Preparation method and application of photonic crystal hydrogel film sensor with elastic modulus self-reporting function

Abstract

This invention discloses a method for fabricating a photonic crystal hydrogel film sensor with a self-reporting elastic modulus function and its application. The method involves fabricating a hydrogel film with a periodic photonic crystal structure and constructing a bio-interface on its surface that can dynamically regulate cell adhesion, detachment, spreading, migration, proliferation, and differentiation. An optical imaging system is used to acquire images of the structural color changes of the photonic crystal hydrogel film. Image processing extracts color parameters, which are then converted into changes in reflected wavelength and the film deformation field. The elastic modulus of the film is calculated by inverting the deformation field generated by microspheres of known mass placed on the film surface, combined with an elasticity model. By combining the cell-interface interaction deformation field, the cell-interface interaction force and its spatial distribution can be obtained. A photonic crystal hydrogel film sensor with a self-reporting hardness function is constructed. This method achieves integrated biomechanical research combining dynamic regulation and real-time monitoring, offering advantages such as non-destructive operation, real-time imaging, and acquisition of mechanical spatial distribution.

Description

Technical Field

[0001] This invention belongs to the field of biointerface mechanics detection and biomaterials technology, specifically relating to a method for preparing a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus and its application. It utilizes the structural color changes of the photonic crystal hydrogel thin film to invert interface deformation and calculate interfacial forces, combined with dynamic interface effects to achieve active regulation and real-time dynamic monitoring of cell interface behavior. Particularly, it relates to the real-time dynamic monitoring of interfacial micromechanics during cell adhesion, detachment, spreading, migration, division, and differentiation at the interface, as well as the self-reporting of the material's own mechanical properties (elastic modulus). Background Technology

[0002] During cell growth, cells undergo a series of interfacial behaviors on the substrate surface, including adhesion, spreading, migration, division, and detachment. All of these behaviors are accompanied by changes in the interfacial mechanical interactions between the cell and the substrate. Therefore, real-time dynamic monitoring of interfacial mechanics during cell-substrate interfacial behaviors is of great significance for cell biology, tissue engineering, drug screening, and biomaterial evaluation.

[0003] Existing methods for detecting cell mechanics mainly include atomic force microscopy (AFM), traction force microscopy (TFM), and microcolumn array methods. AFM is a contact measurement, which can easily damage cells and is difficult to implement for long-term dynamic monitoring. TFM requires fluorescently labeled particles and complex image calculations, making real-time detection difficult. Microcolumn array methods require complex micro / nano fabrication processes and struggle to obtain continuous interfacial stress distributions. In summary, existing methods are insufficient for real-time, non-destructive monitoring of interfacial mechanics throughout the entire process of cell interface behavior.

[0004] Photonic crystal hydrogels possess coupled mechanical and optical response characteristics. When subjected to minute deformations, their structural color or reflection spectrum changes, allowing for the detection of material deformation through optical methods. However, current technologies primarily utilize photonic crystal hydrogels for pressure sensing or environmental stimulus response, lacking a method that simultaneously reports the material's elastic modulus and monitors cell interface mechanics by leveraging changes in photonic crystal structural color. Furthermore, existing cell mechanics monitoring substrates are mostly static interfaces, unable to actively regulate cell adhesion and detachment behaviors, making it difficult to simulate the dynamic microenvironment in vivo or intervene in cell behavior.

[0005] To address the aforementioned issues, this invention introduces dynamic interface effects (such as reversibly modulated interfaces based on dynamic covalent bonds of phenylboronic acid and photo / electric / thermal / magnetic responsive materials), enabling the sensor not only to monitor cell interface mechanics in real time, but also to actively control cell adhesion and detachment and adjust the elastic modulus of the substrate itself through external stimuli, thereby realizing an integrated cell mechanics research platform of "dynamic regulation-self-reporting-real-time monitoring". Summary of the Invention

[0006] To address the technical challenges of existing cell mechanics detection methods, such as contact damage, difficulty in real-time dynamic monitoring, inability to actively regulate cell behavior, and lack of self-reporting functionality for material mechanical properties, this invention proposes a method for fabricating a photonic crystal hydrogel film sensor with self-reporting elastic modulus based on the dynamic interface characteristics of photonic crystal hydrogel films, along with its applications. By calibrating the elastic modulus of the hydrogel film using microspheres, a quantitative relationship is established between changes in the photonic crystal structure color and interface deformation and stress. Combined with the active regulation of cell adhesion and other behaviors by the dynamic interface layer, real-time, non-destructive, and dynamic monitoring of the interfacial micromechanics throughout the entire process of cell adhesion, spreading, migration, division, and detachment at the interface is achieved.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for fabricating a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus includes the following steps:

[0009] Step 1: Prepare a photonic crystal hydrogel film with a periodic photonic crystal structure;

[0010] Step 2: Graft a reversible dynamic response material onto the surface of the hydrogel film to construct a biodynamic interface layer for dynamically regulating cell behavior.

[0011] Furthermore, the preparation process of the photonic crystal hydrogel film is as follows:

[0012] Step 1.1: Pre-treat the glass substrate by introducing hydrophobic and hydrophilic groups onto the surface of the glass substrate.

[0013] Step 1.2: Mix sulfonated silica particles with a periodic structure, crosslinking agent, photoinitiator and water in proportion to prepare a prepolymer solution; drop the prepolymer solution onto a glass substrate, cover it with a hydrophilic glass slide, and obtain a film with uniform thickness through polymerization reaction.

[0014] Furthermore, the reversible dynamic response material contains phenylboronic acid groups, which form dynamic crosslinking points with diol groups on adjacent molecular chains. By changing the pH value or sugar concentration, the crosslinking density can be adjusted, thereby dynamically regulating the elastic modulus of the hydrogel film.

[0015] By utilizing the interaction between phenylboronic acid and the diol groups of cell surface glycoproteins, cell adhesion and detachment can be reversibly controlled: cell adhesion is enhanced under alkaline / sugar-free conditions, and cell detachment occurs under acidic / high-sugar conditions.

[0016] Furthermore, the reversible dynamic response material includes, but is not limited to, photoresponsive azobenzene / cyclodextrin, electroresponsive polypyrrole, thermally responsive poly(N-isopropylacrylamide), and magnetically responsive Fe3O4 nanoparticles to modify the surface, thereby controlling the connection and breakage of dynamic covalent bonds or changes in molecular conformation in the interface layer, thereby dynamically regulating the adhesion, detachment, spreading, migration, proliferation or differentiation behavior of cells, and / or dynamically regulating the elastic modulus of the hydrogel film.

[0017] Furthermore, the specific steps for introducing polymerizable active phenylboronic acid groups onto the surface of the photonic crystal hydrogel film sensor to give it further grafting polymerization capability are as follows:

[0018] The first step involved introducing vinyl groups onto the surface of PCHFs using Sulfo-SANPAH and NH2-PEG5000-Acrylamide. The azide groups of Sulfo-SANPAH reacted with PCHFs under UV light (405 nm, 50 mW, 30 min) to form covalent bonds. Subsequently, its NHS ester groups reacted with NH2-PEG5000-Acrylamide to graft vinyl groups onto the PCHFs surface. After washing, a 10 mg mL−1 solution of NH2-PEG5000-Acrylamide was added dropwise to the PCHFs surface, and crosslinking was completed overnight at room temperature. The surface was rinsed three times with PBS before polymerization.

[0019] In the second step, 436 mg of HEAAm, 56 mg of AFPBA and 10 mg of HHMP were dissolved in 8 mL of DMSO, filtered and deoxygenated with N2 for 30 min; 50 μL of the above mixture was dropped onto the surface of PCHFs, irradiated with 365 nm UV lamp for 30 min, and then washed three times each with anhydrous ethanol and PBS to enable cells to adhere to the surface of the hydrogel film and form a cell-base interface.

[0020] A method for dynamic monitoring of cell interface behavior micromechanics and self-reporting of elastic modulus based on dynamic interfaces includes the following steps:

[0021] S1. Based on the prepared photonic crystal hydrogel film, a gradient thickness is prepared;

[0022] S2. Perform hue-wavelength calibration on the photonic crystal hydrogel film;

[0023] S3. Based on the structural color photograph of the hydrogel film under arbitrary loading, the structural color information is converted into reflection wavelength information, and then the deformation field of the film is reconstructed.

[0024] S4. Based on the known deformation field of the thin film under the load, calculate the elastic modulus of the thin film using an elastic mechanics model, and further obtain the cell-interface interaction force and stress distribution to achieve real-time dynamic monitoring of cell-interface behavior.

