufactured is used as a DNA logic computing chip in the microfluidic chip system; the manufacturing method includes: pre-setting a polydimethylsiloxane chip that has been molded and a glass sheet that has been cleaned with deionized water and blown dry; placing the polydimethylsiloxane chip and the glass sheet together in a plasma machine for cleaning; and using a bonding method to form a chemical bond between the polydimethylsiloxane chip and the glass sheet.

[0016] As described above, the microfluidic chip system and chip manufacturing method of the present application have the following beneficial effects:

[0017] (1) Quantifiable and measurable: DNA logic gates are used to read the molecular logic relationships of cell signals, and the calculated DNA molecular signals are converted into current signals through electrochemical sensor chips. This conversion process makes the molecular logic relationships of cell signals, which were originally difficult to read directly, quantifiable and measurable.

[0018] (2) Help researchers directly observe: Visualizing the logical relationships between cells makes it easier for researchers to directly observe the interactions and communication methods between cells, thereby gaining a deeper understanding of the complex networks between cells.

[0019] (3) Real-time monitoring: Since electrochemical sensor chips have the ability to respond quickly and monitor in real time, this technology can realize real-time and dynamic monitoring of the logical relationship between cells, and can be extended to study life processes such as cell growth, differentiation, and apoptosis.

[0020] (4) High sensitivity and high specificity: Since the electrochemical sensor chip has high sensitivity and specificity, it can accurately detect specific DNA molecular signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is a structural schematic diagram of a microfluidic chip system in one embodiment of the present application.

[0022] Figure 2A Shown is a schematic diagram of current signals before and after amplification in one embodiment of the present application.

[0023] Figure 2B Shown is a schematic diagram of the target protein concentration detected by the technical solution of the present application compared with the target protein concentration detected by the ELISA method in one embodiment of the present application.

[0024] Figure 3A Shown is a schematic diagram of the change of current curves with time at different miRNA concentrations in one embodiment of the present application.

[0025] Figure 3B Shown is a schematic diagram of the linear relationship between current and miRNA concentration in one embodiment of the present application.

[0026] Figure 4A Shown is a schematic diagram of the change of current curves over time at different target protein concentrations in one embodiment of the present application.

[0027] Figure 4B Shown is a schematic diagram of the linear relationship between current and target protein concentration in one embodiment of the present application. DETAILED DESCRIPTION

[0028] The following describes the embodiments of the present application through specific examples. Those skilled in the art can easily understand the other advantages and effects of the present application from the content disclosed in this specification. The present application can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0029] It should be noted that in the following description, reference is made to the accompanying drawings, which describe several embodiments of the present application. It should be understood that other embodiments may also be used, and that mechanical, structural, electrical, and operational changes may be made without departing from the spirit and scope of the present application. The following detailed description should not be considered restrictive, and the scope of the embodiments of the present application is limited only by the claims of the published patents. The terms used herein are only for describing specific embodiments and are not intended to limit the present application. Spatially related terms, such as "upper", "lower", "left", "right", "below", "below", "lower", "above", "upper", etc., may be used in the text to facilitate the description of the relationship between one element or feature shown in the figure and another element or feature.

[0030] In this application, unless otherwise specified or limited, the terms "mounted," "connected," "connect," "fixed," "holding," and the like should be understood broadly. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediary; or internal communication between two components. Those skilled in the art will understand the specific meanings of these terms in this application based on specific circumstances.

[0031] Furthermore, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context indicates otherwise. It should be further understood that the terms "comprise", "include" indicate the presence of the described features, operations, elements, components, items, kinds, and / or groups, but do not exclude the presence, occurrence, or addition of one or more other features, operations, elements, components, items, kinds, and / or groups. The terms "or" and "and / or" used herein are interpreted as inclusive, or mean any one or any combination. Thus, "A, B, or C" or "A, B, and / or C" means "any of the following: A; B; C; A and B; A and C; B and C; A, B, and C". Exceptions to this definition occur only when the combination of elements, functions, or operations is inherently mutually exclusive in some way.

[0032] To address the aforementioned issues, the present invention provides a systematic method for visualizing intercellular logical relationships. The system comprises a DNA logic gate calculation module, a microfluidic chip module, and an electrochemical sensing module. This method uses DNA logic gates to calculate and read the logical relationships between cellular signaling molecules. Electrochemical sensors convert the calculated DNA molecular signals into current signals, visualizing the logical relationships.

