Detection method for rapid real-time drug effect evaluation and application thereof

Abstract

The invention discloses a detection method for rapid real-time drug effect evaluation and application of the detection method. The detection method comprises the following steps: providing a quantitative corresponding relation between a same-generation calibration change value of electrical characteristics caused by physiological changes such as cell same-representative characteristics and the like and the cell proliferation ability; adhering to-be-detected cells to the surface of the same membrane electrode; applying the medicine to be detected, and observing the same generation measurement change value of the electrical characteristics in a cell proliferation cycle; and based on the quantitative corresponding relationship, obtaining the drug effect of the corresponding real-time proliferation characteristic evaluation drug. According to the detection method provided by the invention, the physiological changes such as the same-representative characteristics of the adherent cells are monitored through the membrane electrode, and after the quantitative corresponding relation between the change indexes of the same-representative characteristics of the cells and the proliferation capacity of the cells is established, the cell proliferation capacity is detected; the change of the cell proliferation capacity can be predicted through physiological changes such as the same representative characteristics of the cells before proliferation and apoptosis of the cells, so that the judgment period of the influence of the medicine on the cells can be greatly shortened, and the progress of medicine evaluation and research and development can be remarkably accelerated.

Description

Technical Field

[0001] This invention relates to the field of drug efficacy evaluation technology, and in particular to a detection method for rapid real-time drug efficacy evaluation and its application. Background Technology

[0002] Pharmacodynamic evaluation is a core step in drug development, aiming to verify the mechanism of action and therapeutic effect of candidate drugs on target diseases, and to provide key efficacy evidence for subsequent clinical trials and clinical applications. Analyzing and evaluating drug efficacy is an indispensable part of the drug development process, and it is divided into in vitro pharmacodynamic evaluation, animal testing, and clinical trials. Among these, in vitro pharmacodynamic evaluation has the advantages of avoiding ethical controversies and facilitating the study of drug mechanisms of action, and is widely used in the field of drug development.

[0003] At the cellular level, cell proliferation inhibition is a key indicator for assessing drug efficacy. However, existing detection methods generally suffer from limitations such as cumbersome operation, insufficient sensitivity, and difficulty in real-time dynamic monitoring, and cannot achieve early and rapid prediction of drug efficacy. There is an urgent need to develop efficient and accurate new detection technologies.

[0004] The existing evaluations mainly include:

[0005] Microscopic counting, a method that typically involves observing and counting cells under a microscope after drug application to assess the drug's effect on cell proliferation, requires frequent microscopic observation and manual counting under specific environmental conditions (such as shockproof, temperature and humidity-controlled environments), making it complex and time-consuming. Cell proliferation / inhibition is a dynamic process, and key stages of change are easily missed. Achieving observable changes in cell number requires at least 10-24 hours, necessitating multiple sampling observations, resulting in low efficiency. Improper observation timing (such as insufficient or excessive changes in cell number) or optical interference during cell counting can lead to inaccurate results. This method relies heavily on manual operation, is cumbersome, and prone to subjective errors; it cannot achieve real-time, continuous monitoring; and the results are significantly delayed (requiring more than 10-24 hours to observe noticeable changes).

[0006] Enzyme activity assays (such as the MTT assay): This method requires pre-setting multiple fixed time points for endpoint detection, and cannot reflect continuous changes in cell state. Each time point requires a certain number of cells (usually 10³-10⁴ cells / well) to generate a detectable signal, limiting sensitivity. Multi-point sampling is cumbersome, prolonging the experimental cycle, and data from discrete time points cannot accurately capture the dynamic process of drug action. This multi-point sampling and discrete detection result in significant response delays, leading to lengthy experimental cycles and a lack of real-time performance and accuracy.

[0007] The fundamental limitation of these methods is that they can only obtain a single or a few discrete indicators reflecting the cell state (e.g. final cell number, metabolic activity at a certain time point). However, the response of cells to drugs is a complex biological process, which is manifested through a series of interrelated "phenotypic characteristics". These characteristics encompass a variety of observable biological properties of cells under a specific environment (e.g. normal culture vs. drug treatment), including: i cCharacteristics) performance. These characteristics encompass a variety of observable biological properties of cells under a specific environment (e.g. normal culture vs. drug treatment), including:

[0008] Contemporaneous indicators: morphological structure (e.g. cell shape, size, arrangement), physical properties (viscoelastic characteristics, cell mechanical characteristics), physiological and biochemical reactions (e.g. metabolic products, enzyme activity, nutritional requirements), antigenicity, and ecological behavior, etc.

[0009] Iterative indicators: growth characteristics (e.g. growth rate, optimal temperature pH), etc.

[0010] A comprehensive understanding of the complete picture of drug action requires the comprehensive analysis of these rich and inseparable contemporaneous and iterative indicators. However, the current mainstream detection techniques (such as the above microscope method and MTT method) can usually only indirectly infer the drug efficacy by measuring a single isolated indicator, making it difficult to achieve multi-dimensional, real-time dynamic comprehensive characterization, which is the key reason for the lack of sensitivity, cumbersome operation, and inability to monitor and predict early. SUMMARY

[0011] In view of the deficiencies of the prior art, the purpose of the present application is to provide a detection method for rapid real-time drug efficacy evaluation and its application.

[0012] To achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application comprises:

[0013] In a first aspect, the present application provides a detection method for rapid real-time drug efficacy evaluation, which comprises:

[0014] providing a quantitative correspondence between the contemporaneous calibration change value of the electrical property of the thin film electrode caused by the change in the contemporaneous phenotypic characteristics of the selected cell variety and the cell proliferation ability;

[0015] adhering the to-be-tested cells to the surface of the same thin film electrode, wherein the to-be-tested cells belong to the selected cell variety;

[0016] applying a to-be-tested drug to the to-be-tested cells, and observing the contemporaneous measurement change value of the electrical property of the thin film electrode caused by the change in the contemporaneous phenotypic characteristics of the to-be-tested cells within one cell proliferation cycle;

[0017] Based on the quantitative correspondence, a real-time proliferation characteristic of the selected cell variety exhibited by the cells is obtained, and the drug efficacy is evaluated based on the real-time proliferation characteristic.

[0018] In a second aspect, the present application further provides an application of the above-mentioned detection method in the field of drug research and development.

