A real-time monitoring method for cell-substrate interactions in photodynamic therapy

Abstract

The present invention discloses a real-time monitoring method for photodynamic therapy cell-substrate interaction, including: exciting monitoring signals based on a surface plasmon resonance microbial sensing system, and calibrating parameters of the surface plasmon resonance microbial sensing system; setting a cell culture well of a sensor chip, and performing a sealing test on the cell culture well; monitoring the cell-substrate adhesion of the photodynamic therapy process in real time based on the surface plasmon resonance microbial sensing system, and outputting the cell-substrate interaction results. The present invention can realize the label-free detection of the dynamic process of biomolecular interaction on the gold film surface, thereby comprehensively reflecting the complex interaction between cells and substrates, and improving the accuracy of the analysis of the effect of PDT on tumor cell adhesion.

Description

Technical Field

[0001] The invention relates to the technical field of cell monitoring, and in particular to a real-time monitoring method for photodynamic therapy cell-substrate interaction. Background Art

[0002] One of the emerging methods for tumor treatment is photodynamic therapy (PDT), which is gradually being used in clinical tumor treatment due to its high efficiency, non-invasiveness and low side effects. This is a therapy that treats tumors and other diseases through the combined action of photosensitizers, light irradiation of specific wavelengths and oxygen. When the photosensitizer is injected into the body, it selectively accumulates at the tumor site. By irradiating these areas with a light source of a specific wavelength, the photosensitizer is excited into a high-energy state and produces highly reactive reactive oxygen species (ROS). These reactive oxygen species induce irreversible damage to tumor cells, ultimately inducing cell apoptosis or necrosis and destroying the tumor tissue structure. In addition, ROS not only directly attack key components of cells, such as cell membranes, mitochondria and DNA, but also further weaken the adhesion ability of tumor cells by destroying the extracellular matrix (ECM) and cell-matrix adhesion complexes (such as adhesion plaques). This mechanism not only helps to inhibit the growth and spread of tumors, but may also affect processes such as cell migration, invasion and metastasis. Therefore, cell-extracellular matrix adhesion is not only a key factor in PDT-induced cell apoptosis or necrosis, but also an influencing factor in further inhibiting tumor invasion and metastasis.

[0003] At present, the characterization methods of cell-substrate adhesion during photodynamic therapy (PDT) mainly rely on technologies such as fluorescence imaging and flow cytometry. However, these traditional methods have significant limitations, which restrict the depth and breadth of PDT research. First, traditional methods require fluorescent labeling of samples and cannot achieve real-time monitoring of cell fixation, which may not only affect the natural state of cells and lead to deviations in results, but also fail to capture the dynamic changes in tumor cell adhesion ability during PDT treatment. More importantly, traditional methods can only characterize certain cell adhesion molecules and cannot fully reflect the complex interactions between cells and substrates. This indirect characterization method may lead to an incomplete and inaccurate understanding of the effects of PDT on tumor cell adhesion, especially when exploring the effects of PDT on tumor metastasis potential and therapeutic efficacy. This limitation is particularly prominent.

[0004] Therefore, the prior art needs to be improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is that, in view of the defects of the prior art, the present invention provides a real-time monitoring method for cell-substrate interaction in photodynamic therapy to solve the problem that the prior art cannot fully reflect the complex interaction between cells and substrates, resulting in an incomplete and inaccurate analysis of the effect of PDT on tumor cell adhesion.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0007] In a first aspect, the present invention provides a method for real-time monitoring of cell-substrate interactions in photodynamic therapy, comprising:

[0008] Exciting a monitoring signal based on a surface plasmon resonance microbial sensing system, and calibrating parameters of the surface plasmon resonance microbial sensing system;

[0009] Setting a cell culture well of the sensor chip, and performing a sealing test on the cell culture well;

[0010] Based on the surface plasmon resonance microbial sensing system, the cell-substrate adhesion during the photodynamic therapy process is monitored in real time, and the cell-substrate interaction results are output.

[0011] In one implementation, the surface plasmon resonance-based microbial sensing system excites and monitors signals, including:

[0012] The laser is emitted by a laser, and the laser is passed through a beam expansion system to perform a beam expansion process of a preset multiple;

[0013] The expanded light beam passes through a half-wave plate and a polarizer to convert the light beam into P-polarized light, and the P-polarized light is reflected by a reflector and then converged to the rear focal plane through a tube lens to control the light beam to irradiate the gold film in parallel at a preset angle, thereby achieving total internal reflection and exciting surface plasma waves.

