Preparation method and detection method of Raman detection sample of cell culture fluid
Through the removal of impurity, cryopreservation and control of the drying environment in the preparation method, combined with aluminum-plated quartz slides and Raman spectroscopy, the problem of high cost and slow speed in cell culture liquid detection is solved, and efficient and stable metabolism is achieved object analysis.
Patent Information
- Application Number
- CN202411608570.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2024-12-27
AI Technical Summary
The existing cell culture solution detection methods are costly, complex sample processing, slow detection speed and high technical requirements, making it difficult to effectively monitor cell metabolism changes. Especially in Raman spectroscopy detection, weak spectrum and small metabolites are difficult to observe. .
A Raman detection sample preparation method of cell culture medium, including collecting the culture medium for impurity removal and cryopreservation, using aluminum-plated quartz slides as a slide, controlling the drying environment humidity ≤60% and the temperature 20℃-25℃ , and samples were prepared by centrifugation and dropping steps, and tested in combination with Raman spectroscopy.
It improves the efficiency and stability of sample detection, reduces experimental errors, and realizes rapid and non-invasive analysis of metabolites in cell culture medium, reducing detection costs.
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Abstract
Description
Technical Field
[0001] The present application relates to the field of chemical analysis and detection, and specifically to a method for preparing a Raman detection sample of a cell culture fluid and a method for detecting the same. Background Art
[0002] Cell culture medium provides various nutrients required for cell development and reproduction, and contains metabolic information during cell growth. Studying cell metabolism can monitor cell reactions and accurately discover the characteristics of cell populations and their metabolic changes. Cell metabolomics has been applied to tumors, immunity, drug effects, etc. Common cell metabolomics analysis methods include mass spectrometry, chromatography, and fluorescence, which have the characteristics of high detection sensitivity and accurate quantification, but have defects such as high cost, complex sample processing, slow detection speed, and high detection technology requirements. In response to these shortcomings, Raman spectroscopy can detect the changes in the intensity and wavelength position of the functional groups of molecules in the sample after absorbing electromagnetic rays. It supports the rapid and non-invasive analysis of biochemical molecules in cells, body fluids, and culture fluids at low cost.
[0003] Raman spectroscopy is a type of scattering spectrum. Its principle is that when the laser interacts with the sample to be tested, the light scattered by the molecules will change in frequency. By analyzing the scattered spectrum with a different frequency from the incident light, the molecular vibration information of the sample can be obtained, and the composition, structure and properties of the sample can be analyzed. The application range of Raman spectroscopy covers various fields such as medicine, physics, chemistry and biology, and it is of great value for purely qualitative analysis, highly quantitative analysis and molecular structure research.
[0004] Raman spectra are weak in nature, and the changes in metabolites in cell culture fluid are small. Direct detection of liquid samples may not be able to observe metabolic changes. Therefore, when Raman detection of complex liquids such as cell culture fluids, liquid sample preparation is particularly important for Raman detection. Summary of the invention
[0005] Based on this, an embodiment of the present application provides a sample preparation method for Raman detection of cell culture fluid, which can improve the efficiency and stability of sample Raman detection.
[0006] The technical solutions include the following:
[0007] A method for preparing a test sample of a cell culture fluid comprises the following steps:
[0008] The culture fluid produced during the cell culture process is collected, impurities are removed, and the culture fluid is dropped onto a slide, and allowed to stand and dry;
[0009] The dry environment has a humidity of ≤60% and a temperature of 20℃-25℃.
[0010] In one embodiment, the dry environment has a humidity of 35%-55% and a temperature of 20.3°C-22.5°C.
[0011] In one embodiment, the slide is an aluminum-coated quartz glass slide.
[0012] In one embodiment, the aluminum-coated quartz glass slide is prepared by a vacuum evaporation coating method.
[0013] In one embodiment, the sample preparation method has a drying time of 20 min-30 min.
[0014] In one embodiment, in the sample preparation method, the impurity removal step includes centrifuging the collected culture fluid and taking the middle layer liquid.
[0015] In one embodiment, the sample preparation method further comprises a step of freezing and preserving the culture fluid produced during the cell culture process after collecting the culture fluid;
[0016] In one embodiment, after cryopreservation, a thawing step is further included before centrifugation.
[0017] In one embodiment, the centrifugal conditions include: a rotation speed of 5000 rpm-5500 rpm and a time of 25s-35s.
[0018] In one embodiment, in the sample preparation method, the volume of the cell culture fluid dropped onto the slide is 1.0 μL-2.0 μL.
[0019] A method for detecting a cell culture fluid, the method comprising the following steps:
[0020] preparing a sample using the preparation method; and
[0021] The samples were tested using Raman spectroscopy.