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following embodiments and the accompanying drawings are used to further describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] Before further explaining the present invention in detail, the nouns and terms involved in the embodiments of the present invention are explained. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations:

[0035] <1> DNA logic gates: A computing element based on DNA molecules and biotechnology that emulates the logic gate operations used in traditional electronic computers. DNA logic gates utilize the specific structure of DNA molecules and biochemical reactions to perform logical operations, thereby logically processing input signals and generating corresponding outputs. The design of DNA logic gates is typically based on reaction mechanisms such as hybridization and strand displacement in DNA molecules. By tailoring the DNA sequence and reaction conditions, functions similar to electronic logic gates can be achieved, such as basic logical operations like AND, OR, and NOT. These logic gates can be combined to construct more complex logic circuits and systems.

[0036] <2> Microfluidic chip: also known as Lab-on-chip, is a scientific technology for manipulating fluids in micron-scale space. It integrates a variety of basic operating units in fields such as chemistry and biology, such as sample preparation, reaction, separation, detection, cell culture, sorting, lysis, etc., onto a chip of a few square centimeters or even smaller. These basic operating units form a network of microchannels, and controllable fluids run through the entire system, thereby realizing various functions of different laboratories such as conventional chemistry, biology, materials, and optics. In microfluidic chips, fluid driving methods can generally be divided into two categories: mechanical driving methods (including pneumatic micropumps, piezoelectric micropumps, reciprocating micropumps, centrifugal force drive, etc.) and non-mechanical driving methods (including electroosmotic drive, gravity drive, etc.).

[0037] <3> A cell culture chip: A microfluidic chip used for cell culture within a tiny space, designed to combine traditional cell culture methods with modern microfluidic technology. Cell culture chips typically consist of a multilayer structure, including a cell culture layer, fluid channels, and a control layer. They utilize a network of microchannels to introduce culture medium and cells into the chip. By precisely controlling the flow of fluids and cell culture conditions, cells can grow and reproduce on the chip.

[0038] <4> ELISA (Enzyme-Linked Immunosorbent Assay) is a qualitative and quantitative method for measuring immune reactions based on the specificity of antigen-antibody binding. ELISA methods include: Direct ELISA: The antigen is directly immobilized on a solid support, and an enzyme-labeled primary antibody is added to measure the total amount of antigen. In this method, the specificity of the primary antibody is crucial. Indirect ELISA: Similar to the direct ELISA, the primary antibody is not enzyme-labeled. Instead, an enzyme-labeled secondary antibody is used to recognize the primary antibody to measure the amount of antigen. Unlabeled primary antibodies retain much of their immunoreactivity. Sandwich ELISA: The antigen to be detected is sandwiched between two antibodies. One antibody immobilizes the antigen on a solid support (the capture antibody), while the other antibody (the detection antibody) can be enzyme-labeled to directly measure the amount of antigen, or left unlabeled and then an enzyme-labeled secondary antibody is introduced to measure the amount of antigen. This method is primarily used to detect large antigens. Competitive ELISA: The antigen in the sample (free antigen) and the antigen purified and immobilized on a solid support (immobilized antigen) compete for the same antibody. The more free antigen in the sample, the more antibody it can bind to, while the immobilized antigen can bind to less antibody, and vice versa.

[0039] like Figure 1FIG2 shows a schematic diagram of the structure of a microfluidic chip system in an embodiment of the present application. The microfluidic chip system includes a first cell culture chip 11, a second cell culture chip 12, a DNA logic computing chip 13, and an electrochemical sensor chip 14. The first cell culture chip 11 is connected to the second cell culture chip 12 via a liquid circulation tube, the second cell culture chip 12 is connected to the DNA logic computing chip 13 via a liquid circulation tube, and the DNA logic computing chip 13 is connected to the first cell culture chip 11 and the electrochemical sensor chip 14 via a liquid circulation tube. Two types of cells grown for different days on the first cell culture chip 11 and the second cell culture chip 12 are stained for live or dead, and the target cell signal molecules secreted by the cells are amplified and transmitted to the DNA logic computing chip 13. The DNA logic computing chip 13 calculates the DNA molecule signals. The electrochemical sensor chip 14 converts the calculated DNA molecule signals into current signals and outputs the logical relationship visually.