[0014] In one implementation, the parameter calibration of the surface plasmon resonance microorganism sensing system includes:

[0015] The surface plasmon resonance microorganism sensing system is detected by using sodium chloride solutions of different concentrations, and the refractive index resolution of the surface plasmon resonance microorganism sensing system is calculated by using a refractive index calculation formula to obtain calibrated parameters.

[0016] In one implementation, the refractive index calculation formula is:

[0017] ;

[0018] In the formula, represents the refractive index resolution, represents the change in the sample's refractive index, represents the change in the surface plasmon resonance signal, Indicates the noise level of the system.

[0019] In one implementation, the refractive index resolution of the surface plasmon resonance microbial sensing system is 5.13×10 -5 RIU.

[0020] In one implementation, the step of setting up a cell culture well of a sensor chip and performing a sealing test on the cell culture well comprises:

[0021] Using a dry wound healing insert to bond with a gold film to obtain the cell culture well;

[0022] A 3D model with a hole in the center is obtained by 3D printing, and the cell culture well is placed in the center hole position of the 3D model for sealing;

[0023] After adding liquid into the cell culture well, the well was left to stand for 24 hours, and a sealing test was performed by observing whether liquid seepage occurred.

[0024] In one implementation, the surface plasmon resonance microbial sensing system monitors the cell-substrate adhesion during photodynamic therapy in real time and outputs the cell-substrate interaction results, including:

[0025] Planting cells in the cell culture wells on the sensor chip and incubating photosensitizers;

[0026] The cell-substrate adhesion of cells incubated with photosensitizer during photodynamic therapy is monitored in real time, and the cell-substrate interaction results are output.

[0027] In one implementation, the step of planting cells in the cell culture wells on the sensor chip and incubating the photosensitizer comprises:

[0028] HeLa cells in the logarithmic growth phase were taken and digested with trypsin, the remaining cells were collected and counted, and the cells were seeded on the gold film at 2000 cells / well in the cell culture well, and the cell culture well was placed in an incubator for incubation.

[0029] In one implementation, the real-time monitoring of the cell-substrate adhesion of cells incubated with photosensitizer during photodynamic therapy includes:

[0030] The gold film in the cell culture well is irradiated with a laser to excite the surface plasma wave of the gold film, the excited surface plasma wave is collected through an objective lens, and after being collimated by a lens, it is collected by a camera to obtain a surface plasma resonance image;

[0031] The cell-substrate adhesion of the cells during the photodynamic therapy process is analyzed according to the surface plasmon resonance image.

[0032] In one implementation, analyzing the cell-substrate adhesion of the cells during the photodynamic therapy according to the surface plasmon resonance image includes:

[0033] Based on the image grayscale value of the reflected light response of the gold film surface, the dynamic process of cell-substrate adhesion of the cells during photodynamic therapy is analyzed label-free, and the cell-substrate interaction result is output; wherein the cell-substrate interaction result is used to reflect the position of the cell detaching from the substrate and the quantitative degree of detachment.

[0034] The present invention adopts the above technical solution to achieve the following effects:

[0035] The present invention excites monitoring signals based on a surface plasmon resonance microbial sensing system, and performs parameter calibration on the surface plasmon resonance microbial sensing system; sets a cell culture well of a sensor chip, and performs a sealing test on the cell culture well; and monitors the cell-substrate adhesion during photodynamic therapy in real time based on a surface plasmon resonance microbial sensing system, and outputs the cell-substrate interaction results. The present invention can realize label-free detection of the dynamic process of biomolecular interaction on the gold film surface, thereby comprehensively reflecting the complex interaction between cells and substrates, and improving the accuracy of analysis of the effect of PDT on tumor cell adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0037] Figure 1 It is a flow chart of the method for real-time monitoring of cell-substrate interaction in photodynamic therapy of the present invention.

[0038] Figure 2 It is a schematic diagram of the optical path of the SPRM system in the present invention.

[0039] Figure 3It is a schematic diagram of the test results of the SPRM system indicators in the present invention.