[0025] Furthermore, photographs of the photonic crystal hydrogel film were taken, and the hue values ​​of the image pixels were extracted using Matlab software. Combined with the reflection spectrum information collected by the reflection spectrometer, the correspondence between the image hue value H and the center wavelength λ of the reflection peak was established, resulting in the calibration curve: λ = a·H + b, where a· and b are the slope and intercept of this linear calibration equation, respectively (generally the initial peak at 450 nm).

[0026] Furthermore, using optical structural color photographs and the correspondence between the hue values ​​of photograph pixels and the reflected wavelength, the deformation field of the photonic crystal hydrogel film is calculated:

[0027] According to Bragg's law of diffraction, when the lattice spacing decreases, the reflected wavelength undergoes a blue shift; when the lattice spacing increases, the reflected wavelength undergoes a red shift; the change in the reflection peak wavelength... With changes in film thickness The following relationship must be satisfied:

[0028]

[0029] in, peak L0 is the initial reflection peak wavelength of the thin film, and L0 is the initial thickness of the thin film. The change in film thickness after being subjected to force is represented by compression (positive) and stretching (negative). Based on this, the local height of the hydrogel film corresponding to each pixel in the image is obtained, and the two-dimensional deformation field of the entire film is reconstructed.

[0030] Furthermore, based on the gravity of the microspheres and the local deformation field they generate on the film surface, the elastic modulus E of the film is calculated using an elasticity model:

[0031] Let the mass of the microsphere be m and the gravity be F=mg. The indentation depth δ generated in the contact area between the microsphere and the film is directly read from the deformation field, using the Hertzian contact model:

[0032] ;

[0033] ;

[0034] Where R is the radius of the microsphere, E* is the reduced elastic modulus, and ν is the Poisson's ratio of the hydrogel.

[0035] Furthermore, cells are seeded onto the biodynamic interface layer, where they undergo interfacial behaviors such as adhesion, spreading, migration, division, and desorption. The structural color change images or reflectance spectral signals on the surface of the photonic crystal hydrogel film sensor are acquired throughout the process using an optical imaging system.

[0036] Based on the calibrated elastic modulus E and the film deformation field, the cell-interface interaction forces and stress distribution are further obtained:

[0037] The strain-stress relationship of the material follows Hooke's law, and the strain ε is calculated from the change in reflected wavelength.

[0038] ;

[0039] Where, λ peak Let Δλ be the initial reflection peak wavelength of PCHFs. peak This represents the difference between the reflected peak wavelength of PCHFs after being subjected to force and its initial value. The initial lattice spacing of PCHFs; L0 is the difference between the lattice spacing of PCHFs after being subjected to force and its initial value; L0 is the initial height of PCHFs; ΔL is the change in height of PCHFs after being subjected to force; E is the elastic modulus of PCHFs. The strain of PCHFs is calculated. A stress distribution map is obtained by calculating the stress at each pixel in the deformation field.

[0040] Based on the deformation field, the interface stress distribution and interface forces are calculated using a hydrogel material mechanical model. According to the change of interface stress over time, the interface mechanical changes during cell adhesion, migration, division and detachment are obtained, realizing dynamic monitoring of the interface micromechanics throughout the entire process of cell interface behavior.

[0041] Beneficial effects

[0042] 1. Elastic modulus self-reporting function: The elastic modulus of the hydrogel film can be inverted non-contactly through the indentation deformation field of microspheres with known mass, without the need for additional mechanical testing equipment, and can be calibrated in real time during cell culture.

[0043] 2. Dynamic Interface Active Regulation: By introducing dynamic covalent bonds of phenylboronic acid or light, electricity, heat and magnetism responsive materials, cell adhesion and detachment can be reversibly controlled, and the elastic modulus of the film itself can be dynamically adjusted, providing a programmable microenvironment for cell mechanics research.

[0044] 3. Non-destructive real-time monitoring: Based on structural color imaging, it does not require fluorescent labels or contact probes, does not damage cells, and can achieve long-term, full-process dynamic monitoring.

[0045] 4. High spatial resolution mechanical imaging: By observing the color changes of each pixel, a two-dimensional distribution map of interface stress can be obtained, with a spatial resolution down to the micrometer level.

