Multi-temperature gradient heating device used in oleanolic acid cell thermal shift assays
The multi-temperature gradient heating device addresses the challenge of inconsistent data by allowing simultaneous heat treatment at multiple temperatures, thus shortening experimental cycles and improving data consistency.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Utility models
- Current Assignee / Owner
- Filing Date
- 2026-02-06
- Publication Date
- 2026-04-02
AI Technical Summary
Conventional heating devices for cellular thermal shift assays often lack the capability to synchronously control multiple temperature gradients, leading to extended experimental cycles and inconsistent data due to batch-based temperature control methods.
A multi-temperature gradient heating device with independently controllable heating blocks in separate compartments, insulated by low-conductivity materials, allowing simultaneous heat treatment at multiple pre-set temperatures.
This device enables synchronized heat treatment at multiple temperatures, reducing experimental time and enhancing data consistency and comparability by minimizing variations in experimental conditions.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical analysis instruments, and particularly relates to a multi-temperature gradient heating device used for the cell thermal shift assay of oleanolic acid.
Background Art
[0002] According to the 2020 World Cancer Statistics, breast cancer has surpassed lung cancer for the first time and is the cancer with the highest number of diagnoses in the world. The number of new cases of breast cancer in women reached 2.26 million, accounting for 11.7% of all new cancer cases. In recent years, the diagnostic and treatment levels of breast cancer have improved in conventional treatment methods such as surgery, radiotherapy, chemotherapy, and new treatment methods such as endocrine therapy and targeted therapy, achieving certain results. However, problems such as recurrence, metastasis, and drug resistance still exist.
[0003] In recent years, the role of single compounds derived from Chinese herbal medicines (natural product isolates) in tumor treatment has attracted attention. According to research, these single compounds derived from Chinese herbal medicines exert anti-cancer effects through multiple targets and pathways and have the advantages of low toxicity and low occurrence of resistance. For example, oleanolic acid (hereinafter referred to as OA) is a pentacyclic triterpenoid compound with efficient and low-toxic pharmacological effects, having multiple anti-tumor mechanisms and affecting the growth and proliferation of tumor cells. According to research, OA can induce apoptosis and autophagy of breast cancer cells and can selectively damage tumor cells. In addition, OA can inhibit the growth of tumor cells through the mitochondrial pathway and induce cell cycle arrest and cell death. Therefore, as a candidate molecule with anti-tumor potential, OA has broad prospects for clinical conversion.
[0004] Cellular thermal shift assay (CETSA) is an experimental technique for studying drug-target protein interactions, evaluating the binding ability of drug molecules to target proteins by detecting changes in the thermal stability of proteins under different temperatures. This technique has significant application value in drug screening, mechanism of action studies, and lead compound optimization. In practice, it is usually necessary to heat-treat a sample containing a drug and cells under a series of precisely set temperature conditions and observe the thermal denaturation behavior of the protein. Therefore, the interaction between oleanolic acid and target proteins in breast cancer cells can be verified using the CETSA technique.
[0005] However, conventional heating devices often have only a single temperature range or employ a batch-based temperature control method that sequentially sets different temperatures, making it difficult to synchronize multiple continuous or discrete temperature gradient processes within the same experimental procedure. These limitations necessitate multiple repeated operations, extending the experimental cycle and potentially affecting data consistency and comparability due to differences in conditions between batches. Therefore, there was room for improvement in devices that could synchronously and stably heat multiple temperature gradients in cell thermal shift assays. [Overview of the project] [Problems that the invention aims to solve]
[0006] This invention solves the above-mentioned problems and provides a multi-temperature gradient heating device for use in oleanolic acid cell thermal shift assays. This enables synchronous stable heating in multiple different temperature ranges during a single experimental operation, satisfying the multi-temperature gradient synchronization requirements of cell thermal shift assays and improving the consistency and comparability of experimental data. [Means for solving the problem]
[0007] To achieve the above objective, this invention employs the following technical solution: The multi-temperature gradient heating device used in the oleanolic acid cell thermal shift assay is pedestal; A partition frame, installed on the aforementioned base and composed of a plurality of partition walls arranged in parallel, forming an independent heating compartment between two adjacent partition walls; A plurality of heating blocks, the same number as the number of heating compartments, wherein each heating block is installed in a corresponding heating compartment and each heating block is independently temperature controllable; An insulating sheet installed vertically between two adjacent partition walls to block heat exchange between adjacent heating compartments; A positioning cover that covers the top of a compartment frame, wherein the positioning cover is provided with through holes for positioning reaction tubes corresponding to the positions of each heating compartment; It is characterized by including.
[0008] In a preferred embodiment, the heating block is made of a metal material with high thermal conductivity, a resistance wire for generating heat is embedded inside the heating block, and a sample groove for accommodating a reaction tube is provided on the upper surface of the heating block.