[0040] Figure 4 It is a schematic diagram and a physical diagram of the chip cell culture well in the present invention.

[0041] Figure 5 It is a schematic diagram of the SPR image and bright field image of the control group without incubation of photosensitizer in the present invention and the SPR signal.

[0042] Figure 6 It is a schematic diagram of the SPR image and bright field image and SPR signal of the experimental group incubated with 10 μM Ce6 molecules as a photosensitizer in the present invention.

[0043] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and advantages of the present invention clearer and more specific, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] Exemplary Methods

[0046] At present, the characterization methods of cell-substrate adhesion during photodynamic therapy (PDT) mainly rely on technologies such as fluorescence imaging and flow cytometry. However, these traditional methods have significant limitations, which restrict the depth and breadth of PDT research. First, traditional methods require fluorescent labeling of samples and cannot achieve real-time monitoring of cell fixation, which may not only affect the natural state of cells and lead to deviations in results, but also fail to capture the dynamic changes in tumor cell adhesion ability during PDT treatment. More importantly, traditional methods can only characterize certain cell adhesion molecules and cannot fully reflect the complex interactions between cells and substrates. This indirect characterization method may lead to an incomplete and inaccurate understanding of the effects of PDT on tumor cell adhesion, especially when exploring the effects of PDT on tumor metastasis potential and therapeutic efficacy. This limitation is particularly prominent.

[0047] Among various technologies, surface plasmon resonance (SPR) sensing technology has been widely used in the study of molecular interactions and tumor diagnosis because of its ability to achieve real-time, label-free detection. The development of SPR microscopy in recent years has led to the application of the SPRM system (surface plasmon resonance microscopy sensing system). The SPRM system combines the advantages of traditional SPR and microscopic imaging technology, and can perform high-resolution real-time monitoring of living cells without labeling. Based on the ability of the SPRM system to study cell-matrix interactions, it has become a powerful tool for studying cell adhesion, migration, and interaction with the matrix.

[0048] Therefore, this embodiment proposes a novel method of applying the SPRM system to the photodynamic therapy (PDT) process to characterize the cell-substrate adhesion, so as to solve the problem that the existing technology cannot fully reflect the complex interaction between cells and substrates, resulting in incomplete and inaccurate analysis of the effect of PDT on tumor cell adhesion; the method in this embodiment can achieve real-time, label-free, dynamic monitoring of cell-substrate adhesion during PDT, and has important research significance and application prospects for improving the accuracy of PDT efficacy evaluation and in-depth study of the mechanism of PDT on tumor biology.

[0049] In this embodiment, a real-time monitoring method for cell-substrate interaction in photodynamic therapy is proposed. The method is based on the surface plasmon resonance microbial sensor system to excite the monitoring signal, and the surface plasmon resonance microbial sensor system is calibrated; the cell culture well of the sensor chip is set, and the sealing of the cell culture well is detected; the cell-substrate adhesion during the photodynamic therapy process is monitored in real time based on the surface plasmon resonance microbial sensor system, and the cell-substrate interaction results are output. This embodiment can realize the label-free detection of the dynamic process of biomolecular interaction on the gold film surface, thereby fully reflecting the complex interaction between cells and substrates, and improving the analysis accuracy of the effect of PDT on tumor cell adhesion.

[0050] like Figure 1 As shown, an embodiment of the present invention provides a real-time monitoring method for photodynamic therapy cell-substrate interaction, comprising the following steps:

[0051] Step S100, exciting a monitoring signal based on a surface plasmon resonance microorganism sensing system, and calibrating parameters of the surface plasmon resonance microorganism sensing system.

[0052] In this embodiment, the real-time monitoring method for photodynamic therapy cell-substrate interaction is implemented based on the SPRM system (surface plasmon resonance microbial sensing system) designed in this embodiment; Figure 2 As shown, the SPRM system includes:

[0053] The optical path is formed in sequence: a femtosecond laser 1, a first half-wave plate 2, a polarization beam splitter 3, a light collector 4, a first reflector 5, an attenuation plate 6, a second reflector 7, a first lens 8, a second lens 9, a second half-wave plate 10, a third lens 11, a dichroic mirror 12, an objective lens 13, a fourth lens 14, a beam splitter 15, a first CMOS camera 16, and a second CMOS camera 17.

