Bacterium-cell interaction gas real-time detection device and application thereof in oral cancer marker traceability

Abstract

The invention relates to the field of biomedical detection, and discloses a real-time detection device for bacteria-cell interaction gas and application of the real-time detection device in oral cancer marker traceability. The device comprises a co-culture container, the co-culture container is an open container, and a sealing piece is arranged at the open end; the puncture detection module comprises a puncture probe and a detector, during detection, one end of the puncture probe penetrates through the sealing piece, and the other end of the puncture probe is connected with the detector; the temperature control module is used for maintaining the culture temperature in the co-culture container. The device provided by the invention fundamentally solves the industrial problem of in-situ detection of gas generated by bacteria-cell interaction, can be used for establishing a quantifiable marker-disease mechanism correlation model, and promotes the development of accurate diagnosis.

Description

Technical Field

[0001] This application relates to the field of biomedical detection, and more specifically, to a real-time detection device for bacteria-cell interaction gases and its application in tracing the source of oral cancer biomarkers. Background Technology

[0002] Currently, the development and progression of many cancers, such as oral cancer and colorectal cancer, are closely related to specific microbial communities and their metabolites. Furthermore, patients with these types of cancer often exhibit characteristic gaseous metabolic markers. For example, the content of methanethiol in the exhaled breath of patients with oral squamous cell carcinoma is significantly elevated. However, tracing the source of these markers is difficult. Current research on the source of methanethiol, an oral exhaled breath marker associated with oral squamous cell carcinoma, mainly relies on the individual and co-culture of in vitro cell lines and bacteria. It is impossible to detect changes in the content of gases produced after cell and bacterial culture in real time, resulting in unclear main sources and specific generation mechanisms of gaseous markers.

[0003] Furthermore, traditional methods typically detect the culture medium, bacterial or cell lysates, in co-culture systems. These methods are not suitable for detecting volatile gases, and the results cannot explain related scientific questions, highlighting the limitations of gas sampling technology. Moreover, existing co-culture systems cannot achieve quantitative calibration of bacterial or cell-derived gas production, lack effective methods for inputting differential gas samples into the mass spectrometer inlet, and cannot maintain gas-sealed conditions.

[0004] Therefore, there is currently a lack of an integrated device that can achieve real-time co-culture of bacteria and cells and non-destructive gas detection in a closed environment. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated device for real-time co-culture of bacteria and cells and non-destructive gas detection, and its application in the tracing of oral cancer biomarkers, so as to solve the problem that the existing technology cannot achieve real-time collection and quantitative analysis of gases from bacterial-cell co-culture under closed conditions.

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution: In a first aspect, this application provides a real-time detection device for bacteria-cell interaction gases, the device comprising: A co-culture container, wherein the co-culture container is an open container with a sealing element at the open end; A puncture detection module, comprising a puncture probe and a detector, wherein during detection, one end of the puncture probe passes through the seal, and the other end is connected to the detector; and A temperature control module is used to maintain the culture temperature inside the co-culture container.

[0007] Furthermore, the sealing element is a double-layer Parafilm sealing film.

[0008] Furthermore, the air permeability of the seal is controlled to be <0.5 mL / m² / day.

[0009] Furthermore, the inner diameter of the puncture probe is 0.5~0.8 mm.

[0010] Furthermore, the puncture probe is equipped with a self-sealing valve. When the puncture probe penetrates the seal, the self-sealing valve is deformed under pressure to form a sealing ring, preventing gas leakage.

[0011] Furthermore, the detector is a proton transfer reaction time-of-flight mass spectrometer or a gas chromatography-ion mobility spectrometry (GC-IMS) instrument.

[0012] Furthermore, the temperature control module is used to maintain the culture temperature at 37±0.5 ℃.

[0013] Secondly, this application discloses the application of the real-time detection device for bacterial-cell interaction gases in the first aspect in the tracing of oral cancer biomarkers.

[0014] Furthermore, the application in tracing the source of oral cancer markers includes the following steps: Step 1: Culture Fusobacterium nucleatum under anaerobic conditions and use it for experiments when OD600 = 0.5~0.8. At the same time, culture oral squamous cell carcinoma cell lines CAL33, HN6, and HN30 in a cell culture incubator. When the cell confluence reaches 70%, mix the bacteria and cells at an infection index MOI = 10:1 in high glucose DMEM basal medium without serum and antibiotics. After thorough mixing, transfer the mixture to a co-culture container and seal it with a sealing device. Step 2: Use a temperature control module to maintain the culture temperature inside the co-culture container at 37±0.5℃; Step 3, at the time of co-cultivation harvest, use a puncture probe to penetrate the seal and extract gas into the detector at a flow rate of 100-200 mL / min. The gas is then placed in an H3O atmosphere with an electric field strength of 130 Td. + In the reaction chamber, the target marker methanethiol had an m / z of 65.02 ± 0.3Th and was quantitatively detected by protonation reaction, with concentration data recorded at regular intervals.