[0009] In a preferred embodiment, the heat insulating sheet is made of a high-temperature resistant material with low thermal conductivity, and a reflective layer is provided on the surface of the heat insulating sheet facing the heated compartment to reduce heat radiation transfer.
[0010] In a preferred embodiment, the positioning cover is made of an engineering plastic with a low thermal conductivity. [Effects of the Invention]
[0011] This invention enables the synchronous construction of multiple non-interfering temperature environments on the same experimental platform by arranging multiple independent heating blocks in separate heating compartments enclosed by partitions and insulating sheets. Each heating block can be connected to an external, independently controllable power supply for output adjustment, thereby setting different target temperatures. The insulating sheets effectively block heat conduction and radiation between adjacent heating compartments, and the positioning covers, with their low thermal conductivity, ensure that significant heat is not transferred between heating compartments. This structural design allows the heat treatment of oleanolic acid and breast cancer cell samples at multiple pre-set temperature points to be completed synchronously during a single experimental operation, meeting the core needs of multi-temperature gradient synchronous processing required by cell thermal shift assay technology.
[0012] This invention employs a design concept that combines physical separation and independent heating, significantly shortening the experimental cycle of cell thermal shift assays. While conventional batch temperature control methods require multiple repeated heating operations, this invention allows for the synchronized completion of sample processing at all temperature points within a single experimental process. This avoids the time spent on multiple setup, waiting, and operation steps. This not only improves experimental efficiency but also reduces the time burden on the operator, making the workflow for screening or validation experiments of target protein interactions with drug molecules such as oleanolic acid more compact and rapid.
[0013] This invention effectively enhances the comparability and consistency of experimental data by eliminating variations in experimental conditions caused by batch processing. Since all samples under a temperature gradient are processed under the same apparatus, for the same amount of time, and in the same external environment (excluding the set temperature), it minimizes potential differences in ambient temperature between batches, minute changes in instrument condition, and systematic errors introduced by the operator. These highly consistent processing conditions provide stronger direct comparability for protein thermal stability data obtained from different temperature points, offering a reliable basis for accurately evaluating the binding ability of oleanolic acid to target proteins. [Brief explanation of the drawing]
[0014] [Figure 1] Figure 1 is a schematic diagram showing the overall structure of the present invention. [Figure 2] Figure 2 is a schematic diagram showing the local structure of the present invention. [Figure 3] Figure 3 is a schematic diagram showing the local structure of the present invention. [Modes for carrying out the invention]
[0015] The embodiments of this invention will be described in detail below with reference to the drawings. Please refer to Figures 1 to 3.
[0016] This invention provides a multi-temperature gradient heating apparatus for use in oleanolic acid cell thermal shift assays. The apparatus as a whole has a rectangular plate structure, and its main components include a base 1, a partition frame 2, a heating block 3, an insulating sheet 4, a positioning cover 5, a terminal block 6, a heat-insulating casing 7, and support legs 8. The physical assembly between each component achieves thermal isolation, independent temperature control, and sample positioning functions, making it applicable to synchronized heat treatment experiments of breast cancer cells and oleanolic acid under multiple set temperatures.
[0017] Base 1 is a rectangular, flat plate structure made of a thermal insulation material with good mechanical strength. The top surface of base 1 is provided with multiple parallel mounting grooves along its length, the number of which corresponds to the number of heating compartments to be installed subsequently. Each mounting groove has a rectangular cross-section, and its inner bottom surface is flat and smooth, used to support and tightly seal the lower surface of the heating block 3. Support legs 8 are fixedly connected to each of the four corners of the base 1, and rubber pads are attached to the lower end of each support leg 8 to enhance the stability of the entire apparatus when it is placed on the laboratory bench and to effectively block external vibrations from affecting the cell sample.
[0018] The heat insulation casing 7 surrounds the outside of the partition frame 2. The heat insulation casing 7 is integrally molded from a polyurethane foam material and has a low thermal conductivity coefficient and good structural strength. A plurality of terminal blocks 6 are fixedly installed on the heat insulation casing 7. The terminal block 6 is made by integrally sintering and molding a ceramic insulating material, and terminal posts are provided on its surface. The terminal posts are electrically connected to the ends of the resistance wires drawn out from the inside of the heating block 3 through wires, realizing power supply to the heating block 3 by an external power source.
[0019] The partition frame 2 is installed on the upper surface of the pedestal 1 and is composed of a plurality of adjacent partitions installed in parallel. All the partitions are made by integrally injection molding a high-temperature resistant engineering plastic. An independent heating compartment is formed between two adjacent partitions. The width of each heating compartment is 20 mm, the length is 50 mm, and the height is constant, ensuring that each heating section has the same heat capacity characteristics. The bottom of each heating compartment faces one mounting groove on the pedestal 1 and is used to accommodate the corresponding heating block 3.