[0019] Based on the above technical solution, compared with the prior art, the present application has at least the following beneficial effects:

[0020] The detection method provided by the present application detects the isogenic type characteristic change of the adherent cells by using a thin film electrode, and establishes a quantitative correspondence between the isogenic type characteristic change index of the cells and the cell proliferation capacity, so that the change of the cell proliferation capacity can be predicted by the isogenic type characteristic of the cells before the cells proliferate or undergo apoptosis (i.e. the number of cells changes) under the action of the drug. The method significantly shortens the determination period of the effect (especially the proliferation / inhibition effect) of the drug on the cells, thereby accelerating the drug evaluation and research and development process.

[0021] The above description is only a summary of the technical solutions of the present application. In order to enable those skilled in the art to more clearly understand the technical means of the present application, and to implement the content of the specification, the following describes the preferred embodiments of the present application with reference to the detailed description and accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 FIG. 1 is a structural schematic diagram of a bottom chip of a thin film electrode provided by a typical embodiment of the present application;

[0023] Figure 2 FIG. 2 is a structural schematic diagram of a top chip of a thin film electrode provided by a typical embodiment of the present application;

[0024] Figure 3 FIG. 3 is a structural schematic diagram of the overall structure of a thin film electrode provided by a typical embodiment of the present application;

[0025] Figure 4 FIG. 4 is a drug efficacy evaluation curve diagram of a cell drug provided by another typical embodiment of the present application;

[0026] Figure 5 FIG. 5 is another type of drug efficacy evaluation curve diagram of a cell drug provided by another typical embodiment of the present application;

[0027] Figure 6 FIG. 6 is still another type of drug efficacy evaluation curve diagram of a cell drug provided by another typical embodiment of the present application;

[0028] Figure 7 FIG. 7 is an IC50 consistency evaluation result of an ADCC drug provided by another typical embodiment of the present application;

[0029] Figure 8 Figure 4 is a comparative chart of the efficacy evaluation of the ADCC drug provided by another exemplary embodiment of the present application;

[0030] Figure 9 Figure 5 is a chart of the impedance-frequency variation relationship of the impedance test provided by yet another exemplary embodiment of the present application;

[0031] Figure 10 Figure 6 is a chart of the impedance-capacitance distribution curve of the impedance test at high and low frequencies provided by yet another exemplary embodiment of the present application. DETAILED DESCRIPTION

[0032] The morphological structure (such as cell shape, size, arrangement), physical characteristics (viscoelastic characteristics, cell mechanics characteristics), physiological and biochemical reactions (such as metabolic products, enzyme activity, nutritional requirements), antigenicity, and ecological behavior of cells under the action of drugs often change significantly earlier (within several hours or even shorter time) than the iterative indicators such as cell proliferation inhibition, and the inventors of the present application have confirmed through experiments that this same representative characteristic change trend has a high quantitative consistency with subsequent drug efficacy results (such as proliferation inhibition rate). However, the drug efficacy evaluation and detection methods provided by the prior art cannot capture such early and sensitive physiological change signals, which seriously restricts the rapid prediction of drug efficacy and the efficiency of mechanism research.

[0033] In view of the deficiencies in the prior art, the inventors of the present application have long-term research and a large amount of practice, and have finally proposed the technical solutions of the present application. The technical solutions, the implementation process and principles thereof will be further explained as follows.

[0034] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, and therefore, the scope of protection of the present application is not limited by the specific embodiments disclosed below.

[0035] Moreover, the relationship terms such as "first" and "second" are only used to distinguish one from another of the components or method steps having the same name, and do not necessarily require or imply any such actual relationship or sequence between the components or method steps.

[0036] In view of the above technical bottlenecks, especially the problem that the existing method has a lagging response and cannot achieve early prediction, the present application designs and develops a new type of cell culture and real-time dynamic sensing and detection method and device based on flexible nanobiological chip technology or other equivalent bioelectrical detection technology. The core function and advantage of this technical solution is "single signal source, double time-effect analysis".

[0037] Based on the technical idea, the embodiment of the present application provides a detection method for rapid real-time drug efficacy evaluation, which comprises the following steps:

[0038] A quantitative corresponding relationship between a same-generation calibration change value of an electrical characteristic of a thin film electrode caused by a same-generation characteristic change of a selected cell variety and a quantification of cell proliferation ability is provided;

[0039] The to-be-detected cells, which belong to the selected cell variety, are adhered to the surface of the same thin film electrode;

[0040] A to-be-tested drug is applied to the to-be-detected cells, and within one cell proliferation cycle, a same-generation measurement change value of the electrical characteristic of the thin film electrode caused by a same-generation characteristic change of the to-be-detected cells is observed;

[0041] Based on the quantitative corresponding relationship, a real-time proliferation characteristic of the selected cell variety exhibited by the to-be-detected cells corresponding to the same-generation measurement change value is obtained, and the drug efficacy is evaluated based on the real-time proliferation characteristic.

[0042] The technical solution provided by the present application uses a flexible nanobiological chip as a unified cell-electrical characteristic sensing platform. When cells grow, migrate or respond to drugs on the thin film electrode, the continuous changes in their physiological states such as morphological structure (such as cell shape, size, arrangement), physical characteristics (viscoelastic characteristics, cell mechanics characteristics), physiological and biochemical reactions (such as metabolic products, enzyme activity, nutritional requirements), antigenicity and ecological behavior will cause real-time changes in the chip electrical signal (impedance / capacitance). The electrical signal constitutes the core raw data stream of monitoring. On the chip surface, the capacitive signal is more sensitive than the traditional impedance-based sensor, and can timely capture the physiological changes of the same-generation characteristics of cells. Therefore, the representative embodiment of the present application adopts a capacitive signal as the monitoring data stream. Since the electrical characteristic detection of the thin film electrode is real-time, high-sensitivity and high-resolution, the thin film electrode can accurately capture the changes in the same-generation characteristics of adherent cells when the same-generation characteristics of adherent cells are affected by drugs, and can observe the influence of drugs on cells before the cells are affected by drugs and proliferate or undergo apoptosis. According to the extensive experiments of the inventor, the early physiological changes such as the same-generation characteristics of cells have a sufficient quantitative corresponding relationship with the changes in the proliferation ability of cells, so that by exploring the relevant data through pre-experiments to establish this corresponding relationship, the proliferation / inhibition of cells can be predicted in advance by detecting the physiological changes such as the same-generation characteristics of cells, so as to preliminarily evaluate the drug efficacy.