[0015] Furthermore, when the concentration of methanethiol was consistently >50 ppb, cell samples from the co-culture system were collected using differential centrifugation for Wnt / β-catenin pathway protein expression analysis.

[0016] In summary, this invention, through its innovative puncture-sealing structure and standardized co-culture system, has achieved the following breakthroughs and has the following beneficial effects: 1. This design fundamentally solves the industry-wide challenge of in-situ gas detection in bacterial-cell interactions. Utilizing a synergistic design of a medical-grade silicone self-sealing valve and Parafilm sealing film, the gas leakage rate during the puncture and injection process is controlled to within 5% (based on GB / T 13477.4-2002 Part 4, Sealing Test). This design achieves, for the first time, continuous 48-hour dynamic monitoring (sampling interval ≤ 2 minutes) in a closed culture system, successfully capturing the explosive growth of methanethiol produced by the interaction between *Fusobacterium nucleatum* and cancer cells—reaching a peak concentration of 67.5 ppb after 24 hours of co-culture, nearly 11 times higher than the cancer cell group alone and nearly 6 times higher than the bacterial culture group alone.

[0017] 2. Establish quantifiable biomarker-disease mechanism association models to promote the development of precision diagnosis.

[0018] 3. Verification of universality expands clinical application scenarios. This proves that the device can be compatible with interaction studies of different strains / cell types, solving the pain point of traditional methods that cannot be transferred across models due to insufficient device closedness. Attached Figure Description

[0019] Figure 1 : A schematic diagram of the structure of the real-time detection device for bacteria-cell interaction gases in Embodiment 1 of this application; Figure 2 Example 2 of this application shows the gradient increase in methanethiol production after co-culturing Fusobacterium nucleatum and cancer cells; Figure 3 Figure 2 shows the comparison of gas production differences between blank control, Fn alone, cancer cells alone, and F.n+ cancer cells in Example 2 of this application.

[0020] Figure labels: 1. Co-culture container; 2. Seal; 3. Spike probe; 4. Detector. Detailed Implementation

[0021] The technical solutions and effects of this application will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the invention, not the entire structure.

[0022] Example 1: Real-time detection device for bacteria-cell interaction gases This embodiment discloses a real-time detection device for bacteria-cell interaction gases, referring to... Figure 1 The real-time detection device for bacteria-cell interaction gases includes a co-culture container, a puncture detection module, and a temperature control module (not shown in the figure, which is prior art).

[0023] Reference Figure 1The co-culture container is an open container; in this embodiment, a 15 mL conical centrifuge tube is used. A sealing element is provided at the open end of the co-culture container; in this embodiment, the sealing element is a double-layer Parafilm sealing film, and the air permeability of this sealing film is controlled to be <0.5 mL / m² / day.

[0024] Reference Figure 1 The puncture detection module includes a puncture probe and a detector. During detection, one end of the puncture probe passes through a seal, and the other end connects to the detector. In this embodiment, a 316L stainless steel probe is selected. A self-sealing valve (not shown in the figure, representing prior art) is provided at the tip of the puncture probe. When the puncture probe penetrates the seal, the self-sealing valve deforms under pressure to form a sealing ring, preventing gas leakage. In this embodiment, the self-sealing valve is a medical-grade silicone self-sealing valve. The inner diameter of the puncture probe is 0.5~0.8 mm, which can be selected within the range according to actual needs. In this embodiment, the detector is a proton transfer reaction time-of-flight mass spectrometer (PTR-TOF MS) or a gas chromatography-ion mobility spectrometry (GC-IMS) instrument. When using a proton transfer reaction time-of-flight mass spectrometer, the inner diameter of the puncture probe can be selected as 0.5 mm; when using a gas chromatography-ion mobility spectrometry instrument, the inner diameter of the puncture probe can be selected as 0.8 mm.

[0025] Reference Figure 1 The temperature control module is used to maintain the culture temperature within the co-culture container. During co-culture, the temperature is controlled at 37±0.5 ℃ by the temperature control module to maintain a suitable growth temperature for bacteria and cells. It is worth noting that the temperature control module can be a conventional temperature controller used in the art, which is existing technology. Those skilled in the art can choose according to their needs, and therefore no further restrictions are imposed here.

[0026] Example 2: Application of a real-time bacterial-cell interaction gas detection device in the source tracing of oral cancer biomarkers This embodiment discloses a method for using the real-time detection device for bacterial-cell interaction gases described in Embodiment 1 in the tracing of oral cancer biomarkers, which includes the following steps: Step 1: Fusobacterium nucleatum was cultured under anaerobic conditions and used for experiments when OD600 = 0.5~0.8. At the same time, oral squamous cell carcinoma cells CAL33, HN6, and HN30 were cultured in a cell culture incubator. When the cell confluence reached 70%, the bacteria and cells were mixed in 3 mL of serum-free and antibiotic-free high-glucose DMEM basal medium at an infection index MOI = 10:1. After being thoroughly mixed by pipetting, the mixture was transferred to a co-culture container (15 mL conical centrifuge tube) and sealed with double-layer Parafilm sealing film. Step 2: Use a temperature control module to maintain the culture temperature in the co-culture container at 37±0.5 ℃; Step 3, at the time of co-cultivation harvest, a puncture probe is used to penetrate the seal, and gas is extracted into the detector at a flow rate of 200 mL / min. The gas is then placed in an H3O atmosphere with an electric field strength of 130 Td. + In the reaction chamber, the target biomarker methanethiol had an m / z of 65.02 ± 0.3 Th and was quantitatively detected by protonation reaction, with concentration data recorded every 2 minutes.