[0020] The number of heating blocks 3 is the same as the number of heating compartments. Each heating block 3 is installed in the corresponding heating compartment, and its outer dimensions match the internal space of the heating compartment. The heating block 3 is made by processing an aluminum alloy material with high thermal conductivity. A spiral resistance wire is embedded inside each heating block 3. This resistance wire is arranged in a layered spiral along the thickness direction of the heating block 3, realizing uniform distribution of heat quantity inside the heating block 3. Both ends of the resistance wire are drawn out from the side walls of the heating block 3 respectively, penetrate through the heat insulation casing 7, and finally are connected to the terminal posts on the corresponding terminal block 6. A sample groove is provided at the center position of the upper surface of the heating block 3. The sample groove is a circular concave groove with a depth of 5 mm and a diameter of 12 mm, and is used to place a reaction tube containing a mixture of oleanolic acid and breast cancer cells.
[0021] The number of the heat insulation sheets 4 is equal to the number of the gaps between adjacent partition walls. Each heat insulation sheet 4 is installed perpendicular to the gap between two adjacent partition walls. The heat insulation sheet 4 is made of mica material, with a thickness of 2 mm, and has excellent high temperature resistance performance and low thermal conductivity. Furthermore, reflective aluminum films are coated on both side surfaces of the heat insulation sheet 4, and this reflective layer significantly reduces the energy exchange by the heat radiation method between adjacent heating sections, and improves the temperature independence of each heating section.
[0022] The positioning cover 5 covers the top of the section frame 2. Its outer dimensions match the top surface contour of the section frame 2. The positioning cover 5 is made by processing polyether ether ketone (PEEK) engineering plastic with a low thermal conductivity coefficient, and has good mechanical strength and heat resistance. The lower surface of the positioning cover 5 contacts the top surface of the section frame 2, and its main function is to provide vertical positioning and support for the reaction tubes to be inserted. Due to its low thermal conductivity characteristics, it does not form an effective heat conduction path between adjacent heating blocks 3 with different temperatures, thereby avoiding heat interference between heating sections. The positioning cover 5 is provided with through holes corresponding to the positions of each sample groove. The diameter of the through hole is slightly larger than the outer diameter of a standard reaction tube, enabling the reaction tube to be inserted vertically into the sample groove and maintaining stable positioning, and preventing clogging phenomena due to thermal expansion.
[0023] An opening is provided at the top of the heat preservation casing 7, and the edge of this opening is in the same plane as the outer edge of the positioning cover 5, which not only does not affect the insertion operation of the reaction tube, but also maximally reduces the heat loss from the entire device to the environment, improves the energy utilization efficiency, and assists in maintaining the temperature stability of the heating section.
[0024] In actual use, the experimenter first inserts multiple reaction tubes containing a mixture of oleanolic acid and breast cancer cells into their respective through-holes on the positioning cover 5, ensuring that the bottoms of the reaction tubes are fully fitted into the corresponding sample grooves. Then, the output terminals of external, independently controllable power supplies are connected to their corresponding terminal posts on the terminal block 6, supplying power independently to each heating block 3. Based on the experimental design requirements, pre-calibration or adjustment of the output of each power supply allows each heating block 3 region to be adjusted to different target temperatures, such as 37°C, 42°C, or 45°C. After power is supplied, the resistance wires inside each heating block 3 generate heat, and the heat is conducted through the heating block 3 body to the sample groove region on the upper surface. Thermal insulation between each heating section is achieved primarily by the insulating sheet 4 and reflective layer. The low thermal conductivity positioning cover 5 ensures that the cover itself does not cause significant lateral heat conduction. The heat-insulating casing 7 effectively suppresses heat loss throughout the apparatus, and the support legs 8 ensure the apparatus remains stable during the experimental process.
[0025] This device can synchronously construct multiple independent, relatively stable temperature environments during a single experimental operation, meeting the technical requirements of multi-temperature gradient synchronization processing demanded by cell thermal shift assays. Each component integrates its function through physical structural design, achieving multi-temperature gradient synchronization processing primarily through physical separation and independent heating. This significantly shortens the experimental cycle of cell thermal shift assays, eliminates variations in experimental conditions caused by batch operations, and enhances data comparability.
[0026] The following describes an example of an experimental method for elucidating the direct molecular target and mechanism of oleanolic acid's anti-breast cancer effect.