[0054] In this embodiment, the SPRM system is an intensity-type SPRM system, which is mainly divided into a light source module, a sensor module and an imaging detection module; specifically, the light source module is a femtosecond laser, the sensor module is a gold film (i.e., a sensor chip) of a TIR (Total Internal Reflection Fluorescence) microscope, and the imaging detection module is composed of two CMOS cameras; in this embodiment, the SPRM system is used to excite the laser signal, and then the laser is controlled through the optical path to irradiate the gold film in parallel at a certain angle, thereby achieving total internal reflection and exciting surface plasma waves, thereby obtaining a monitoring signal of the interaction between biomolecules.

[0055] Specifically, in one implementation of the present embodiment, the surface plasmon resonance-based microbial sensing system excites a monitoring signal, comprising: emitting laser light through a laser, and passing the laser light through a beam expansion system to perform beam expansion processing with a preset multiple; passing the expanded light beam through a half-wave plate and a polarizer to convert the light beam into P-polarized light, and reflecting the P-polarized light through a reflector, and then converging it onto a rear focal plane through a tube lens, so as to control the light beam to irradiate the gold film in parallel at a preset angle, thereby achieving total internal reflection and exciting surface plasma waves.

[0056] In this embodiment, a femtosecond laser emits laser light (the laser center wavelength is 720nm and the full width at half maximum is 8nm), which is expanded by a beam expansion system, which consists of two lenses of f=25 mm and f=400 mm, to achieve 16 times beam expansion. The expanded light beam is converted into P polarized light by a half-wave plate and a polarization beam splitter; then, the P polarized light is reflected by a series of mirrors and then converged to the back focal plane by a tube lens, so that the light beam can irradiate the gold film in parallel at a certain angle, achieve total internal reflection and excite the surface plasma wave SPW (Surface Plasmons Wave, SPW).

[0057] Finally, the reflected light after the gold film is collected by the objective lens and then collimated by the lens and received by the CMOS camera. In the CMOS camera, when the refractive index of the gold film surface changes, the reflected light responds by changing the grayscale value within a certain linear range (that is, within the linear range, the change in the grayscale value of the reflected light linearly maps the change in the refractive index of the gold film surface). In this way, label-free detection of the dynamic process of biomolecular interaction on the gold film surface can be achieved.

[0058] In this embodiment, after the intensity-based SPRM system is built and the trial run is successful, it is necessary to calibrate the parameters of the SPRM system; the most important thing is to calibrate the refractive index resolution of the SPR sensor.

[0059] Specifically, in an implementation of this embodiment, the parameter calibration of the surface plasmon resonance microorganism sensing system includes: using sodium chloride solutions of different concentrations to detect the surface plasmon resonance microorganism sensing system, and calculating the refractive index resolution of the surface plasmon resonance microorganism sensing system by a refractive index calculation formula to obtain calibrated parameters.

[0060] In this embodiment, the refractive index resolution of the SPR sensor refers to the minimum refractive index that the SPRM system can distinguish. In this embodiment, during the SPRM system index test, the SPRM sensor system is tested with NaCl solutions of different concentrations; Figure 3 As shown, Figure 3 (a) is the linear relationship diagram of the refractive index of salt water. Figure 3 (b) is a schematic diagram of the SPR signal changes of the SPRM system in response to different salt water concentrations.

[0061] The principle of the SPRM system index test in this embodiment is to use the linear relationship of the refractive index of salt water and the SPR signal change of the SPRM system for salt water of different concentrations, and calculate the refractive index resolution of the system to be 5.13×10 -5 RIU. The refractive index calculation formula is:

[0062] ;

[0063] In the formula, represents the refractive index resolution, represents the change in the sample's refractive index, represents the change in the surface plasmon resonance signal, Indicates the noise level of the system.

[0064] In this embodiment, the performance calibration of the SPRM system can be completed through the above-mentioned SPRM system construction and refractive index resolution calibration process; then, a cell culture chip (i.e., a sensor chip) is set, and a cell culture well of the sensor chip is set.

[0065] like Figure 1 As shown, an embodiment of the present invention provides a real-time monitoring method for photodynamic therapy cell-substrate interaction, comprising the following steps:

[0066] Step S200, setting a cell culture well of the sensor chip and performing a sealing test on the cell culture well.