[0043] Specifically, in the early stage of drug action (for example, within several hours), the changes in cell isogenic characteristics and the like (reflecting the interaction between the cell and the membrane electrode) will cause specific high-frequency or weak time-domain characteristics of the electrical characteristic signals of the membrane electrode. Through data analysis, these early characteristic signals can be analyzed and quantified in real time and with high sensitivity, and they can be converted into physiological indicators of cell isogenic characteristics and the like; the core advantage of the present application lies in the "early warning" capability of this physiological indicator. Experiments have shown that this physiological dynamic change is significantly earlier than the observable change in the number of proliferating / suppressed cells, and its change trend is highly consistent with the subsequent final drug efficacy results (such as the proliferation inhibition rate). By continuously monitoring and analyzing these early electrical signal characteristics, the drug efficacy can be prospectively predicted in a very short time after the drug is applied, which greatly shortens the evaluation period and provides an opportunity for drug screening decisions.

[0044] In some embodiments, the process of establishing the quantitative correspondence relationship comprises:

[0045] The calibration cells of the selected cell variety are planted on the surface of the membrane electrode;

[0046] An external disturbance is applied to the calibration cells to change the cell proliferation ability of the calibration cells;

[0047] The isogenic electrical characteristic changes of the calibration cells within one proliferation cycle and the iterative electrical characteristic changes greater than one cell proliferation cycle are recorded by the membrane electrode;

[0048] The isogenic electrical characteristic changes are taken as the detection data reflecting the isogenic characteristics of the cells, and the iterative electrical characteristic changes are taken as the evaluation indicators of the cell proliferation ability, so as to establish the quantitative correspondence relationship.

[0049] In some embodiments, the process of establishing the quantitative correspondence relationship further comprises:

[0050] After a period of time greater than one cell proliferation cycle, biological detection of cell proliferation inhibition is performed on the calibration cells to verify and / or correct the quantitative correspondence relationship.

[0051] Based on this, in addition to the many advantages analyzed above, the technical method provided by the present application also has the advantage of whole-process monitoring: in the middle and late stages of drug action (for example, after several hours to several days), the main change trend of the electrical property signal is dominated by macro factors such as cell population number and adhesion area (i.e., traditional proliferation inhibition effect), the same capacitance data stream can be used, the signal characteristics in this stage can be analyzed by an algorithm, and the real-time, label-free and quantitative evaluation of the cell proliferation / inhibition state can be continued, the subsequent evaluation is optional, and the iterative data obtained can be used to verify the corresponding relationship between the cell isotype characteristic data and the cell proliferation data in the early stage, or after accumulating a large amount of data, the relationship can be continuously corrected.

[0052] Therefore, the unique value of the technical scheme provided by the present application lies in that: based on the same platform and the same continuous capacitance signal stream, the early cell isotype characteristic change can be analyzed to realize rapid prediction, and the late proliferation inhibition signal can be analyzed to realize final effect verification. This provides a dynamic pharmacodynamic evaluation system with complete time domain coverage (from early drug use to late drug use) and strong index correlation (isotype characteristic→proliferation inhibition), which can not only accelerate early decision-making, but also deeply reveal the dynamic process and dose-effect relationship of drug action.

[0053] In some embodiments, the selected cell species is an adherent cell.

[0054] In some embodiments, the electrical property of the thin film electrode includes impedance or capacitance

[0055] In some embodiments, the thin film electrode is selected from a variable capacitance micro-nano biological detection chip, which includes a fixed electrode plate and an elastic electrode plate, and the cells to be detected adhere to the side of the elastic electrode plate opposite to the fixed electrode plate.

[0056] The cell isotype characteristic is a comprehensive physiological change including morphological structure (such as cell shape, size, arrangement), physical property (viscoelasticity characteristic, cell mechanics characteristic), physiological and biochemical reaction (such as metabolic product, enzyme activity, nutritional requirement), antigenicity and ecological behavior, etc. These factors will change the property of the thin film electrode, resulting in a change in the capacitance of the variable capacitance micro-nano biological detection chip.

[0057] In specific embodiments, reference can be made to Chinese invention patent CN107192747A “A variable capacitance micro-nano biological detection chip and a processing method thereof” and SZ2024H0474U “A cell culture and detection device for rapid screening of antibodies” to prepare a flexible nano biological chip and a matching cell culture and detection device, and the basic structure is as follows Figures 1-3The cells are usually required to be surface-activated on the chip for cell adhesion, which is usually achieved by modifying extracellular matrix (ECM) proteins (e.g. fibronectin, actin, laminin, collagen, etc.) to facilitate cell adhesion, but the specific surface-activation method and the protein type are not limited to the examples shown here.

[0058] The above-mentioned capacitive chip has a high sensitivity to the isotype characteristics of cells, especially in the measurement of cell mechanical characteristics such as cell migration force, which is difficult for other bioelectric devices (e.g. many impedance-type devices).

[0059] In some embodiments, the process of surface-activation of the chip can include: according to the instructions of the commercial dithiobis(succinimidyl propionate) (DSP) crosslinking agent, prepare a solution by dissolving it in DMSO in proportion. Take 150 μL of the solution and add it to the chip, and incubate at room temperature for 30 minutes. Then, wash the chip with DMSO and pure water respectively for 5 times. Next, add 250 μL of sterile cell adhesion-promoting ECM protein solution (e.g. recombinant human fibronectin (FN) solution) with a concentration of 100 μg / mL to the chip, and incubate at room temperature or in a 37°C cell incubator for 4 hours. Finally, wash the chip with sterile 1X PBS buffer for 10 times to remove the by-products of the crosslinking agent and the unbound proteins. After carefully removing most of the washing solution, place the chip on a clean bench for 30 minutes, and air dry the residual liquid in the micro-reaction chamber.

[0060] Of course, the specific surface-activation method is not limited to the above, as long as it can help the cells to adhere.

[0061] Regarding the specific experimental process and data acquisition, in some embodiments, the detection method specifically includes:

[0062] After the culture solution containing the cells to be tested is brought into contact with the variable capacitive micro-nano biological detection chip, and the cells to be tested are completely adhered, the instantaneous capacitance after the application of the drug to be tested is taken as the initial capacitance value. When the drug acts on the cells, the isotype characteristics of the cells to be tested change, and the change amount of the measured capacitance value compared with the initial capacitance value or the capacitance value of the control group is taken as the isotype determination change value.