[0022] In one embodiment, the detected parameters include one or more of the following:
[0023] Laser wavelength 532 nm,
[0024] Laser exposure time 4 s-10 s,
[0025] Cumulative number of times 6-15 times, and
[0026] The laser power is 5 mW-15 mW.
[0027] Compared with the traditional technology, this application has the following beneficial effects:
[0028] The preparation of Raman spectroscopy detection samples of cell culture fluid provided in the present application standardizes the environmental conditions when preparing Raman drying samples, reduces experimental errors caused by different sample preparation conditions, and greatly increases the efficiency and stability of sample detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.
[0030] Figure 1 Schematic diagram of an aluminum-coated quartz glass slide;
[0031] Figure 2 A schematic diagram of a sample preparation and detection method for Raman detection of cell culture fluid;
[0032] Figure 3 The spectrum of cell culture fluid detected when the ambient temperature for sample preparation was 20.34 °C and the air humidity was 36%;
[0033] Figure 4 The spectrum of the culture solution obtained when the sample preparation environment temperature was 22.4 ℃ and the air humidity was 46%;
[0034] Figure 5 The spectrum of the culture solution obtained when the sample preparation environment temperature was 20.4 ℃ and the air humidity was 55%;
[0035] Figure 6 The spectrum of cell culture medium is at 1003 cm -1 The peak intensity at , wherein 1-1 to 1-5 are the Raman spectra of Example 1, 2-1 to 2-5 are the Raman spectra of Example 2, and 3-1 to 3-5 are the Raman spectra of Example 3. DETAILED DESCRIPTION
[0036] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.
[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.
[0038] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.
[0039] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.
[0040] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.
[0041] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".
[0042] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.
[0043] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.
[0044] In the present application, exemplary descriptions such as "in a specific example" and "in one of the embodiments" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form a new technical solution.
[0045] In this application, the terms "first", "second", etc. in "the first aspect", "the second aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.
[0046] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.
[0047] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.
[0048] In this article, metabolomics is a part of systems biology, which is a qualitative and quantitative analysis of all low molecular weight metabolites of a certain organism or cell in a specific physiological period, and a study method to find the relative relationship between metabolites and physiological and pathological changes. It mainly studies small molecule metabolites that are substrates and products of various metabolic pathways.
[0049] In this article, molecular vibration is one of the forms of internal motion of molecules. Raman is a spectroscopic technique that studies molecular structure by analyzing the motion of molecules such as rotation, vibration and stretching.
[0050] In this article, vacuum evaporation coating refers to a method in which a high-purity coating material is heated and evaporated into a gaseous state at a high temperature under certain vacuum conditions, and the vapor flow flies and sputters onto the surface of a glass substrate to condense into a solid thin film.
[0051] Raman spectra are weak in themselves, and the changes in metabolites in cell culture fluids are small. Direct detection of liquid samples may not allow for the observation of metabolic changes. In addition, in Raman detection, substrates are frequently used as detection consumables. It is particularly important to select substrates with low cost and little substrate interference. Therefore, when detecting complex liquids such as cell culture fluids, the liquid samples can be concentrated and prepared, and it is further required that the supporting substrate does not interfere with the scattering spectrum.
[0052] Therefore, one embodiment of the present application provides a method for preparing a test sample of a cell culture fluid, comprising the following steps:
[0053] The culture fluid produced during the cell culture process is collected, treated to remove impurities, dropped onto a slide, and allowed to stand to dry.
[0054] In a specific example, the method for detecting the sample is Raman spectroscopy.
[0055] In a specific example, the dry environment has a humidity of ≤60% and a temperature of 20°C-25°C. This environmental condition is consistent with the use environment of the instrument, and the inventors have found through multiple experiments that when the humidity of the sample preparation environment exceeds 60% and the temperature exceeds 20°C-25°C, the detection efficiency of the sample is affected. The significance of preparing and testing under this environmental condition is that after standardizing the preparation environment, the efficiency and stability of sample detection are greatly increased.
[0056] In a specific example, the humidity of the dry environment can be, but is not limited to, 5%, 10%, 20%, 30%, 35%, 36%, 37%, 38%, 39%, 40%, 42%, 44%, 45%, 46%, 48%, 50%, 52%, 55%, 56%, 58%, 60%, or a range between any two of the above values. In a specific example, the temperature can be, but is not limited to, 20°C, 20.4°C, 20.5°C, 21°C, 21.4°C, 21.5°C, 22°C, 22.4°C, 22.5°C, 23°C, 23.4°C, 23.5°C, 24.5°C, 25°C, or a range between any two of the above values.
[0057] In a specific example, the dry environment has a humidity of 35%-55% and a temperature of 20.3°C-22.5°C.
[0058] In a specific example, the cell culture solution is allowed to stand in a dry environment for 20 min to 30 min.