[0067] In this embodiment, since the SPRM system uses a high NA (magnification) TIR objective lens with a magnification of 100, a thinner gold film chip is specially customized in this embodiment; then, it is sealed by plug-in bonding and 3D printing model.

[0068] Specifically, in one implementation of the present embodiment, the cell culture well of the sensor chip is provided, and the sealing test of the cell culture well is performed, including: using a dry wound healing plug-in to bond with a gold film to obtain the cell culture well; obtaining a 3D model with a hole in the center by 3D printing, and placing the cell culture well at the center hole position of the 3D model for sealing; adding liquid to the cell culture well and letting it stand for 24 hours to perform a sealing test to observe whether there is liquid seepage.

[0069] In this embodiment, after a thinner gold film is customized, a wound healing plug-in (plug-in model: Ibidi, culture-insert 2-well, 80209) is used to bond with the gold film to obtain a cell culture pool; the two-hole plug-in not only has good self-adhesive sealing properties, but can also be reused.

[0070] After the plug-in is used to form a cell culture pool, this embodiment uses SolidWorks software to design a 3D model with a hole in the center (matching the length and width of the two-hole plug-in), and uses 3D printing technology to make the 3D model. The hole in the center of the 3D model is used to place the plug-in to ensure that the cells can grow in it. Figure 4 As shown, Figure 4 (a) is a schematic diagram of a cell culture well. Figure 4 (b) is a physical picture of the cell culture well. Figure 4 (c) is a diagram of the cell culture well being loaded on a TIR microscope; in this embodiment, the cell culture well design of the sensor chip enables the cell culture to have good biocompatibility and can be stably placed on the stage without being affected in adjusting the field of view and focal length.

[0071] In this embodiment, after setting up the chip cell culture wells, the SPRM system can be used to monitor the PDT process of cell-substrate adhesion in real time by planting cells and incubating photosensitizers in the cell culture wells.

[0072] like Figure 1 As shown, an embodiment of the present invention provides a real-time monitoring method for photodynamic therapy cell-substrate interaction, comprising the following steps:

[0073] Step S300: monitor the cell-substrate adhesion during the photodynamic therapy in real time based on the surface plasmon resonance microbial sensing system, and output the cell-substrate interaction result.

[0074] In this embodiment, the SPRM system is used to monitor the changes in cell-matrix adhesion and cell death caused by the production of reactive oxygen species by the commercial photosensitizer Chlorin e6 (Ce6), so as to obtain high-quality HeLa single-cell SPR image / bright field image dual-mode data in real time. Compared with traditional methods, real-time monitoring of cell-substrate adhesion is achieved faster, earlier and more sensitively, which can provide more comprehensive and real-time information on changes in cell-substrate interactions during PDT treatment.

[0075] Specifically, in an implementation of this embodiment, the surface plasmon resonance-based microbial sensing system monitors the cell-substrate adhesion during photodynamic therapy in real time and outputs the cell-substrate interaction results, including: planting cells in the cell culture wells on the sensor chip and incubating photosensitizers; monitoring the cell-substrate adhesion of the cells after incubation with the photosensitizer during photodynamic therapy in real time, and outputting the cell-substrate interaction results.

[0076] In this embodiment, in the process of real-time monitoring of cell-substrate adhesion during photodynamic therapy, HeLa cells are first planted in a cell culture well and incubated with a photosensitizer; wherein the photosensitizer is a chlorin e6 (Ce6) photosensitizer; then, real-time image acquisition is performed using a CMOS camera of the SPRM system, and the acquired images are used to monitor changes in cell-substrate adhesion of cells after incubation with the photosensitizer during the photodynamic therapy process.

[0077] Specifically, in one implementation of this embodiment, cells are planted on the cell culture well and incubated with a photosensitizer, including: taking HeLa cells in the logarithmic growth phase and digesting them with trypsin, collecting and counting the remaining cells, planting the cells on a gold film in the cell culture well at 2000 cells / well, and placing the cell culture well in an incubator for incubation.