[0046] 5. Full-process coverage: It can continuously monitor the mechanical changes of cells throughout the entire interfacial behavior process from adhesion, spreading, migration, division to detachment, making up for the shortcomings of existing technologies that can only measure a single time point. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0048] Figure 1 This is a schematic diagram of the photonic crystal hydrogel film preparation process of the present invention.

[0049] Figure 2 This is a photograph of the photonic crystal hydrogel film of the present invention.

[0050] Figure 3 The following are the color calibration methods and supporting data diagrams, where (a) is the color calibration method of the microscope coupling device with gradient color spectrum, (b) is the color calibration diagram and reflection peak, and (c) is the Hue correspondence diagram.

[0051] Figure 4 This is a graph showing the elastic modulus of the photonic crystal hydrogel film of the present invention measured by the structural color change caused by microspheres. Among them, (ac) is the bright field image of glass microspheres of three sizes, (df) is the reflection image of microspheres of three sizes, (gi) is the combined image of 6 sets of data in the reflection mode, (j) is the force matrix distribution image corresponding to the 217μm microsphere, (k) is the three-dimensional deformation image corresponding to the 217μm microsphere, and (l) is the elastic modulus of the film measured by nanoindentation.

[0052] Figure 5 This is a schematic diagram of the construction of a dynamic biological interface on the surface of the hydrogel film of the present invention.

[0053] Figure 6 This is a schematic diagram of the coupling device between the microscope camera and the optical fiber of the present invention, wherein (a) is a diagram of the microscope reflectance spectrum coupling measurement device, and (b) is a diagram of the microscope imaging.

[0054] Figure 7The diagram illustrates the interfacial behavior of cells on the surface of a hydrogel, from adhesion to detachment, regulated by fructose solution. (a) shows the cell adhesion state immediately after the fructose solution is added, (b) shows the cell adhesion state after 3 minutes, and (c) shows the cell adhesion state after 12 minutes.

[0055] Figure 8 for Figure 7 The structural color changes corresponding to the three dynamic adjustment time points are converted into interface deformation and interface strain degree diagrams. Detailed Implementation

[0056] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0057] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0058] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0059] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0060] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0061] Example 1: A method for fabricating a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus, comprising the following steps:

[0062] Step 1: Prepare a hydrogel film with a photonic crystal periodic structure. The specific steps are as follows:

[0063] Step 1.1 Glass substrate pretreatment

[0064] Ordinary glass slides were ultrasonically cleaned sequentially with acetone, ethanol, and deionized water for 15 min each, and then dried with nitrogen. They were then immersed in a 1:1 mixture of concentrated sulfuric acid and hydrogen peroxide (piranha solution) for 30 min, rinsed thoroughly with deionized water, and dried with nitrogen to obtain a hydrophilic substrate rich in hydroxyl groups. To introduce hydrophobic groups, the cleaned slides could be immersed in a toluene solution containing 5 vol% trimethylchlorosilane for 2 h, then washed with toluene and ethanol, and dried with nitrogen.

[0065] Step 1.2 Prepolymer Solution Preparation and Film Forming

[0066] Sulfonated silica microspheres (150-200 nm in diameter, monodispersity index <5%) were dispersed in deionized water at a mass fraction of 10-80 wt%. The microsphere dispersion, crosslinking agent (N,N′-methylenebisacrylamide, 2 wt% aqueous solution), and photoinitiator (2-hydroxy-2-methyl-1-phenylpropanone, 1 wt% ethanol solution) were added sequentially at a volume ratio of 100:1:1, and the mixture was stirred thoroughly to obtain a prepolymer solution.

[0067] 20 μL of prepolymer solution was dropped onto the pretreated glass substrate, and a hydrophilically treated (plasma treatment for 5 min) ordinary glass slide was gently covered on top, with the thickness controlled by spacers (e.g., using 50 μm thick polyimide tape as a gasket). The solution was then incubated under a UV lamp (365 nm, power 10 mW / cm²). 2 Irradiate the substrate under light for 10 minutes to allow the prepolymer to polymerize and solidify. Carefully peel off the top glass slide to obtain a photonic crystal hydrogel film of uniform thickness.

[0068] Step 2: Graft one or more reversible dynamic response materials onto the surface of the hydrogel film to form a biodynamic interface layer.