[0059] In a specific example, a cell culture fluid is collected after the cells have been cultured for a period of time. The culture fluid is a waste culture fluid in the process of cell development. The culture fluid contains essential nutrients such as amino acids, glucose, and serum proteins in the process of cell development.
[0060] In a specific example, the collected cell culture fluid cannot be immediately subjected to Raman detection. In order to prevent the metabolite components of the sample from deteriorating, the collected cell culture fluid may be frozen and stored. Before preparing the test sample, the cell culture fluid may be completely thawed and then centrifuged after thawing.
[0061] In a specific example, the cryopreservation temperature is -72°C to 86°C, which can be -72°C, -80°C or -86°C.
[0062] In a specific example, the temperature of thawing the cell culture solution is 20° C.-25° C., which can be 20° C., 21° C., 22° C., 23° C., 24° C. or 25° C.
[0063] In a specific example, the step of removing impurities includes centrifuging the collected cell culture fluid, separating the cell culture fluid from the mineral oil and impurity layers in the sample, and taking the middle layer liquid as the cell culture fluid to be tested.
[0064] In a specific example, the volume of the collected culture fluid is 3 μL-10 μL.
[0065] In a specific example, the centrifugal conditions include a rotation speed of 5000 rpm-5500 rpm and a time of 25s-35s. The above rotation speed and time can effectively remove impurities or grease in the sample to prevent impurities or grease from interfering with the detection. In a specific example, the centrifugal conditions include a rotation speed of 5500 rpm and a time of 30s.
[0066] In a specific example, the middle layer of the cell culture fluid after centrifugation is dropped vertically onto a slide (Raman substrate).
[0067] In a specific example, the volume of the cell culture fluid dropped on the slide is 1.0 μL-2.0 μL, which can be 1.0 μL, 1.2 μL, 1.3 μL, 1.4 μL, 1.5 μL, 1.6 μL, 1.7 μL, 1.8 μL, 1.9 μL, 2.0 μL, or a range between any two of the above values. In a specific example, using 1.0 μL-2.0 μL of liquid sample can support Raman detection of trace samples. And greatly increase the utilization rate of the substrate slide, one slide can complete the preparation of 60-90 samples.
[0068] In a specific example, the slide is an aluminum-coated quartz glass slide. Using an aluminum-coated quartz glass slide as a Raman detection substrate reduces the substrate background of Raman scattering, has low cost, is not fragile, and can be applied to Raman detection of a large number of liquid samples.
[0069] Glass slides, as Raman detection substrates, have strong fluorescence bands and strong background noise, which will interfere with the detection and may cover up some peak information. However, quartz slides covered with tin / aluminum foil have poor fixation, low flatness, and low reflection efficiency.
[0070] Characteristics of aluminum-coated quartz glass slides: Compared with ordinary glass slides, aluminum-coated glass slides have stronger light reflection ability and can detect as many different wavelengths of light as possible. The surface is easy to clean and contaminants attached to the substrate can be easily removed. They can provide a clear background and reduce the interference of background signals.
[0071] In a specific example, an aluminum-coated quartz glass slide is prepared by vacuum evaporation coating method, which has a highly reflective mirror surface, reduces the refraction loss of light, and enables the Raman scattered light of the sample to be incident on the detector to the greatest extent. A dark mark is placed on a corner of the aluminum-coated glass slide to distinguish the front and back of the glass slide, such as Figure 1 As shown, the black dots in the figure are used to distinguish the front and back sides of the slide.
[0072] The method for preparing a sample for cell culture fluid detection based on Raman technology of the present application uses an aluminum-plated quartz glass slide as a substrate and concentrates the liquid sample to ensure the standardization and stability of Raman detection results by controlling the preparation process and environmental conditions.
[0073] This embodiment provides a method for detecting a cell culture fluid, the method comprising the following steps:
[0074] preparing a sample using the above-mentioned preparation method; and
[0075] The sample is detected by Raman spectroscopy.
[0076] There are clear requirements for the humidity and temperature of the testing environment to reduce batch errors during the testing process and ensure the consistency and stability of Raman testing. Ambient humidity has an important impact on Raman testing results: when the ambient humidity is too high (over 60%), it affects sample drying and testing efficiency.
[0077] In one specific example, the detecting includes acquiring a Raman spectrum of the sample using a confocal Raman instrument.
[0078] In a specific example, the acquisition parameters are a laser wavelength of 532 nm, a laser exposure time of 4 s-10 s, a cumulative number of 6-15 times, and a laser power of 5 mW-15 mW.
[0079] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.
[0080] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
[0081] Example 1
[0082] A method for preparing a sample for Raman detection of a cell culture fluid comprises the following steps:
[0083] (1) Collection of cell culture medium: Collect 3 μL-10 μL of discarded culture medium from mouse embryonic cell culture.