[0027] First, using chemical proteomics technology, functional probes containing photoaffinity labeling and alkynyl groups are designed and synthesized. After activity verification, specific binding proteins are screened by competitive affinity pull-down binding quantitative mass spectrometry. Next, drug-target interaction dynamics parameters are quantitatively measured using surface plasmon resonance technology, intracellular target binding is verified using a cell thermal shift assay, and target expression regulation is detected by Western blotting. Finally, target knockdown / overexpression cell models are constructed, and techniques such as CCK-8, flow cytometry, Transwell, and Western blotting are comprehensively applied through a four-group comparison system to systematically evaluate the central role of the target in cell proliferation, apoptosis, migration / invasion ability, and mitochondrial function.
[0028] The specific method is as follows: 1. Target search Functional OA probes are designed and structurally validated. Then, in a cell system, probe-treated groups, a competition group (probe + free OA), and a control group are established. OA-specific binding proteins are screened through UV crosslinking, click reaction labeling, affinity enrichment, and quantitative mass spectrometry. Finally, through bioinformatics comparison, specific binding proteins significantly reduced in the competition group are screened, thereby identifying highly reliable candidate targets of action.
[0029] 2. Interaction Verification 2.1. Surface Plasmon Resonance Technology: In a cell-free system, purified target proteins are immobilized on the surface of a chip, an oleanolic acid solution is flowed over the chip, and the binding dynamics parameters and affinity between the two are monitored and quantitatively analyzed in real time to obtain the equilibrium dissociation constant.
[0030] 2.2. Cellular thermal shift assay: The process is as described above, after which the thermal shift curve is analyzed and the change in melting point temperature (ΔTm) is calculated. A significant increase in thermal stability can be strong evidence of direct binding.
[0031] 2.3. Verification of Target Expression Levels: Breast cancer cells are treated with a concentration gradient of oleanolic acid, and changes in target protein expression are detected using Western blotting techniques. Quantitative protein expression levels are analyzed to evaluate the effect of drug treatment on target expression.
[0032] 3. Functionality check 3.1. Genetically engineered cell line construction: Using siRNA technology, we will construct breast cancer cell lines that stably knock down or overexpress candidate targets, providing a model for subsequent loss-of-function / gain-of-function experiments.
[0033] 3.2. The following four-group experimental comparisons will be established: NC+Vehicle (negative control + solvent), NC+OA-L (negative control + low dose of oleanolic acid), NC+OA-H (negative control + high dose of oleanolic acid), and si / OE-X+OA-H (targeted knockdown / overexpression + high dose of oleanolic acid). By comparing the third and fourth groups, the target dependence of drug efficacy will be directly investigated.
[0034] 3.3.Cell phenotypic analysis Growth detection: A growth curve is created using the CCK-8 method, and the 50% inhibitory concentration is calculated. Apoptosis detection: The Annexin V-FITC / PI double staining method is employed, and the rate of cell apoptosis is quantitatively detected using a flow cytometer. Assessment of migration / invasion ability: The healing rate is calculated using a cell scratch test, and the number of membrane-penetrating cells is counted to evaluate migration and invasion ability, respectively.
[0035] 3.4. Assessment of Mitochondrial Function The JC-1 probe is used to detect dynamic changes in mitochondrial membrane potential (red / green fluorescence ratio), the DCFH-DA probe is used to evaluate total intracellular reactive oxygen species levels, and the MitoSOX probe is used to specifically detect mitochondrial superoxide production.
[0036] 3.5. Protein and gene-level detection The expression and activation levels of apoptosis-related key proteins are detected through Western blotting and quantitative PCR. [Explanation of Symbols]
[0037] 1 base 2-section frame 3. Heating Block 4. Insulation sheet 5 Positioning cover 6 Terminal block 7. Insulated casing 8 Support legs
Claims
1. A multi-temperature gradient heating device used in a cell thermal shift assay for oleanolic acid, The base and A partition frame installed on a base, having multiple partition walls arranged in parallel, forming an independent heating compartment between two adjacent partition walls, A plurality of heating blocks, the same number as the number of heating compartments, wherein each heating block is installed in the corresponding heating compartment and each heating block is independently temperature controllable, An insulating sheet is installed vertically between two adjacent partition walls to block heat exchange between adjacent heating compartments, A positioning cover that covers the top of the compartment frame, the positioning cover having through holes for positioning reaction tubes corresponding to the position of each heating compartment, A multi-temperature gradient heating device characterized by comprising the following features.
2. The heating block is made of a metal material with high thermal conductivity, a resistance wire for generating heat is embedded inside the heating block, and a sample groove for accommodating a reaction tube is provided on the upper surface of the heating block. The multi-temperature gradient heating apparatus according to claim 1.
3. The aforementioned heat insulating sheet is made of a high-temperature resistant material with low thermal conductivity, and a reflective layer is provided on the surface of the heat insulating sheet facing the heated compartment to reduce heat radiation transfer. The multi-temperature gradient heating apparatus according to claim 1.
4. The positioning cover is made of engineering plastic with a low thermal conductivity. The multi-temperature gradient heating apparatus according to claim 1.