[0040] Illustratively, the connection hole at the first preset position ① on the first cell culture chip is connected to the connection hole at the second preset position ② on the second cell culture chip through a liquid circulation tube; the connection hole at the third preset position ③ on the second cell culture chip is connected to the connection hole at the fourth preset position ④ on the DNA logic computing chip through a liquid circulation tube; the connection hole at the fifth preset position ⑤ on the DNA logic computing chip is connected to the connection hole at the sixth preset position ⑥ on the first cell culture chip through a liquid circulation tube; and the connection hole at the seventh preset position ⑦ on the DNA logic computing chip is connected to the connection hole at the eighth preset position ⑧ on the electrochemical sensor chip through a liquid circulation tube.

[0041] In the embodiments of the present application, the liquid flow tubes used to connect the chips are preferably soft capillaries. Soft capillaries can be easily connected to experimental equipment due to their flexibility. Such soft capillaries can be made of a variety of materials, such as silicone, polytetrafluoroethylene (PTFE), stainless steel, etc.

[0042] In an embodiment of the present application, the two cells grown on the first cell culture chip and the second cell culture chip for different days were stained for live and dead, and the specific method includes the following: using calcein acetoxymethyl ester and iodide to perform live and dead staining on the two cells grown on the first cell culture chip and the second cell culture chip for 1 day and 3 days.

[0043] Live-dead staining using calcein-AM and iodide (PI) is a cell viability assay. Calcein-AM is a fluorescent dye selective for live cells. It can enter live cells and be hydrolyzed by intracellular esterases to calcein, which emits strong green fluorescence. PI, on the other hand, is a fluorescent dye selective only for dead cells. It cannot enter live cells, but it can enter dead cells with damaged cell membranes and bind to nucleic acids there, emitting red fluorescence.

[0044] Exemplarily, the steps of live-dead staining using calcein acetoxymethyl ester and iodide include:

[0045] 1) Prepare the cell sample: Plant the cells to be tested in an appropriate culture vessel and ensure that the cells adhere to the vessel wall. This can be done using a culture plate, dish, or cell slide.

[0046] 2) Prepare the staining solution: Dissolve calcein-AM and pyridinium iodide (PI) in an appropriate solvent, typically serum-free culture medium or saline, according to the recommended concentrations in the reagent instructions. If needed, add an appropriate amount of buffer and preservative to maintain staining stability and cell viability.

[0047] 3) Staining: Add the prepared staining solution to the cell culture vessel, ensuring that the staining solution covers the cell surface. Staining time may vary depending on the cell type and stain concentration. Typically, staining is performed at room temperature for 15-30 minutes.

[0048] 4) Washing: After staining is complete, remove the staining solution and wash the cells with an appropriate washing solution (such as PBS) to remove unbound dye and background fluorescence. The washing step can be repeated several times to improve the signal-to-noise ratio.

[0049] 5) Observation and Analysis: Use a fluorescence microscope or flow cytometer to observe the cell staining. Live cells will emit green fluorescence, while dead cells will emit red fluorescence. Cell viability can be assessed by comparing the intensity and distribution of these two fluorescence signals.

[0050] It should be understood that live-dead staining is a biotechnology used to distinguish between live and dead cells, usually using specific dyes that react with specific structures or components in live or dead cells, thereby presenting different colors or fluorescent signals. Commonly used dyes include fluorescent dyes and live cell dyes; fluorescent dyes can be detected by fluorescence microscopy, while live cell dyes can produce color changes in live cells. The main purpose of live-dead staining is to determine the status of cells. Since cells may die due to various reasons during the experiment, such as hypoxia, malnutrition, toxin exposure, etc., live-dead staining can be used to understand the extent and cause of cell death, thereby better understanding cell biology and the mechanisms of disease.

[0051] In the embodiments of the present application, two types of cells grown on the first cell culture chip and the second cell culture chip for different days were stained for live and dead, and the target cell signaling molecules secreted by the cells were amplified. This is because the concentration of the target cell signaling molecules secreted by the cells is low. Taking the target miRNA signaling molecule as an example, by adding an amplification reagent to the miRNA secreted by the cells, its detection limit meets the detection capability of the electrochemical sensor. After the cells grow on the chip for 3 days, the secreted miRNA is amplified and added to the DNA logic computing chip, and the output signal is detected using the electrochemical sensor chip.