[0069] The reversible dynamic response materials can be used alone or in combination to meet the requirements of regulation under a specific condition or under multiple conditions. Specific material types are as follows:

[0070] An interface layer based on dynamic covalent bonds of phenylboronic acid. Construction method: The above-mentioned photonic crystal hydrogel film was immersed in a PBS solution (pH 8.5) containing 3-acrylamidophenylboronic acid (AAPBA, 10 mM), a crosslinking agent (N,N'-methylenebisacrylamide, 0.5 mM), and a photoinitiator (Irgacure 2959, 1 mM). The film was then irradiated with UV light for 15 min under nitrogen protection, allowing phenylboronic acid groups to be grafted onto the surface or internal network of the hydrogel. The resulting film surface contains phenylboronic acid groups, which can form dynamic borate ester bonds with diol groups on adjacent molecular chains (such as hydroxyl groups in the hydrogel backbone).

[0071] Dynamically controlling the elastic modulus of hydrogel films: By adjusting the environmental pH (boronic acid ester bonds break under acidic conditions and form under alkaline conditions) or adding glucose (glucose preferentially binds to phenylboronic acid, competitively destroying the original crosslinks), the crosslink density can be reversibly controlled, thereby dynamically changing the elastic modulus of the film (e.g., the modulus decreases at pH 5.0 and increases at pH 8.5).

[0072] Dynamic regulation of cell behavior: By utilizing the interaction between phenylboronic acid and cell surface glycoproteins (containing diol groups), cell adhesion and detachment can be reversibly controlled: cell adhesion is enhanced under alkaline / sugar-free conditions, and cell detachment occurs under acidic / high-sugar conditions.

[0073] Other reversible dynamic response materials that can be reversibly controlled by light, electricity, heat or magnetic field.

[0074] Photoresponsive interface: Azobenzene-modified polyacrylic acid is grafted onto the surface of a hydrogel, and a cyclodextrin-modified cell adhesion peptide (RGD-cyclodextrin) is introduced. UV irradiation (365 nm) converts azobenzene to the cis form, reducing its binding affinity to cyclodextrin, causing RGD to detach from the surface and cells to desorb. Visible light (450 nm) restores the trans form, allowing RGD to rebind and cells to re-adhere.

[0075] Thermal response interface: The surface is grafted with poly(N-isopropylacrylamide) (PNIPAM) brush. When the temperature is below 32℃, the surface is hydrophilic and cells easily adhere to it; when the temperature is above 32℃, the surface is hydrophobic and cells detach.

[0076] Electro-responsive interface: Conductive polymer polypyrrole is deposited on the surface of the thin film. When a negative potential (-0.6 V) is applied, the polypyrrole is reduced and the surface becomes neutral, allowing cell adhesion. When a positive potential (+0.4 V) is applied, the polypyrrole is oxidized and becomes positively charged, which changes protein adsorption and regulates cell behavior.

[0077] Magnetic response interface: Fe3O4 magnetic nanoparticles are immobilized on the surface of a thin film. The cells are stimulated by mechanical force generated by an external magnetic field, and the cell response is monitored by the structural color change of the thin film.

[0078] More specifically, the specific steps for introducing polymerizable active phenylboronic acid groups onto the surface of a photonic crystal hydrogel film sensor to enable further graft polymerization are as follows:

[0079] The first step involved introducing vinyl groups onto the surface of PCHFs using Sulfo-SANPAH and NH2-PEG5000-Acrylamide. The azide groups of Sulfo-SANPAH reacted with PCHFs under UV light (405 nm, 50 mW, 30 min) to form covalent bonds. Subsequently, its NHS ester groups reacted with NH2-PEG5000-Acrylamide to graft vinyl groups onto the PCHFs surface. After washing, a 10 mg mL−1 solution of NH2-PEG5000-Acrylamide was added dropwise to the PCHFs surface, and crosslinking was completed overnight at room temperature. The surface was rinsed three times with PBS before polymerization.

[0080] In the second step, 436 mg of HEAAm, 56 mg of AFPBA, and 10 mg of HHMP were dissolved in 8 mL of DMSO, filtered, and deoxygenated with N2 for 30 min. 50 μL of the mixture was then dropped onto the surface of PCHFs. After irradiation with a 365 nm UV lamp for 30 min, the cells were washed three times each with anhydrous ethanol and PBS to allow them to adhere to the hydrogel film surface and form a cell-base interface.