[0078] In this embodiment, during the cell planting process, HeLa cells in the logarithmic growth phase were digested with trypsin, collected and counted, and the cells were planted on the gold film at 2000 cells / well in the chip culture pool, with 90 μL / well of culture medium. After the chip was placed in an incubator for 24 hours, it was placed under a TIR microscope to observe the cell state and adhesion.

[0079] Specifically, in one implementation of the present embodiment, the real-time monitoring of the cell-substrate adhesion of cells after incubation with a photosensitizer during the photodynamic therapy process includes: irradiating the gold film in the cell culture well with a laser to excite the surface plasma waves of the gold film, collecting the excited surface plasma waves through an objective lens, and collecting them with a camera after being collimated by a lens to obtain a surface plasma resonance image; and analyzing the cell-substrate adhesion of the cells during the photodynamic therapy process according to the surface plasma resonance image.

[0080] In this embodiment, based on the SPRM system, in the process of monitoring the cell-substrate adhesion during the photodynamic therapy process, the surface plasma wave of the gold film is excited by the laser, and the reflected light after the gold film is acted on is collected by the objective lens and then collimated by the lens and received by the CMOS camera. In the CMOS camera, when the refractive index of the gold film surface changes, the reflected light responds in the form of grayscale value changes within a certain linear range (that is, within the linear range, the change in the grayscale value of the reflected light linearly maps the change in the refractive index of the gold film surface). In this way, label-free detection of the dynamic process of biomolecular interaction on the gold film surface can be achieved.

[0081] Specifically, in one implementation of the present embodiment, analyzing the cell-substrate adhesion of the cells during the photodynamic therapy process based on the surface plasmon resonance image includes: performing a label-free analysis of the dynamic process of the cell-substrate adhesion of the cells during the photodynamic therapy process based on the image grayscale value of the reflected light response of the gold film surface, and outputting the cell-substrate interaction results.

[0082] In this embodiment, when monitoring the cell-substrate adhesion of cells after incubation with photosensitizer during the PDT process in real time, a CMOS camera is used to capture images to record SPR images in real time; then, based on these captured SPR images, a dynamic analysis is performed on whether the cells are adhered to the substrate (i.e., the degree of cell-substrate adhesion is reflected by the SPR intensity signal), as well as the cell death process. In this embodiment, the SPRM system is used to monitor in real time the situation of HeLa cells detaching from the substrate and undergoing apoptosis under the action of PDT after being co-incubated with Ce6 molecules for 4 hours. After being irradiated with a white light source, the cells detach from the substrate and undergo apoptosis, and finally the cell-substrate interaction results are output; wherein, the cell-substrate interaction results are used to reflect the position of the cell detaching from the substrate and the quantitative degree of detachment; based on the SPR image, it can be seen that the tighter the adhesion between the cell and the gold film, the higher the grayscale value; conversely, the closer the grayscale value is to the background grayscale value, the more the cell is gradually detaching from the gold film (such as Figure 6 The SPR signal in (c) is shown in the result of marker 1).

[0083] As an example, in this embodiment, the following is set in the experiment: Figure 5 The control group shown in FIG. 1 ; wherein the control group was incubated without adding photosensitizer; Figure 5 As shown, Figure 5 (a) is the SPR image of the control group without incubation of photosensitizer; Figure 5 (b) is a sticky picture of the control group without incubation of photosensitizer; Figure 5 Middle (c) is the SPR signal marker diagram of the control group without incubation of photosensitizer; Figure 5 (d) shows the change in the SPR signal of the control group without incubation of photosensitizer.

[0084] In this embodiment, the following experiment is set up: Figure 6 The experimental groups shown were incubated with medium containing 10 μM Ce6; Figure 6 As shown, Figure 6 (a) is the SPR image of the experimental group incubated with 10 μM Ce6 molecules as photosensitizer; Figure 6 Middle (b) is a sticky picture of the experimental group incubated with 10 μM Ce6 molecules as photosensitizer; Figure 6 Middle (c) is the SPR signal marker diagram of the experimental group incubated with 10 μM Ce6 molecules as photosensitizer; Figure 6 (d) shows the change in SPR signal of the experimental group incubated with 10 μM Ce6 molecules as photosensitizer.