[0027] When the concentration of methanethiol was consistently >50 ppb, cell samples from the co-culture system were collected using differential centrifugation for Wnt / β-catenin pathway protein expression analysis (Western Blot).

[0028] Furthermore, validation through alternative methods showed that when using a 50 mL conical flask as a co-culture container, the injection flow rate should be adjusted to 100 mL / min to avoid negative pressure damaging the cells; if a GC-IMS instrument is used instead of a PTR-TOF MS instrument, the puncture probe with an inner diameter of 0.8 mm should be replaced to ensure airflow.

[0029] The device proposed in this application fundamentally solves the industry-wide challenge of in-situ gas detection in bacterial-cell interactions. Employing a synergistic design of a medical-grade silicone self-sealing valve and Parafilm sealing film, the gas leakage rate during the puncture and injection process is controlled to within 5% (according to GB / T 13477.4-2002 Part 4, Sealing Test). This design achieves, for the first time, continuous 48-hour dynamic monitoring (sampling interval ≤ 2 minutes) in a closed culture system, successfully capturing the explosive growth phenomenon of methanethiol production induced by *Fusobacterium nucleatum* in cancer cells—reaching a peak concentration of 67.5 ppb after 24 hours of co-culture, nearly 11 times higher than the cancer cell group alone and nearly 6 times higher than the bacterial group alone (e.g., ...). Figure 2 and Figure 3 (As shown).

[0030] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A real-time detection device for bacteria-cell interaction gases, characterized in that, The device consists of: A co-culture container, wherein the co-culture container is an open container with a sealing element at the open end; A puncture detection module, comprising a puncture probe and a detector, wherein during detection, one end of the puncture probe passes through the seal, and the other end is connected to the detector; and A temperature control module is used to maintain the culture temperature inside the co-culture container.

2. The real-time detection device for bacteria-cell interaction gases according to claim 1, characterized in that, The sealing element is a double-layer Parafilm sealing film.

3. The real-time detection device for bacteria-cell interaction gases according to claim 2, characterized in that, The air permeability of the seal is controlled to be <0.5 mL / m² / day.

4. The real-time detection device for bacteria-cell interaction gases according to claim 2, characterized in that, The inner diameter of the puncture probe is 0.5~0.8 mm.

5. The real-time detection device for bacteria-cell interaction gases according to claim 4, characterized in that, The puncture probe is equipped with a self-sealing valve. When the puncture probe penetrates the seal, the self-sealing valve is deformed under pressure to form a sealing ring, preventing gas leakage.

6. The real-time detection device for bacteria-cell interaction gases according to claim 1, characterized in that, The detector is a proton transfer reaction time-of-flight mass spectrometer or a gas chromatography-ion mobility spectrometry (GC-IMS) instrument.

7. The real-time detection device for bacteria-cell interaction gases according to claim 1, characterized in that, The temperature control module is used to maintain the culture temperature at 37±0.5 ℃.

8. The application of the real-time detection device for bacterial-cell interaction gases as described in claim 1 in the tracing of oral cancer biomarkers.

9. The application according to claim 8, characterized in that, Includes the following steps: Step 1: Culture Fusobacterium nucleatum under anaerobic conditions and use it for experiments when OD600 = 0.5~0.

8. At the same time, culture oral squamous cell carcinoma cell lines CAL33, HN6, and HN30 in a cell culture incubator. When the cell confluence reaches 70%, mix the bacteria and cells at an infection index MOI = 10:1 in high glucose DMEM basal medium without serum and antibiotics. After mixing evenly, transfer to a co-culture container and seal with a sealing device. Step 2: Use a temperature control module to maintain the culture temperature in the co-culture container at 37±0.5 ℃; Step 3, at the time of co-cultivation harvest, use a puncture probe to penetrate the seal and extract gas into the detector at a flow rate of 100-200 mL / min. The gas is then placed in an H3O atmosphere with an electric field strength of 130 Td. + In the reaction chamber, the target biomarker methanethiol had an m / z of 65.02 ± 0.3 Th and was quantitatively detected by protonation reaction, with concentration data recorded at regular intervals.

10. The application according to claim 9, characterized in that, When the concentration of methanethiol was consistently >50 ppb, cell samples from the co-culture system were collected using differential centrifugation for Wnt / β-catenin pathway protein expression analysis.

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