[0081] Example 2: Based on the photonic crystal hydrogel thin film sensor prepared by the method described in Example 1, this invention proposes a method for dynamic monitoring of cell interface behavior micromechanical dynamics and self-reporting of elastic modulus, comprising the following steps:

[0082] S1, based on the prepared photonic crystal hydrogel film, a gradient thickness is prepared.

[0083] Take a prepared photonic crystal hydrogel film (e.g., 2 cm × 2 cm in size), and place a 200 μm thick polyester spacer below one edge of the film. Then, cover the film and spacer with another clean glass slide. Press the glass slide with a lightweight block near the spacer end, causing the slide to tilt slightly. The film thickness gradually decreases from the spacer end (thick end) to the far end (thin end), forming a continuous gradient. The thickness variation range of this gradient sample is 50–250 μm, and the corresponding photonic crystal lattice spacing also shows a gradient change, producing a continuous structural color from red to blue.

[0084] S2 Hue-Wavelength Calibration

[0085] The gradient sample was placed under an optical microscope (with a color CCD) and a structural color photograph of the entire region was taken in reflectance mode. Simultaneously, a fiber optic reflectance spectrometer (0.5 nm resolution) was used to acquire the reflectance spectra at multiple known locations (0.5 mm intervals) in the photograph, obtaining the center wavelength λ of the reflectance peak at each location. The RGB values ​​of the corresponding pixels in the photograph were read using Matlab, converted to the HSV color space, and the hue value H was extracted. A linear fit was performed between H and λ to obtain the calibration curve: λ = a·H + b.

[0086] In this embodiment, the typical fitting result is λ = 1.2·H + 450 (unit nm), and the correlation coefficient R²>0.99.

[0087] S3 Deformation Field Reconstruction

[0088] According to Bragg's law of diffraction, when the lattice spacing decreases, the reflected wavelength undergoes a blue shift; when the lattice spacing increases, the reflected wavelength undergoes a red shift. (The change in reflection peak wavelength is not explicitly stated in the original text.) With changes in film thickness The following relationship must be satisfied:

[0089]

[0090] in, peak L0 is the initial reflection peak wavelength of the thin film, and L0 is the initial thickness of the thin film. This represents the change in film thickness under stress (compression is positive, stretching is negative). Based on this, the local height of the hydrogel film corresponding to each pixel in the image can be obtained, thereby reconstructing the two-dimensional deformation field of the entire film.

[0091] For a structured color photograph under arbitrary loading, the reflected wavelength λ(x,y) is first calculated using the calibration curve based on the hue value H(x,y) of each pixel.

[0092] According to the differential form of Bragg's law:

[0093] Δλ / λ0 = ΔL / L0

[0094] Where λ0 and L0 are the initial reflection wavelength and thickness of the film when it is undeformed (obtainable from the uncompressed area at the edge of the film). Thus, the thickness variation of each pixel is ΔL(x,y) = L0·(λ(x,y)-λ0) / λ0. If the initial thickness L0 is defined as a constant, then the absolute thickness distribution of the film is L(x,y) = L0 + ΔL(x,y), and the deformation field (displacement field) is u(x,y) = -ΔL(x,y) (compressed to positive). The spatial resolution of the reconstructed deformation field can reach approximately 0.5 μm for a single pixel.

[0095] S4, Elastic modulus self-reporting and cell interface mechanical monitoring.

[0096] S4.1 Microsphere Loading

[0097] A photonic crystal hydrogel film with a uniform color (reflection wavelength λ0 = 560 nm, initial thickness L0 = 20 μm) and a phenylboronic acid dynamic interface was selected and placed on the stage of an inverted optical microscope. Under the microscope, a single glass microsphere (diameter 434 μm, mass 107 μg, confirmed by weighing) was picked up with a micromanipulation needle and gently placed in the central region of the film surface. An initial photomicrograph of the area containing the microsphere was taken (magnification 10×, resolution 5480×3648 pixels).

[0098] In this embodiment, microspheres (glass spheres, metal microspheres, etc.) with smooth surfaces and no internal air bubbles are selected. Microspheres of the same size can be used on the same hydrogel film, or microspheres of different sizes can be used for subsequent self-reporting calibration of elastic modulus.