[0063] The evaluation index of the isotype determination change value includes any one or a combination of two or more of the following: capacitance change percentage (%), capacitance change rate κ, curve area change ΔAUC, consistency evaluation R2, half-effective concentration or half-inhibitory concentration EC50 / IC50.

[0064] In addition, before the experiment, the optimal inoculation density of the cells and the drug administration time usually need to be determined:

[0065] Cell density directly affects drug sensitivity, proliferation rate and cell status at the end of the experiment. Too high density can lead to contact inhibition, nutrient depletion or premature entry into plateau phase; too low density can amplify edge effects and reduce the intensity of the detection signal, which is not conducive to the accurate reflection of the proliferation process of the cells on the thin film electrode. The general operation process for determining the seeding density can be as follows:

[0066] 1) Digest, collect, centrifuge and count the adherent cells for efficacy evaluation, ensuring that the cell viability is >90%.

[0067] 2) Seed the test cells at 5-6 gradient densities (such as 100, 500, 1000, 2000, 5000, 10000 cells / well) in the chip well detection.

[0068] 3) Continuously monitor and record the capacitance changes during the 72-hour growth of the cells. This process can determine the natural growth curve of the cells, and the appropriate seeding density can be determined according to the growth curve.

[0069] After the cells adhere, healthy cells begin to spread and recover morphology (from round to spread), and then enter the cell cycle. This stage is the key period for selecting the optimal density.

[0070] Selection basis:

[0071] 1) Stimulating proliferation drugs: the cell density at 48 hours after seeding in the control group should still be in the middle of the logarithmic growth phase (not yet in the plateau phase), at which time the drug action window is larger.

[0072] 2) Inhibiting proliferation or having cytotoxicity: select the cell density that has entered the plateau phase at 48 hours after seeding in the control group, at which time the window period is relatively large.

[0073] In addition to seeding density, the drug addition time affects the action window of the drug on the cell cycle: too early drug addition (cells not adhered) will reduce the drug contact efficiency; too late drug addition (cells have entered the plateau phase) will mask the proliferation inhibition effect. After completing the cell density optimization experiment, the drug addition time point exploration needs to be carried out. Drug addition time point exploration: select a density to seed cells, and add drugs at 0, 2, 4, 6, 24 hours after seeding, and the final optimal drug addition time selection principle is:

[0074] 1) Proliferation inhibition experiment: add drugs after the cells enter the logarithmic growth phase (such as 24 hours after seeding), to ensure that the drugs act on the active proliferation period.

[0075] 2) Cell killing experiment: drugs can be added immediately after the cells complete adhesion (such as 6 hours after seeding) to evaluate direct toxicity.

[0076] 3) Time-dependent drugs (e.g. S-phase inhibitors): cells need to be synchronized so that the drug acts on the target cell cycle.

[0077] After determining the seeding density of cells and the timing of drug addition, cells are seeded and drugs are added. The specific process includes:

[0078] 1) Take the adherent cells in the logarithmic growth phase for 0.25% trypsin digestion, and after stopping with complete medium, transfer the cells to a centrifuge tube for centrifugation. The speed is 1200 rpm, 5 min.

[0079] 2) After centrifugation, discard the supernatant, and resuspend the cell pellet in complete medium to form a single cell suspension.

[0080] 3) Seed at the optimized density on the chip.

[0081] 4) Seed cells at the appropriate time point after the cells enter the stable platform phase (determine the number of plated chips according to experimental requirements).

[0082] 5) Turn on the bridge, and evaluate whether the cell seeding amount in each chip well is consistent according to the capacitance value.

[0083] 6) Place the chip in a 37°C cell incubator for culture.

[0084] 7) Prepare the test drug into 5-8 concentration gradients.

[0085] 8) Add equal volumes of different concentrations of drug solution to the chip wells with cultured cells: take the initial capacitance at the beginning of drug addition as the initial capacitance value Cp0. Proliferation inhibitors need to be added at the time point when the cells enter the stable platform phase (cell density under the microscope is greater than or equal to 80%), and killing experiments need to be added after the cells are completely adherent. Set up a negative control group (add equal volume of PBS solution instead of drug).

[0086] 9) Connect the external bridge to monitor and record the capacitance value changes in real time, and obtain the data stream of capacitance changes.

[0087] After obtaining the relevant data using the above detection method, data analysis and efficacy evaluation can be performed. The evaluation process can include:

[0088] 1) Drug efficacy pre-evaluation: According to the data flow processing, the change trend of the capacitance within several hours after adding the drug can be used to preliminarily evaluate the inhibitory effect of the drug on cell proliferation. Within the first few hours after adding the drug, the cell phenotype can be observed by the capacitance change curve. According to the change amount (percentage change), the change rate, the area change ΔAUC of the capacitance curve, and the index with statistically significant difference in the statistical method (for example, P<0.01), the sensitivity of the cell to the drug can be evaluated (for example, P<0.01), and the effectiveness of the drug can be preliminarily predicted.

[0089] 2) Drug efficacy post-verification: To verify the accuracy of the pre-evaluation and more accurately evaluate the drug efficacy, the capacitance change under the action of the drug is continuously observed, and the change in cell proliferation inhibition is evaluated according to the change in the capacitance change amount and the change rate caused by the change in the number of cells in the later period, and the drug efficacy is further evaluated.

[0090] 3) Data processing, data processing based on capacitance change:

[0091] Initial capacitance: Cp0, the initial capacitance value of the drug preparation is taken as the initial capacitance Cp0;

[0092] The specific data processing process is set according to the actual needs, and the key technical means of the present application is not in the specific data processing process. Any index with statistically significant difference in the statistical method can be used as an index for evaluating the drug efficacy.

[0093] For example:

[0094] ① Capacitance change percentage (percentage change %):

[0095] ② Capacitance change rate κ (Slope of Capacity Change):

[0096] Technical definition: The instantaneous slope of the capacitance change with time (unit: Δ capacitance / minute), reflecting the contraction speed of the cell population size / activity per unit time.

[0097] The calculation formula is:

[0098] ③ Area under curve (Area Under Curve, AUC):

[0099] Technical definition: The integral area enclosed by the capacitance signal-time curve and the baseline (unit: capacitance unit x time), reflecting the cumulative loss of the total amount of cell activity within the monitoring period.