[0085] The above control group and experimental group were stimulated with SPRM illumination light source and recorded bright field and SPRM images in real time at a frame rate of 30 frames per second to reflect the interaction between cells and substrate. Compared with the control group, the experimental group in this embodiment can use the SPRM system to monitor in real time that HeLa cells are incubated with Ce6 molecules for 4 hours, and then irradiated with white light source, and the cells detach from the substrate and undergo apoptosis under the action of PDT.

[0086] It is worth mentioning that, as a variant solution that can replace the technical solution of this embodiment, different types of photosensitizers such as Rose Bengal (RB) or self-developed photosensitizers can be used to realize the incubation of photosensitizers; other cells such as breast cancer cells can be used for PDT treatment research; other types of SPRM systems can be used as tools for real-time monitoring of PDT, for example, phase-type SPRM systems can be used to monitor cell adhesion, etc., so as to realize dynamic monitoring of cell-substrate adhesion during photodynamic therapy; all these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

[0087] This embodiment achieves the following technical effects through the above technical solution:

[0088] This embodiment excites the monitoring signal based on the surface plasmon resonance microbial sensing system, and calibrates the parameters of the surface plasmon resonance microbial sensing system; sets the cell culture well of the sensor chip, and performs a sealing test on the cell culture well; monitors the cell-substrate adhesion during the photodynamic therapy process in real time based on the surface plasmon resonance microbial sensing system, and outputs the cell-substrate interaction results. This embodiment can realize the label-free detection of the dynamic process of biomolecular interaction on the gold film surface, thereby fully reflecting the complex interaction between cells and substrates, and improving the analysis accuracy of the effect of PDT on tumor cell adhesion.

[0089] Exemplary Devices

[0090] Based on the above embodiments, the present invention further provides a real-time monitoring system for photodynamic therapy cell-substrate interaction, comprising:

[0091] An SPRM system, wherein the SPRM system is used to excite monitoring signals and collect SPR images of cell-substrate adhesion during photodynamic therapy in real time;

[0092] The terminal device is used for real-time and analytical monitoring of the cell-substrate adhesion results during photodynamic therapy and output of the cell-substrate interaction results.

[0093] like Figure 2 As shown, the SPRM system includes:

[0094] The optical path is formed in sequence: a femtosecond laser 1, a first half-wave plate 2, a polarization beam splitter 3, a light collector 4, a first reflector 5, an attenuation plate 6, a second reflector 7, a first lens 8, a second lens 9, a second half-wave plate 10, a third lens 11, a dichroic mirror 12, an objective lens 13, a fourth lens 14, a beam splitter 15, a first CMOS camera 16, and a second CMOS camera 17.

[0095] This embodiment achieves the following technical effects through the above technical solution:

[0096] The system provided in this embodiment can realize label-free detection of the dynamic process of biomolecular interaction on the gold film surface, thereby comprehensively reflecting the complex interaction between cells and substrates and improving the analysis accuracy of the effect of PDT on tumor cell adhesion.

[0097] Those skilled in the art can understand that all or part of the processes in the above-mentioned embodiments can be implemented by instructing related hardware through a computer program, and the computer program can be stored in a non-volatile storage medium. When the computer program is executed, it can include the processes of the embodiments of the above-mentioned methods. Among them, any reference to memory, storage, database or other media used in the embodiments provided by the present invention can include non-volatile and volatile memory.

[0098] In summary, the present invention provides a real-time monitoring method for photodynamic therapy cell-substrate interaction, including: exciting monitoring signals based on a surface plasmon resonance microbial sensor system, and calibrating parameters of the surface plasmon resonance microbial sensor system; setting a cell culture well of a sensor chip, and performing a sealing test on the cell culture well; real-time monitoring of cell-substrate adhesion during photodynamic therapy based on the surface plasmon resonance microbial sensor system, and outputting the cell-substrate interaction results. The present invention can realize label-free detection of the dynamic process of biomolecular interaction on the gold film surface, thereby comprehensively reflecting the complex interaction between cells and substrates, and improving the accuracy of analysis of the effect of PDT on tumor cell adhesion.