[0099] S4.2 Calculation of Elastic Modulus

[0100] The film deformation field induced by the microspheres is reconstructed using the method in step S3, and the maximum indentation depth δmax directly below the microspheres is read. Six measurements are taken and averaged; for example, δmax = 4.3 μm. The microsphere radius is known to be R = 217 μm, and the gravity is F = mg = 1.07 × 10⁻⁶. -7 kg × 9.8 m / s 2 = 1.05×10 -6 N. Using the Hertzian contact model (spherical indenter and elastic half-space):

[0101]

[0102]

[0103] For a hydrogel with a Poisson's ratio ν≈0.5, then E* = 4E / 3. Substituting the values, we get E = 4.49 kPa. This value is very close to the modulus measured by independent compression tests (4.58 kPa), verifying the accuracy of the method.

[0104] S4.3 Cell Seeding and Behavioral Monitoring

[0105] Mouse fibroblasts (L929) were suspended in DMEM medium (containing 10% fetal bovine serum) at a density of 1×10⁻⁶. 5cells / mL. 200 μL of cell suspension was dropped onto the surface of the above-mentioned membrane and placed in a 37 ℃, 5% CO2 incubator to allow cell settling and adhesion. Simultaneously, a microscope and imaging system were placed in the incubator or equipped with a temperature-controlled platform for continuous imaging for 24 hours. When the phenylboronic acid interface was in its initial state (pH 7.4, no additional glucose), cells adhered and spread normally. When glucose was added to the culture medium to a final concentration of 50 mM, the dynamic bond of the phenylboronic acid-diol was competitively disrupted, causing the cell adhesion ligands to detach from the surface. Approximately 30 minutes later, the cells began to shrink and gradually detach. After changing to a glucose-free culture medium, the cells adhered again.

[0106] S4.4 Calculation of interfacial forces

[0107] The strain-stress relationship of the material follows Hooke's law, and the strain ε is calculated from the change in reflected wavelength.

[0108] ;

[0109] Where, λ peak Let Δλ be the initial reflection peak wavelength of PCHFs. peak This represents the difference between the reflected peak wavelength of PCHFs after being subjected to force and its initial value. The initial lattice spacing of PCHFs; ΔL represents the difference between the lattice spacing of PCHFs after being subjected to force and its initial value; L0 represents the initial height of PCHFs; ΔL represents the height change of PCHFs after being subjected to force; E represents the elastic modulus of PCHFs; and ε represents the strain of PCHFs. A stress distribution map is obtained by calculating each pixel in the deformation field.

[0110] During cell adhesion or spreading, the film undergoes minute deformations. For the structural color photographs taken at each time point, the deformation field is first reconstructed, and then the interfacial stress is calculated using the elastic modulus E = 4.49 kPa obtained from S4.2, according to Hooke's law.

[0111]

[0112] Where Δλ(x,y,t) is dynamically calculated from the hue value. Integrating the stress distribution over the cell attachment region yields the total traction force Fcell(t) = ∫ dA.

[0113] S4.5 Full-process dynamic monitoring

[0114] Based on the curve of interfacial stress change over time, the initial stage of cell adhesion (rapid increase in stress), the spreading stage (stress distribution expands and tends to stabilize), the migration stage (stress center shifts), and the desorption stage (stress decreases until it reaches zero) can be clearly distinguished. By simultaneously recording the regulatory parameters of the dynamic interface (such as the time point of sugar addition), a direct correlation between "stimulus-cell behavior-mechanical response" is established. The method of this invention achieves non-contact, non-damaging, real-time dynamic monitoring of the entire process of cell interface behavior.

[0115] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for fabricating a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus, characterized in that, Includes the following steps: Step 1: Prepare a photonic crystal hydrogel film with a periodic photonic crystal structure; Step 2: Graft a reversible dynamic response material onto the surface of the hydrogel film to construct a biodynamic interface layer for dynamically regulating cell behavior.

2. The method for fabricating a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus function according to claim 1, characterized in that, The preparation process of the photonic crystal hydrogel film is as follows: Step 1.1: Pre-treat the glass substrate by introducing hydrophobic and hydrophilic groups onto the surface of the glass substrate. Step 1.2: Mix sulfonated silica particles with a periodic structure, crosslinking agent, photoinitiator and water in proportion to prepare a prepolymer solution; drop the prepolymer solution onto a glass substrate, cover it with a hydrophilic glass slide, and obtain a film with uniform thickness through polymerization reaction.