[0100] The calculation formula is: (continuous integration)

[0101] The biological and pharmacodynamic significance of the related concepts are as follows:

[0102]

[0103]

[0104] 4) Analysis of parameter changes to interpret pharmacodynamics:

[0105] Example: If the anti-cancer drug treatment group is measured within the 24-48 hour window, κ ＝ -0.25 pw / min, while the control group κ ＝ -0.02 pw / min, indicating that the drug significantly accelerates the decay of the cell population.

[0106] If drug A group: 0-72 hour AUC = 4200 (pw h)

[0107] Control group: 0-72 hour AUC = 8500 (pw h)

[0108] Relative AUC loss rate = (1-4200 / 8500) x 100% ≈ 50.6%

[0109] Indicating that drug A results in more than half of the cumulative loss of total activity of the cells, equivalent to the traditional endpoint method "inhibition rate", but contains time dimension information.

[0110] 5) The above detection process also supports high-throughput methods: such as screening hundreds of small molecules at the same time, by using array-type thin film electrodes, adding drugs to cells according to certain concentrations, and pre-evaluating the effects of cell proliferation inhibition according to the capacitance changes caused by the migration force of the cells, to perform preliminary screening of small molecules.

[0111] The above detection method is generally applicable to the pharmacodynamic evaluation of molecular drugs, but for cell drugs (such as car-T drugs), the interaction between drug cells and target cells (such as tumor cells) will also cause a certain capacitance change. Specifically, using flexible nanobiological chips for capacitance monitoring, especially when immune cells are co-cultured with adherent tumor cells, their dynamic interaction will significantly affect the capacitance sensing signal, which is different from small molecule / antibody drugs (solvent capacitance interference can be ignored).

[0112] Therefore, in order to accurately evaluate the capacitance changes caused by the pharmacodynamics of cell drugs, certain details need to be optimized, that is, in some embodiments, when the drug to be tested is a cell drug, the detection method specifically comprises:

[0113] Curve difference between the sample to be tested to which the cell drug is applied and the cell control sample to which the cell drug is applied to obtain a measured capacitance curve;

[0114] The first change stage of the determined capacitance curve is taken as a physiological warning period, and the second change stage after the physiological warning period is taken as a cell apoptosis period.

[0115] The curve characteristics of the physiological warning period are evaluated, and the curve parameters are extracted as the same generation determination change values, which are used as indicators for early evaluation of drug efficacy.

[0116] In some more specific embodiments, the cell drug is selected from CAR-T or CAR-NK cell drugs, the test sample is a combination of target tumor cells and corresponding CAR-T cell preparations, and the cell control sample is a first combination of target tumor cells and empty T cell or empty NK cell preparations and a combination of non-target tumor cells and CAR-T or CAR-NK cell preparations.

[0117] The inoculation ratio of the target tumor cells or non-target tumor cells at the time of cell inoculation is determined based on the target efficiency ratio.

[0118] Compared with molecular drugs, the main differences in the evaluation process of cell drugs include:

[0119] 1) Different chip grouping. That is, when the test sample of the cell drug is applied, there are at least three groups of cell drugs: the experimental group: target cells and target cell drugs; two control groups are set: one is target cells and non-target cell drugs, and the other is non-target cells and target cell drugs. Curve difference is performed between the cell control sample without the cell drug to obtain the determined capacitance curve; the former removes non-specific killing, and the latter detects killing specificity.

[0120] Group Treatment Scientific purpose Experimental group CAR-T + target tumor cells Evaluate specific killing efficiency Target control group CAR-T + non-target tumor cells Verify targeting specificity Effector control group NC-T + target tumor cells Exclude non-specific killing

[0121] 2) Data normalization processing. Since cell drugs involve the combination of effector cells and target cells, the combination of effector cells will cause changes in the capacitance of the chip, and these data can be processed by normalization to make drug efficacy evaluation more reasonable and effective.

[0122] In some embodiments, the cell drug is selected from CAR-T / CAR-NK cell drugs, the test sample is a combination of target tumor cells and corresponding CAR-T cell preparations, and the cell control sample is a combination of target tumor cells and empty T cell (NC-T) preparations and a combination of non-target tumor cells and the CAR-T cell preparations.

[0123] 3) Different cell inoculation densities. Considering the influence of the combination of cell drugs on the capacitance changes on the chip surface, it is necessary to explore the appropriate effector-to-target ratio E:T of cell drugs and target tumor cells, that is, the ratio of the number of cells of the applied cell drug to the number of adherent tumor cells, which aims to more clearly obtain the killing activity of the cell drug.

[0124] The technical principle of the above-mentioned efficacy evaluation test for cell drugs is that the scheme utilizes the real-time monitoring function of the flexible nanobiological chip capacitor, analyzes specific signal characteristics, and establishes a dynamic evaluation system for cell treatment drugs:

[0125] Among them, the rate of change of capacitance is positively correlated with the binding efficiency of immune cells (CAR-T targeted binding is faster than empty T cells (NC-T)); the rate of change of capacitance directly reflects the killing rate (CAR-T killing time is about minutes, and apoptosis markers appear about hours); cell isotype changes (such as cell mechanics characteristics) are earlier than cell apoptosis signals, and serve as a pre-judgment of drug efficacy index. The specific process includes:

[0126] ① Cell preparation:

[0127] Tumor cells: adherent target cells transfected with CAR molecules (such as B7H3-293T) and non-target controls (wild-type 293T).

[0128] CAR-T cells: anti-CD3 / CD28 magnetic beads + IL-2 (100 IU / mL) activation, B7H3-CAR constructed by lentiviral transduction.

[0129] NC-T cells: derived from unmodified empty T cells.

[0130] ② Co-culture system:

[0131] According to the optimized E:T (such as 1:1, 5:1, 10:1), inoculate the chip, first inoculate the tumor cells, then add immune cells after the tumor cells adhere, pay attention to the experimental grouping, and take the capacitance of the added immune cell preparation as the initial capacitance Cp0, and monitor the capacitance in real time for 72 hours.

[0132] ③ Result analysis:

[0133] Pre-evaluation: cell isotype characteristics:

[0134] The capacitance after adding cell drugs is the initial capacitance, and thereafter experiences capacitance changes caused by the settlement of immune cells, and changes in capacitance signals caused by cell isotype characteristics changes of cells affected by drugs. The change amount of capacitance can be compared with the change rate of capacitance to preliminarily evaluate whether the drug is effective, and to infer the influence of the drug on cell proliferation ability.