[0052] In the embodiment of the present application, the high expression of miRNA in cells on the first cell culture chip after culturing for 3 days generates a significant current signal as a result of calculation by the DNA logic calculation chip. Figure 2A The current signals before and after amplification are displayed, and it can be seen that the current amplitude is significantly larger after amplification. At the same time, the target protein secreted by the cells on the second cell culture chip is subjected to DNA logic calculation by the DNA logic calculation chip, and its output signal is detected by the electrochemical sensor chip and compared with the enzyme-linked immunosorbent assay (ELISA) method. The two methods have good consistency. Figure 2B The concentration values ​​for different days (day 0, day 1, and day 3) are displayed. The target protein concentration is calculated based on the current signal generated by the target protein secreted by the cells in the DNA logic calculation, and compared with the target protein concentration detected by the ELISA method. It is worth noting that this experiment shows that the two cells cultured on the microfluidic chip crosstalk through the microfluidic chip in series, and this crosstalk can be visualized by the DNA logic calculation to obtain the current signal results.

[0053] It should be understood that the enzyme-linked immunosorbent assay (ELISA) method is a commonly used biological experimental technique used to detect specific molecules in biological samples, such as antigens, antibodies, or hormones. It is based on the specific binding reaction between antigens and antibodies. The enzyme-labeled antibody or antigen binds to the target molecule in the sample to be tested. The addition of a substrate then causes the enzyme to catalyze a color reaction in the substrate. The presence and amount of the target molecule are detected based on the color change.

[0054] In the embodiment of the present application, the DNA logic calculation chip 13 calculates DNA molecular signals; the electrochemical sensor chip 14 converts the calculated DNA molecular signals into current signals and outputs the logical relationship visually. Specifically, the following two methods are included:

[0055] Method 1) Use OR gate DNA logic gate and electrochemical sensor chip to detect different concentrations of target cell signaling molecules.

[0056] In a specific embodiment, an OR gate DNA logic gate is prepared, and by adding different concentrations of target miRNA signal molecules and other DNA chains that undergo DNA reactions, after sufficient mixing, the output results are detected using an electrochemical sensor chip. The detection results show that the concentration values ​​of different target miRNA signal molecules are linearly related to the current values ​​generated.

[0057] MiRNA (microRNA) is a short non-coding RNA molecule that plays a role in regulating gene expression in cells. In the embodiments of the present application, different concentrations of miRNA are used as input signals. In order to realize the logical function of the OR gate, in addition to the target miRNA, other specific DNA chains need to be added. These DNA chains interact with the miRNA and generate specific output signals based on reactions such as DNA hybridization and strand displacement. All DNA chains and target miRNA are fully mixed so that sufficient interaction can occur between them.

[0058] In the examples of this application, analysis of experimental data shows that when the concentration of the target miRNA varies within the range of 20nM to 1000nM, there is a linear relationship between the generated current value and the concentration of the miRNA. This linear relationship means that the concentration of the miRNA can be accurately calculated by measuring the current value, which is very important for quantitative detection of miRNA. Figure 3A and 2B As shown, Figure 3A Shown are the changes in current curves of different miRNA concentrations over time. Figure 3B The linear relationship between current and miRNA concentration is shown. Within the miRNA concentration range of 20nM-1000nM, the concentrations of different target miRNA signal molecules show a good linear relationship with the generated current values.

[0059] Method 2) Use AND gate DNA logic gates and electrochemical sensor chips to detect target protein signal molecules at different concentrations.

[0060] An AND gate DNA logic gate was prepared by adding different concentrations of target protein signal molecules, aptamers, and other reactive DNA chains. After thorough mixing, the output results were detected using an electrochemical sensor chip. The detection results showed that the target protein concentration was linearly related to the current signal.

[0061] In a specific embodiment, an AND gate DNA logic gate is prepared. By adding different concentrations of a target miRNA signaling molecule and other DNA strands undergoing DNA reactions, the resulting signal is thoroughly mixed and then detected using an electrochemical sensor. The resulting current is linearly correlated with the target protein concentration.

[0062] In the examples of this application, by analyzing the experimental data, it can be seen that when the concentration of the target protein changes within the concentration range of 0.5pg / mL-100pg / mL, the current value generated is linearly related to the target protein concentration. This linear relationship means that the target protein concentration can be accurately calculated by measuring the current value, which is very important for quantitatively measuring target protein cell signaling molecules. Figure 4A and 3B As shown, Figure 4A Shown is the change of current curve with time at different target protein concentrations. Figure 4B It is shown that in the concentration range of 0.5pg / mL-100pg / mL, different target protein concentrations show a good linear relationship with the generated current values.