[0084] (2) The culture medium was not tested immediately after collection. To prevent the metabolite composition of the sample from deteriorating, the cell culture collected in step (1) was immediately placed in a -80°C freezer for storage.
[0085] (3) Before preparing the dried sample to be tested, the cell culture medium in step (2) needs to be pretreated. The specific processing steps are as follows:
[0086] a. Thawing cell culture medium: Place the cell culture medium at 20℃-25℃ to completely thaw;
[0087] b. Centrifugation of cell culture medium: Separate the culture medium from the mineral oil and impurity layer in the sample taken in step (1) at a centrifuge speed of 5000 rpm for 30 s.
[0088] (4) Use a pipette to slowly aspirate 1.5 μL of the middle layer of the sample after centrifugation in step (3) and drop it vertically onto the Raman substrate.
[0089] (5) The culture medium sample was placed in an environment of 20.3°C and 36% humidity to dry for 20 min.
[0090] In this environment, a confocal Raman instrument was used to collect Raman spectra of the dried samples. The acquisition parameters were a laser wavelength of 532 nm and a laser power of 5 mW. The laser exposure time for spectrum acquisition was 4 seconds, the number of accumulations was 15 times, and 5 points were randomly selected for detection to obtain 5 Raman spectra.
[0091] The schematic diagram of the sample preparation and detection method of Raman detection of cell culture fluid is as follows Figure 2 shown.
[0092] Example 2
[0093] The contents are substantially the same as those of Example 1, except that in step (5), the culture solution sample is dried at 22.4° C. and 46% humidity and the spectrum is detected.
[0094] Example 3
[0095] The contents are substantially the same as those of Example 1, except that in step (5), the culture solution sample is dried at 20.4° C. and 55% humidity and the spectrum is detected.
[0096] Example 1-Example 3 obtains the average spectrum of 5 spectra in each group, see Figure 3-Figure 5 The shadows represent the standard deviation. -1 Indicates aromatic ring breathing, which usually does not change with the conformation of the substance. Compare 15 Raman spectra at 1003 cm -1 The Raman intensity at Figure 6 ), and the Pearson and Spearman correlations of the 15 full spectra (Table 1) are all above 0.95, indicating that using the sample preparation method of the present application for Raman detection can obtain relatively stable Raman detection results.
[0097] Table 1 Pearson and Spearman correlations of culture medium spectra
[0098]
[0099] Comparative Example 1
[0100] The contents are substantially the same as those of Example 1, except that in step (5), the culture solution sample is dried at 20.3° C. and 65% humidity and the spectrum is detected.
[0101] In this environment, the sample cannot be dried smoothly and cannot be detected.
[0102] Even if the samples are dried in a drying oven (20℃-25℃) and then tested in a 65% humidity environment, the samples in the same batch may not be tested due to moisture regain, affecting the test efficiency and stability of the samples.
[0103] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0104] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.
Claims
1. A method for preparing a test sample of a cell culture fluid, characterized in that: The steps include: The culture fluid produced during the cell culture process is collected, impurities are removed, and the culture fluid is dropped onto a slide, and allowed to stand and dry; The dry environment has a humidity of ≤60% and a temperature of 20℃-25℃.
2. The preparation method according to claim 1, characterized in that: The dry environment has a humidity of 35%-55% and a temperature of 20.3℃-22.5℃.
3. The preparation method according to claim 1, characterized in that: The slide is an aluminum-coated quartz glass slide; Optionally, the aluminum-coated quartz glass slide is prepared by a vacuum evaporation coating method.
4. The preparation method according to any one of claims 1 to 3, characterized in that The drying time is 20min-30min.
5. The preparation method according to any one of claims 1 to 3, characterized in that: The step of removing impurities includes centrifuging the collected culture fluid and taking the middle layer liquid.
6. The preparation method according to any one of claims 1 to 3, characterized in that: After collecting the culture fluid produced during the cell culture process, the method further includes a step of freezing and preserving the culture fluid; Optionally, after cryopreservation, a thawing step is further included before centrifugation.
7. The preparation method according to claim 6, characterized in that: The centrifugal conditions include: a rotation speed of 5000 rpm-5500 rpm and a time of 25 s-35 s.
8. The preparation method according to any one of claims 1 to 3 and 7, characterized in that: The volume of the cell culture solution dropped onto the slide is 1.0 μL-2.0 μL.
9. A method for detecting a cell culture fluid, characterized in that: The method comprises the following steps: Preparing a sample using the preparation method according to any one of claims 1 to 8; and The samples were tested using Raman spectroscopy.
10. The method according to claim 9, characterized in that The parameters tested include one or more of the following: Laser wavelength 532 nm, Laser exposure time 4 s-10 s, Cumulative number of times 6-15 times, and The laser power is 5 mW-15 mW.