[0135] Verification evaluation: cell proliferation inhibition evaluation:

[0136] At this stage, cells undergo pathological changes, apoptosis or cell death. The change amount of capacitance and the area under the curve can be used to evaluate the killing effect of cell drugs on target tumor cells.

[0137] The application also provides an application of the detection method provided by any of the above embodiments in the field of drug research and development.

[0138] Compared with the traditional MTT method, the advantages of the technical solution provided by the application mainly lie in the following table:

[0139] Table 1 Comparison of advantages of flexible nanobiological chip technology and MTT

[0140]

[0141]

[0142] The technical solution of the application will be further described in detail by means of several embodiments and in combination with the accompanying drawings. However, the selected embodiments are only used to illustrate the application, and do not limit the scope of the application.

[0143] Embodiment 1

[0144] Surface activation treatment of the detection capacitance chip

[0145] This embodiment aims to prepare a flexible nanobiological chip according to the method described in the patent CN207198082 “A variable capacitance type micro-nano biological detection chip and a processing method thereof”, and to perform surface activation treatment (protein fixation) on the chip. The specific operation process is as follows:

[0146] DSP treatment of the chip:

[0147] ①According to the use instructions of the commercial dithiobis(succinimidyl propionate) (DSP) crosslinking agent, accurately prepare the DSP solution. Dissolve DSP in dimethyl sulfoxide (DMSO) at a specific ratio recommended by the manufacturer to form a uniform solution.

[0148] ②Uniformly apply 150 μL of the prepared DSP solution to the surface of the detection capacitance chip.

[0149] ③Incubate the treated chip at room temperature for 30 minutes to ensure that the DSP fully reacts with the chip surface.

[0150] Cleaning step:

[0151] ①Use DMSO for preliminary cleaning to remove unreacted DSP.

[0152] ②Then, use deionized water for thorough cleaning to remove DMSO residues.

[0153] ③Repeat the above cleaning process a total of 5 times to ensure that the chip surface is clean and free of residues.

[0154] FN treatment of the chip:

[0155] ①Prepare a sterile recombinant human fibronectin (FN) solution with a concentration of 100 μg / mL.

[0156] ②Uniformly spread 250 μL of the FN solution on the surface of the cleaned capacitor chip.

[0157] ③Place the chip in a cell incubator at room temperature or 37°C for 4 hours to promote adsorption and fixation of the FN on the chip surface.

[0158] Final cleaning:

[0159] ①Clean the chip 10 times using sterile 1X PBS buffer to remove the byproducts of the cross-linking agent and unbound proteins.

[0160] ②After cleaning, carefully aspirate most of the cleaning buffer.

[0161] ③Place the chip in a clean bench and let it stand for 30 minutes to air dry the residual liquid in the micro-reaction cavities.

[0162] ④After the chip surface is completely dry, the surface activation treatment and protein fixation process is complete.

[0163] Example 2

[0164] This example illustrates the process of measuring the effect of a drug on the cell isotype characteristics and evaluating the drug efficacy.

[0165] This example aims to evaluate the drug killing of tumor cells on the chip surface by different small molecule drugs using a flexible nanobiological chip. Changes in the early cell isotype characteristics of the drug cause changes in the capacitance to predict the efficacy in advance, and the changes in the number of proliferating cells are combined to verify the feasibility of the predicted efficacy.

[0166] The specific operation steps are as follows:

[0167] I. Determine the cell seeding density:

[0168] Cell seeding: tumor cell Hela was digested with 0.25% trypsin, and the digestion reaction was terminated with an equal amount of complete medium. The cells were blown into a cell suspension and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in PBS. The cells were stained with trypan blue, and the viability and concentration of the cells were detected using a Countstar BioTech automatic cell counter. The viability of the cells was ensured to be greater than 90%, and the cells in the logarithmic growth phase were diluted into 6 different gradients (100, 500, 1000, 2000, 5000, 10000) and inoculated on the surface of the chip, with 2 duplicate wells for each gradient (considering the edge effect, the holes on the edge were filled with PBS);

[0169] Capacitance recording: when the cells act on the chip, the capacitance change Cp of the cell adherent growth is continuously recorded for 72 hours;

[0170] Result analysis: according to the capacitance change data, the cell seeding density is selected to be 1000 or 2000 cells.

[0171] II. Drug inhibition test on cell proliferation:

[0172] Cell seeding: tumor cell Hela was inoculated at a density of 1000 cells / well;

[0173] Drug addition: the same volume of different concentrations of drugs was added 6 hours after cell inoculation, and the control group was the buffer reagent of the drug. The capacitance at the time of adding the drug was taken as the initial capacitance value Cp0, and the capacitance change was observed and recorded. In the first 10 minutes of the drug acting on the cells, the capacitance changes caused by different concentrations of drugs were different, and the IC50 of the drug was determined according to the capacitance change ΔC when the cells were in the logarithmic decline period but not yet in the plateau period. pre ;

[0174] Continuous monitoring: the effect of the drug on the cells was continuously monitored. The plateau period of the capacitance caused by cell apoptosis until cell death was used as the end point of the drug effect to determine the IC50 of the drug. end ;

[0175] The same cells were tested with 4 different drugs (including DXd, SN-38, MMAE, and DM1).

[0176] Through the consistency analysis of the cell isotype characteristics of the cells with multiple drugs-cell proliferation ability, it is concluded that the change of the cell isotype characteristics can be completely used for the in vitro drug efficacy evaluation of the drug on the cells, and the corresponding relationship with the subsequent change of the cell proliferation ability is very significant, which makes it unnecessary to wait for the cell proliferation behavior during the drug development process, and the drug efficacy can be evaluated according to the isotype index within the same generation cycle.

[0177] Example 3

[0178] This embodiment example evaluates the cell therapy drug B7H3-CAR-T for cell killing experiment of human glioma U251.