[0063] It should be noted that DNA logic gates are computing components based on DNA molecules that generate output signals based on input signals, performing logical operations. In DNA computing, logic gates are typically implemented using biological reaction mechanisms such as DNA strand displacement reactions and DNA ribozymes. The logic principle of an OR DNA logic gate is that it generates an output signal when any input signal is present. The logic principle of an AND DNA logic gate is that it generates an output signal only when all input signals are present.

[0064] In the embodiment of the present application, the manufacturing method and detection principle of the electrochemical sensor chip 14 are as follows.

[0065] Preparation step S11: preparing a nano-gold layer.

[0066] A tetrachloroauric acid solution was dripped onto a 16-channel screen-printed carbon electrode (16-SPCE). Through electrodeposition, the tetrachloroauric acid solution was reduced to gold on the electrode, forming a nano-gold layer. This step provided a well-conducting and metallized surface on the electrode for subsequent connection to the DNA tetrahedron structure.

[0067] Production step S12: Modifying the DNA tetrahedral structure.

[0068] By utilizing the formation of Au-S bonds, DNA tetrahedron structures were modified onto screen-printed electrodes to form capture probes. This step fixed the DNA tetrahedrons onto the electrodes, serving as recognition elements for subsequent DNA detection.

[0069] Fabrication step S13: fabricating a microfluidic channel.

[0070] A PDMS (polydimethylsiloxane) cover slip with an S-shaped channel structure was used. This cover slip, along with electrodes modified with DNA tetrahedrons, was placed in a plasma evaporator to impart a surface charge, facilitating subsequent irreversible encapsulation. The channel was 400 microns wide and 115 microns high.

[0071] Production step S14: packaging the microfluidic chip.

[0072] The electrode with the DNA tetrahedrons was capped with a cap, and then the treated PDMS cover sheet and the electrode were quickly attached to form an irreversible encapsulation between the two. This formed a complete chip containing the microfluidic channel and the DNA tetrahedron-modified electrode.

[0073] Production step S15: injecting DNA tetrahedrons and incubating.

[0074] One micromole of DNA tetrahedrons was injected into the microfluidic channel via a syringe pump until it was full, and then incubated at room temperature overnight to allow the DNA tetrahedrons to be fully immobilized on the chip.

[0075] Production step S06: cleaning and drying.

[0076] After overnight, the chip was gently washed with PBS (phosphate buffered saline) to remove unbound DNA tetrahedrons and other impurities. The liquid was then extracted and nitrogen gas was slowly introduced to dry the remaining liquid, obtaining a dry DNA electrochemical sensor chip.

[0077] In the embodiments of the present application, a process for synthesizing DNA tetrahedral nanostructures starting from single-stranded DNA using specific buffer and temperature conditions is as follows:

[0078] First, dissolve and quantify single-stranded DNA: Use TE buffer (a buffer commonly used to dissolve DNA) to dissolve single-stranded DNA. The dissolved DNA is quantified under a UV spectrophotometer. The UV spectrophotometer can estimate the concentration of DNA by measuring its absorbance at a wavelength of 260nm. In order to more accurately calculate the concentration of DNA, the molar extinction coefficient is used. This coefficient can be obtained from the IDTDNA website and describes the relationship between the absorbance of DNA at a specific wavelength and its concentration. Finally, the DNA is diluted to a 100μM storage solution for subsequent experiments.

[0079] Next, tetrahedral nanostructure formation: The four single-stranded DNA strands forming the tetrahedral nanostructure were mixed in equal proportions in TM buffer. TM buffer is commonly used in DNA hybridization experiments because it stabilizes the double-stranded structure of DNA. The total concentration of the mixed single-stranded DNA strands was adjusted to 1 μM, which was the final concentration of the tetrahedral nanostructure.

[0080] Finally, heating and cooling: The prepared sample is placed in a PCR instrument and first heated at 95°C for 10 minutes. This high temperature allows the double-stranded DNA to unwind and form single strands. The sample is then quickly cooled to 4°C and maintained at 4°C for at least 20 minutes. This process allows the DNA single strands to re-hybridize and form the desired tetrahedral nanostructure.

[0081] In the embodiments of the present application, the first cell culture chip and the second cell culture chip are two different cell culture chips, specifically a microfluidic lung cancer cell culture chip and a microfluidic macrophage culture chip, respectively.

[0082] Preferably, the method for fabricating a microfluidic lung cancer cell culture chip involves combining cell culture and microfluidic technology, with the goal of providing a controlled growth environment for lung cancer cells while facilitating various manipulations and monitoring of the cells. The method for fabricating a microfluidic lung cancer cell culture chip specifically includes the following:

[0083] Production step S21: producing upper and lower PDMS (polydimethylsiloxane) chips.