[0099] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for real-time monitoring of cell-substrate interactions in photodynamic therapy, characterized in that: include: Exciting a monitoring signal based on a surface plasmon resonance microbial sensing system, and calibrating parameters of the surface plasmon resonance microbial sensing system; The surface plasmon resonance microbial sensing system is an intensity-type system, which is mainly divided into a light source module, a sensor module and an imaging detection module; the light source module is a femtosecond laser, the sensor module is a gold film of a total internal reflection fluorescence microscope, and the imaging detection module is composed of two CMOS cameras; Designing a cell culture well of a sensor chip, and performing a sealing test on the cell culture well; Based on the surface plasmon resonance microbial sensing system, the cell-substrate adhesion during the photodynamic therapy process is monitored in real time, and the cell-substrate interaction results are output; The surface plasmon resonance-based microbial sensing system excites and monitors signals, including: The laser is emitted by a laser, and the laser is passed through a beam expansion system to perform a beam expansion process of a preset multiple; The expanded light beam is passed through a half-wave plate and a polarizer to convert the light beam into P-polarized light, and the P-polarized light is reflected by a reflector and then converged to the rear focal plane by a tube lens, so as to control the light beam to irradiate the gold film in parallel at a preset angle, thereby achieving total internal reflection and exciting surface plasma waves; The surface plasmon resonance microbial sensing system monitors the cell-substrate adhesion during photodynamic therapy in real time and outputs the cell-substrate interaction results, including: Planting cells in the cell culture wells on the sensor chip and incubating photosensitizers; Real-time monitoring of the cell-substrate adhesion of cells incubated with photosensitizer during photodynamic therapy, and outputting the cell-substrate interaction results; Planting cells in the cell culture wells on the sensor chip and incubating photosensitizers comprises: HeLa cells in the logarithmic growth phase were taken and digested with trypsin, the remaining cells were collected and counted, and the cells were seeded on the gold film at 2000 cells / well in the cell culture well, and the cell culture well was placed in an incubator for incubation; The refractive index resolution of the surface plasmon resonance microbial sensing system is 5.13×10 -5 RIU; The design of the cell culture well of the sensor chip and the sealing detection of the cell culture well include: The cell culture well is obtained by bonding a dry wound healing plug-in with a gold film; a 3D model with a hole in the center is obtained by 3D printing, and the cell culture well is placed at the center hole position of the 3D model for sealing; after customizing the gold film with a preset thickness, the wound healing plug-in is bonded with the gold film to obtain the cell culture pool; a 3D model with a hole in the center is designed by SolidWorks software, and the 3D model is manufactured by 3D printing technology; wherein the length and width of the 3D model match the length and width of the wound healing plug-in, and the hole in the center of the 3D model is used to place the wound healing plug-in, so as to ensure that the cells can grow adherently in the wound healing plug-in; After adding liquid into the cell culture well, the well is left to stand for 24 hours to perform a sealing test by observing whether there is liquid leakage; The real-time monitoring of the cell-substrate adhesion of cells incubated with photosensitizer during photodynamic therapy includes: The gold film in the cell culture well is irradiated with a laser to excite the surface plasma wave of the gold film, the excited surface plasma wave is collected through an objective lens, and after being collimated by a lens, it is collected by a camera to obtain a surface plasma resonance image; analyzing the cell-substrate adhesion of the cells during the photodynamic therapy process according to the surface plasmon resonance image; The step of analyzing the cell-substrate adhesion of the cells during the photodynamic therapy according to the surface plasmon resonance image includes: Based on the image grayscale value of the reflected light response of the gold film surface, the dynamic process of cell-substrate adhesion of the cells during photodynamic therapy is analyzed label-free, and the cell-substrate interaction result is output; wherein the cell-substrate interaction result is used to reflect the position of the cell detaching from the substrate and the quantitative degree of detachment.

2. The method for real-time monitoring of cell-substrate interaction in photodynamic therapy according to claim 1, characterized in that: The parameter calibration of the surface plasmon resonance microorganism sensing system comprises: The surface plasmon resonance microorganism sensing system is detected by using sodium chloride solutions of different concentrations, and the refractive index resolution of the surface plasmon resonance microorganism sensing system is calculated by using a refractive index calculation formula to obtain calibrated parameters.

3. The method for real-time monitoring of cell-substrate interaction in photodynamic therapy according to claim 2, characterized in that: The refractive index calculation formula is: ； In the formula, represents the refractive index resolution, represents the change in the sample's refractive index, represents the change in the surface plasmon resonance signal, Indicates the noise level of the system.

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