[0179] According to the experimental requirements, a pre-experiment is first carried out to explore the inoculation density of human glioma U251. According to the growth curve of U251 on the biochip, the inoculation density of U251 is determined to be 5000 cells per well. After 20 hours of culture on the biochip, the empty immune T cells and the experimental group B7H3-CAR-T cells (effector to target ratio E:T is 2.5:1 (high concentration) and 1:1 (low concentration)) are introduced, and the control group is the natural growth group of tumor. The biochip is placed in a cell culture incubator (37°C, 5% CO2). The change of capacitance value is continuously observed and the data is recorded. According to the physiological early warning period of the initial effect of the drug, the change of capacitance caused by the change of cell isotype characteristics, it is concluded that within the first 30 minutes of the effect of CAR-T cell drug on cells, the physiological changes such as cell isotype characteristics are weakened with the increase of the effector to target ratio E:T of immunity, and the change of capacitance caused by the weakening of cell isotype characteristics, after normalizing the initial capacitance, the initial value is unified to 100 fF, when E:T = 1:1, the capacitance change is 55%, when E:T = 2.5:1, the capacitance change is 88%, and when E:T = 4:1, the capacitance change is 100%. The evaluation result shows that the E:T of immune cells is close to 100% when E:T is 2.5:1 and 4:1, thus evaluating the killing characteristics of the drug on tumor cells (see Figure 5 ), and finally evaluating the tumor killing activity of the cell therapy drug B7H3-CAR-T through the actual tumor killing effect (see Figure 6 ).

[0180] The specific analysis process can refer to the above embodiment, the difference is that after the addition of effector cells, a process of capacitance trend change will occur, which is not only the combination of effector cells and target cells, but also the process of changes such as cell mechanical properties caused by the combination of effector cells and target cells, etc. cell isotype characteristics, thus the killing effect of effector cells on target cells can be evaluated without waiting for the proliferation behavior of cells, and by continuously observing the capacitance, the apoptosis period can be entered, and a larger change of capacitance can be found, such as the continuous rise of capacitance value, and the specific curve is shown in Figure 4 The results show that the killing efficiency is predicted by cell phenotype characteristics change within 1 hour after the addition of cell drug B7H3-CAR-T, and the quantitative evaluation is completed by capacitance dynamics within 4 hours (as a comparison, the traditional flow method needs 24h+), and the change of cell isotype characteristics and the subsequent quantitative relationship of cell proliferation inhibition behavior / apoptosis behavior are very strong, which can directly predict the effect of drug action based on the physiological changes of cell isotype characteristics.

[0181] The method provided by the application still has excellent performance when applied to the functional evaluation of antibody-dependent cellular cytotoxicity (ADCC) antibody drugs, and the evaluation results can be seen from the examples of Figure 7 and Figure 8 , wherein Figure 7 shows the results of the IC50 consistency of ADC drugs, Figure 8 shows the ADCC effect of the antibody. By monitoring the FcR-mediated immune cell activation and the subsequent killing to cause two different capacitance changes in the electrical characteristics, the ADCC activity of the antibody drug (antibody concentration gradient vs. capacitance change slope) can be evaluated, and the specific process will not be repeated here.

[0182] Example 4

[0183] This embodiment compares and analyzes the existing impedance-based bioelectrical detection method and the capacitance-based detection method used in the preferred embodiment of the application to clarify the performance differences of the two methods in bio-detection applications.

[0184] Impedance and capacitance collection and detection are usually achieved by means of an alternating electric field of a certain frequency. The specific detection principle can be referred to in related literature. In the field of semiconductors, the curve of impedance (Z) changing with frequency is essentially different from the curve of capacitance (C) changing with frequency: impedance reflects the “overall hindering characteristics” of a semiconductor device or material to an alternating electric signal at different frequencies, including the joint contribution of resistance and capacitance, while capacitance only represents the direct relationship between its “charge storage capacity” and frequency.

[0185] Specifically, impedance is the vector superposition of resistance (R, reflecting energy loss) and capacitive reactance (Xc, reflecting frequency-dependent capacitive hindering), and its value changes with frequency, which can be used to evaluate the overall hindering performance of a semiconductor structure (such as a PN junction or a dielectric layer) under an alternating signal, and contains comprehensive information of energy loss and charge storage; capacitance only reflects the ability of the semiconductor to store electric charge, and its change with frequency reflects the response speed of internal charges (such as interface state charges or polarization charges) to the electric field, and does not directly contain resistive loss.

[0186] In terms of typical frequency response curves, there are significant differences between the two.

[0187] Impedance-frequency curve (Z-f: overall decreases with increasing frequency, which can be divided into two stages:

[0188] A. Low frequency band (such as 1 Hz-1 kHz): capacitive reactance dominates, and impedance decreases rapidly with increasing frequency (because Xc is inversely proportional to frequency);

[0189] B. High frequency range (e.g., 10kHz-1MHz): Resistance dominates, impedance decreases slowly and gradually approaches the resistance value (at this time, capacitive reactance can be ignored).

[0190] The capacitance-frequency curve (Cf) exhibits complex behavior, is closely related to the semiconductor structure, and can be broken down into three key stages:

[0191] A. Low frequency band: The capacitance value is relatively high because the interface state charge and polarization charge can fully respond to low frequency signals;

[0192] B. Mid-to-high frequency range: The capacitance value decreases and gradually approaches the "inherent capacitance" (such as the oxide layer capacitance Cox of a MOS capacitor) because the interface state charge and other factors cannot respond in time at high frequencies.

[0193] C. Extremely high frequency band: The capacitance value tends to be stable, reflecting only the minimum intrinsic capacitance value contributed by the fastest responding charge component.

[0194] In the semiconductor field, the two also have different application focuses:

[0195] Impedance-frequency sensing is mainly used to evaluate the overall electrical performance and energy loss of semiconductors, such as measuring the leakage current characteristics and dielectric constant of the dielectric layer to determine whether there are defects in the dielectric layer; or analyzing the leakage current and junction capacitance of the PN junction to evaluate the switching performance of diodes / transistors.

[0196] Capacitance-frequency detection is more suitable for characterizing material or structural parameters related to charge storage, such as the oxide thickness of a MOSFET, the semiconductor interface state density, or the polarization behavior of ferroelectric materials.

[0197] When the above methods are applied to the life sciences, the applicable scenarios and considerations for impedance and capacitance detection need to be adjusted accordingly, as the detection objects become biological samples with more complex structures.

[0198] like Figure 9 As shown, impedance measurements at different frequencies can reflect information at different levels of the cell: low-frequency response is mainly related to cell size, mid-frequency response reflects cell membrane capacitance, and high-frequency response reveals more about the electrical properties of the cytoplasm and subcellular structures.