[0084] First, the mold for the microfluidic chip is designed and fabricated using computer-aided design (CAD) software. The mold is then produced through photolithography or machining. Next, the PDMS prepolymer and curing agent are mixed in a specific ratio and then poured onto the mold. Finally, the PDMS is cured at a specific temperature (typically 70°C). Once fully cured, it is peeled from the mold, yielding the upper and lower PDMS chips.

[0085] Production step S22: preparing a polycarbonate porous membrane.

[0086] Polycarbonate porous membranes are used as support layers for cell culture. They provide surface area for cell attachment and growth while allowing culture medium and other solutions to flow through microfluidic channels. Polycarbonate porous membranes can be cut into appropriate sizes and shapes as needed.

[0087] Production step S23: plasma cleaning.

[0088] The upper and lower PDMS chips and polycarbonate porous membranes are plasma cleaned separately. Plasma cleaning removes organic contaminants and hydrophobicity from the surface, increasing surface hydrophilicity, improving biocompatibility, and facilitating subsequent bonding. Plasma cleaning is typically performed in a plasma cleaner. The cleaning time is typically 1 minute, but this may vary depending on the specific equipment and material properties.

[0089] Production step S24: alignment and bonding.

[0090] After plasma cleaning, the upper and lower PDMS chips are aligned with the polycarbonate porous membrane. Precision in alignment is crucial to ensuring connectivity of the microfluidic channels and uniform distribution of cells. A suitable bonding method is used to firmly bond the PDMS chip to the polycarbonate porous membrane. This typically involves applying a certain amount of pressure at a certain temperature to promote close contact and chemical bonding between the materials.

[0091] Production step S25: chip post-processing and cell culture.

[0092] After bonding is complete, the chip can be further processed, such as surface treatment within the channels and the addition of cell culture medium. Lung cancer cells are then inoculated onto the chip and placed in a cell culture incubator for culture. The microfluidic system allows for precise control of the flow of culture medium, cell culture conditions, and interactions with other cells, thereby simulating the in vivo growth environment.

[0093] Preferably, the method for fabricating a microfluidic macrophage culture chip focuses on providing a controlled microenvironment for macrophages to facilitate cell culture, observation, and experimental manipulation. The specific process includes:

[0094] Production step S31: producing upper and lower PDMS chips.

[0095] Design the mold for the microfluidic chip, which is usually done through computer-aided design software (CAD). The design of the mold will take into account the needs of cell culture, including cell culture chambers, inlet and outlet channels, etc. To make the mold, you can use methods such as photolithography, machining or 3D printing. Mix the PDMS prepolymer and curing agent in a certain proportion, and then pour the mold onto the prepared mold. Cure the PDMS at a certain temperature (usually 70°C), and peel it off from the mold after it is completely cured to obtain the upper and lower PDMS chips.

[0096] Production step S32: plasma cleaning.

[0097] The purpose of plasma cleaning is to remove organic contaminants and hydrophobicity from the PDMS chip surface, increase its hydrophilicity, improve cell attachment efficiency, and promote bonding between the upper and lower PDMS chips. The upper and lower PDMS chips are each plasma cleaned for one minute. During the cleaning process, active particles in the plasma react with the material surface, introducing oxygen-containing polar groups, thereby improving its wettability and biocompatibility.

[0098] Production step S33: alignment and bonding.

[0099] After plasma cleaning, the upper and lower PDMS chips are aligned. Precision in alignment is crucial to ensuring connectivity of the microfluidic channels and uniform distribution of cells. An appropriate bonding method is used to securely bond the upper and lower PDMS chips together. This typically involves applying a certain amount of pressure at a certain temperature to promote close contact and chemical bonding between the materials.

[0100] Production step S34: chip post-processing and cell culture.

[0101] After bonding is complete, the chip can be further processed, such as surface treatment within the channels and the addition of cell culture medium. Macrophages are then seeded onto the chip and placed in a cell culture incubator for culture. Macrophages are highly sensitive to stimuli in their microenvironment, so microfluidic chips can provide a highly controlled environment for studying macrophage responses and functions.

[0102] Production step S35: cell observation and experimental operation.

[0103] The design of microfluidic chips makes it easy to observe the growth, migration, and interaction of macrophages with other cells. Microfluidic systems can also be used to precisely control the flow of culture medium and add stimuli or drugs to study the responses of macrophages to these stimuli.