[0199] Since the detection frequency directly affects the biological characteristics and spatial scale under study, and given the special structure of the nanobiochip used in this invention, its impedance changes significantly with frequency. For example... Figure 10 As shown in a and b, the chip impedance is approximately 550.7kΩ at 10kHz, while it drops to 58.68kΩ at 100kHz.

[0200] In contrast, when using capacitance sensing, the response change caused by frequency variation is smaller: for exampleFigure 10 In the formula, c and d are shown in the formula, the chip capacitance value is about 28.91 pF at 10 kHz; and at 100 kHz, the chip capacitance only decreases slightly to 27.12 pF, without a dramatic change in the order of magnitude as the impedance.

[0201] In summary, in the detection of biological materials, especially in the application of early electrical characteristics for drug efficacy prediction, the capacitive detection can more sensitively and reliably reflect the initial changes of cell physiological state due to stable response and smooth changes, and avoid prediction deviation caused by large fluctuations in detection indicators. Therefore, the present application preferably adopts a capacitive sensing element to realize bioelectricity detection.

[0202] Based on the above embodiments, it can be clearly understood that the present application realizes revolutionary speed improvement by providing real-time dynamic monitoring of physiological and physical indicators such as cell isotype characteristics: the "prediction window period" of drug efficacy evaluation is greatly advanced, significantly accelerating the drug screening process. The use of pure electrical detection simplifies the experimental process, performs real-time monitoring, eliminates a large amount of invalid waiting time and detection time caused by traditional microscopic imaging technology, and improves the evaluation efficiency of drug efficacy; has the characteristics of high accuracy and sensitivity: through early and late full-time domain real-time monitoring, multidimensional and bidirectional evaluation of drug efficacy, the results are more accurate, and early subtle physiological changes that cannot be detected by traditional methods are captured, and drug efficacy evaluation is more sensitive.

[0203] At the same time, the method provided by the present application also has the advantage of high throughput, and can be expanded into a microarray chip. The cells on the chip are cultured in droplets, and hundreds of drug screening experiments can be performed at the same time. At the same time, the chip can be recycled to reduce costs: the cell device of the used chip can be digested and cleaned with a cell digestion solution, and then sterilized by irradiation for reuse, saving the cost of consumables.

[0204] It should be understood that the above embodiments are only for illustrating the technical concepts and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A detection method for rapid real-time drug efficacy evaluation, characterized in that, include: Provides a quantitative correlation between the syngeneic calibration changes in the electrical properties of thin-film electrodes caused by syngeneic phenotypic characteristics of selected cell varieties and cell proliferation capacity; The cells to be tested are attached to the surface of the same thin-film electrode, and the cells to be tested belong to the selected cell species. The test drug is applied to the test cells, and within one cell proliferation cycle, the changes in the electrical properties of the thin-film electrode caused by the phenotypic changes of the test cells are observed as phenotypic measurements. Based on the quantitative correspondence, the real-time proliferation characteristics of the selected cell varieties corresponding to the same generation measurement change value are obtained, and the efficacy of the drug is evaluated based on the real-time proliferation characteristics.

2. The detection method according to claim 1, characterized in that, The process of establishing the quantitative correspondence includes: The selected cell species and their corresponding labeled cells are seeded on the surface of the thin-film electrode; External interference is applied to the labeled cells to alter their cell proliferation capacity; The thin-film electrode was used to record the changes in the electrical properties of the calibrated cells within one cell proliferation cycle, as well as the changes in the electrical properties over a period of more than one cell proliferation cycle. The changes in the electrical properties of the same generation are used as detection data to reflect the phenotypic characteristics of the cells, and the changes in the iterative electrical properties are used as evaluation indicators of the cell proliferation capacity to establish the quantitative correspondence.

3. The detection method according to claim 2, characterized in that, The process of establishing the quantitative correspondence also includes: After a period of time greater than one cell proliferation cycle, biological assays for cell proliferation inhibition are performed on the labeled cells to verify and / or correct the quantitative correspondence.

4. The detection method according to claim 1, characterized in that, The selected cell species are adherent cells.

5. The detection method according to claim 1, characterized in that, The electrical characteristics of the thin-film electrode include impedance or capacitance.

6. The detection method according to claim 1, characterized in that, The thin-film electrode is selected from a variable capacitance micro / nano biodetector chip, which includes a fixed electrode plate and an elastic electrode plate, and the cell to be tested is attached to the side of the elastic electrode plate that is opposite to the fixed electrode plate. And / or, the syntypic characteristics are comprehensive physiological changes including morphological structure, physical properties, physiological and biochemical reactions, antigenicity, and ecological behavior.

7. The detection method according to claim 6, characterized in that, The detection method specifically includes: The culture medium containing the test cells is brought into contact with the variable capacitance micro / nano biodetector chip. After the test cells are completely adhered to the chip, the instantaneous capacitance after the application of the test drug is used as the initial capacitance value. When the drug acts on the cells and the phenotypic characteristics of the test cells change, the change in the measured capacitance value compared with the initial capacitance value or the capacitance value compared with the control group is used as the phenotypic change value. The evaluation indicators for the changes in capacitance measured in the same generation include any one or a combination of two or more of the following: percentage change in capacitance (%), rate of change in capacitance κ, area under the curve ΔAUC, consistency assessment R2, half-maximal effective concentration or half-maximal inhibitory concentration EC50 / IC50.

8. The detection method according to claim 7, characterized in that, When the drug to be tested is a cell-based drug, the detection method specifically includes: The test sample treated with the cell drug and the cell control sample treated with the cell drug are subjected to curve difference analysis to obtain the capacitance curve. The first stage of change in the measured capacitance curve is taken as the physiological warning period, and the second stage of change after the physiological warning period is taken as the apoptosis period. The curve characteristics of the physiological warning period are evaluated, and the curve parameters are extracted as the changes measured in the same generation, serving as an indicator for early pre-evaluation of drug efficacy.

9. The detection method according to claim 8, characterized in that, The cell drug is selected from CAR-T or CAR-NK cell drugs. The test sample is a combination of target tumor cells and corresponding CAR-T cell preparations. The cell control sample is a first combination of target tumor cells and empty T cells or empty NK cell preparations, and a combination of non-target tumor cells and CAR-T or CAR-NK cell preparations. The inoculation ratio of the target tumor cells or non-target tumor cells during cell inoculation is determined based on the target-efficacy ratio.

10. The application of the detection method according to any one of claims 1-8 in the field of drug research and development.

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