[0104] In the embodiment of the present application, the manufacturing method of the DNA logic computing chip 13 is as follows:

[0105] Production step S41: prepare materials.

[0106] First, you need a PDMS (polydimethylsiloxane) chip that has been molded. At the same time, prepare a glass slide that has been cleaned with deionized water and blown dry.

[0107] Production step S42: plasma cleaning.

[0108] The PDMS chip and glass sheet are placed together in a plasma cleaning machine. Plasma cleaning is a surface treatment technology that uses high-energy ions to bombard the surface of a material, removing organic contaminants, increasing its hydrophilicity, and introducing polar functional groups, thereby changing the chemical and physical properties of the surface. The purpose of plasma cleaning is to enhance the adhesion between the PDMS chip and the glass sheet, promoting a tight bond between the two.

[0109] Production step S43: producing bonding.

[0110] After plasma cleaning, the PDMS chip is immediately bonded to the glass sheet. Due to the surface activation caused by plasma cleaning, a chemical bond is formed between the PDMS chip and the glass sheet, achieving irreversible encapsulation.

[0111] In summary, the present application provides a microfluidic chip system and a chip manufacturing method thereof, and the technical effects include the following: (1) Quantifiable and measurable: Based on the DNA logic gate, the logical relationship of cell signal molecules is read, and the calculated DNA molecular signal is converted into an electric current signal through the electrochemical sensor chip. This conversion process makes the DNA molecular signal that was originally difficult to detect directly quantifiable and measurable. (2) Helping researchers to observe directly: The logical relationship between cells is visualized, which makes it easier for researchers to directly observe the interaction and communication between cells, thereby gaining a deeper understanding of the complex network between cells. (3) Realizing real-time monitoring: Since the electrochemical sensor chip has the ability to respond quickly and monitor in real time, this technology can realize real-time and dynamic monitoring of the logical relationship between cells, which can be extended to study life processes such as cell growth, differentiation, and apoptosis. (4) High sensitivity and high specificity: Since the electrochemical sensor chip has high sensitivity and specificity, it can accurately detect specific DNA molecular signals. Therefore, the present application effectively overcomes the various shortcomings of the existing technology and has high industrial utilization value.

[0112] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed in this application shall be covered by the claims of this application.

Claims

1. A microfluidic chip system, characterized in that: include: a first cell culture chip, a second cell culture chip, a DNA logic computing chip, and an electrochemical sensor chip; The first cell culture chip is connected to the second cell culture chip via a liquid circulation tube; the first cell culture chip, The second cell culture chip is connected to the first and second logic gates of the DNA logic computing chip through liquid circulation tubes, and the DNA logic computing chip is connected to the electrochemical sensor chip through liquid circulation tubes; Among them, the two types of cells grown on the first cell culture chip and the second cell culture chip for different days are stained for live or dead, and the target cell signal molecules secreted by the cells are amplified and transmitted to the DNA logic computing chip; the DNA logic computing chip calculates the DNA molecular signals; the electrochemical sensor chip converts the calculated DNA molecular signals into current signals and performs a visual output of the logical relationship.

2. The microfluidic chip system according to claim 1, characterized in that: The two cells grown on the first cell culture chip and the second cell culture chip for different days are subjected to live-dead staining, which includes: using calcein acetoxymethyl ester and iodide to perform live-dead staining on the two cells grown on the first cell culture chip and the second cell culture chip for 1 day and 3 days.

3. The microfluidic chip system according to claim 1, characterized in that: The target cell signaling molecule secreted by the cell is amplified, including: using an amplification reagent to amplify the target miRNA signaling molecule secreted by the cell.

4. The microfluidic chip system according to claim 1, characterized in that: The first and second level logic gates of the DNA logic chip include an OR gate DNA logic gate and an AND gate DNA logic gate; the target cell signal molecule includes a target miRNA signal molecule; the DNA logic calculation chip calculates DNA molecule signals; and the electrochemical sensor chip converts the calculated DNA molecule signals into current signals and performs a visual output of the logical relationship, including any one of the following: OR gate DNA logic gate and electrochemical sensor chip are used to detect target miRNA signal molecules with different concentrations; AND gate DNA logic gate and electrochemical sensor chip are used to detect target protein signal molecules with different concentrations.

5. The microfluidic chip system according to claim 4, characterized in that: The method uses an OR gate DNA logic gate and an electrochemical sensor chip to detect target cell signaling molecules of different concentrations. The detection process includes: An OR gate DNA logic gate was prepared by adding different concentrations of target miRNA signal molecules and other DNA chains that underwent DNA reactions. After thorough mixing, the output results were detected using an electrochemical sensor chip. The detection results showed that the concentration value of the target miRNA signal molecule was linearly related to the generated current value.

6. The microfluidic chip system according to claim 4, characterized in that: The use of an AND gate DNA logic gate and an electrochemical sensor chip to detect target protein signal molecules of different concentrations includes: An AND gate DNA logic gate was prepared by adding different concentrations of target protein signal molecules, aptamers, and other reactive DNA chains. After thorough mixing, the output results were detected using an electrochemical sensor chip. The detection results showed that the target protein concentration was linearly related to the current signal.

7. A method for manufacturing an electrochemical sensor chip, characterized in that: The fabricated chip is used as an electrochemical sensor chip in a microfluidic chip system according to any one of claims 1 to 6; the fabrication method comprises: Adding tetrachloroauric acid solution to a screen-printed carbon electrode, and reducing the tetrachloroauric acid solution to gold on the electrode by electrodeposition to form a nano-gold layer; Using gold-sulfur bonds, DNA tetrahedron structures were modified on screen-printed carbon electrodes to form capture probes, thereby immobilizing the DNA tetrahedrons on the electrodes. The polydimethylsiloxane cover sheet with an S-shaped channel structure and the electrode modified with DNA tetrahedron were placed in a plasma machine for treatment; The electrode portion with the DNA tetrahedrons is covered with a cap, and a treated polydimethylsiloxane cover sheet and the electrode are attached to each other so as to irreversibly encapsulate the two, thereby forming a chip containing a microfluidic channel and a DNA tetrahedron-modified electrode; DNA tetrahedrons are injected into the microfluidic channel and incubated to immobilize the DNA tetrahedrons on the chip, and the chip is then washed and dried.

8. A method for preparing a microfluidic lung cancer cell culture chip, characterized in that: The fabricated chip is used as the first cell culture chip in the microfluidic chip system according to any one of claims 1 to 6; the fabrication method comprises: To make a chip mold, polydimethylsiloxane prepolymer and curing agent are mixed in a preset ratio and then poured onto the chip mold. The dimethylsiloxane is cured at a certain temperature. After curing, the upper and lower polydimethylsiloxane chips are peeled off from the chip mold to obtain the upper and lower polydimethylsiloxane chips. preparing a polycarbonate porous membrane; The upper and lower polydimethylsiloxane chips and polycarbonate porous membranes were plasma cleaned respectively; Aligning the upper and lower polydimethylsiloxane chips and the polycarbonate porous membrane after plasma cleaning, and forming a chemical bond between the upper and lower polydimethylsiloxane chips and the polycarbonate porous membrane using a bonding method; After bonding is completed, the chip is post-processed and lung cancer cells are inoculated on the chip, and the chip is placed in a cell culture incubator for culture.

9. A method for manufacturing a microfluidic macrophage culture chip, characterized in that: The fabricated chip is used as a second cell culture chip in the microfluidic chip system according to any one of claims 1 to 6; the fabrication method comprises: To make a chip mold, polydimethylsiloxane prepolymer and curing agent are mixed in a preset ratio and then poured onto the chip mold. The dimethylsiloxane is cured at a certain temperature. After curing, the upper and lower polydimethylsiloxane chips are peeled off from the chip mold to obtain the upper and lower polydimethylsiloxane chips. Plasma cleaning of the upper and lower polydimethylsiloxane chips; After plasma cleaning, the upper and lower polydimethylsiloxane chips are aligned, and a bonding method is used to form a chemical bond between the upper and lower polydimethylsiloxane chips; After bonding is completed, the chip is post-processed and macrophages are inoculated on the chip, and the chip is placed in a cell culture incubator for culture.

10. A method for manufacturing a DNA logic computing chip, characterized in that: The fabricated chip is used as a DNA logic computing chip in a microfluidic chip system according to any one of claims 1 to 6; the fabrication method comprises: A pre-molded polydimethylsiloxane chip and a glass sheet that has been cleaned with deionized water and blown dry are provided; The polydimethylsiloxane chip and the glass sheet are placed together in a plasma machine for cleaning; A bonding method was used to form a chemical bond between the polydimethylsiloxane chip and the glass slide.

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