3. The method for fabricating a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus function according to claim 1, characterized in that, The reversible dynamic response material contains phenylboronic acid groups, which form dynamic crosslinking points with diol groups on adjacent molecular chains. The crosslinking density can be adjusted by changing the pH value or sugar concentration, thereby dynamically regulating the elastic modulus of the hydrogel film. By utilizing the interaction between phenylboronic acid and the diol groups of cell surface glycoproteins, cell adhesion and detachment can be reversibly controlled: cell adhesion is enhanced under alkaline / sugar-free conditions, and cell detachment occurs under acidic / high-sugar conditions.

4. The method for fabricating a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus function according to claim 1, characterized in that, The reversible dynamic response materials include, but are not limited to, photoresponsive azobenzene / cyclodextrin, electroresponsive polypyrrole, thermally responsive poly(N-isopropylacrylamide), and magnetically responsive Fe3O4 nanoparticles that modify the surface to control the connection and breakage of dynamic covalent bonds or changes in molecular conformation in the interface layer, thereby dynamically regulating the adhesion, detachment, spreading, migration, proliferation or differentiation behavior of cells, and / or dynamically regulating the elastic modulus of the hydrogel film.

5. The method for fabricating a photonic crystal hydrogel thin film sensor with self-reporting elastic modulus function according to claim 1, characterized in that, The specific steps for introducing polymerizable active groups onto the surface of a photonic crystal hydrogel thin film sensor to enable further graft polymerization are as follows: The first step involved introducing vinyl groups onto the surface of PCHFs using Sulfo-SANPAH and NH2-PEG5000-Acrylamide. The azide groups of Sulfo-SANPAH reacted with PCHFs under UV light (405 nm, 50 mW, 30 min) to form covalent bonds. Subsequently, its NHS ester groups reacted with NH2-PEG5000-Acrylamide to graft vinyl groups onto the PCHFs surface. After washing, a 10 mg mL−1 solution of NH2-PEG5000-Acrylamide was added dropwise to the PCHFs surface, and crosslinking was completed overnight at room temperature. The surface was rinsed three times with PBS before polymerization. In the second step, 436 mg of HEAAm, 56 mg of AFPBA and 10 mg of HHMP were dissolved in 8 mL of DMSO, filtered and deoxygenated with N2 for 30 min; 50 μL of the above mixture was dropped onto the surface of PCHFs, irradiated with 365 nm UV lamp for 30 min, and then washed three times each with anhydrous ethanol and PBS to enable cells to adhere to the surface of the hydrogel film and form a cell-base interface.

6. A method for dynamic monitoring of cell interface behavior micromechanical dynamics and self-reporting of elastic modulus based on dynamic interfaces, characterized in that, Includes the following steps: S1. Based on the prepared photonic crystal hydrogel film, a gradient thickness is prepared; S2. Perform hue-wavelength calibration on the photonic crystal hydrogel film; S3. Based on the structural color photograph of the hydrogel film under arbitrary loading, the structural color information is converted into reflection wavelength information, and then the deformation field of the film is reconstructed. S4. Based on the known deformation field of the thin film under the load, calculate the elastic modulus of the thin film using an elastic mechanics model, and further obtain the cell-interface interaction force and stress distribution to achieve real-time dynamic monitoring of cell-interface behavior.

7. The method for dynamic monitoring of cell interface behavior micromechanics and self-reporting of elastic modulus based on dynamic interface as described in claim 6, characterized in that, Photographs of the photonic crystal hydrogel film were taken, and the hue values ​​of the image pixels were extracted using Matlab software. Combined with the reflection spectrum information collected by the reflection spectrometer, the correspondence between the image hue value H and the center wavelength λ of the reflection peak was established, and the calibration curve was obtained: λ = a·H + b, where a· and b are the slope and intercept of this linear calibration equation, respectively (generally the initial peak is 450 nm).

8. The method for dynamic monitoring of cell interface behavior micromechanical dynamics and self-reporting of elastic modulus based on dynamic interface as described in claim 6, characterized in that, Using optical structural color photographs and the correspondence between the hue values ​​of photograph pixels and the reflected wavelength, the deformation field of the photonic crystal hydrogel film is calculated: According to Bragg's law of diffraction, when the lattice spacing decreases, the reflected wavelength undergoes a blue shift; when the lattice spacing increases, the reflected wavelength undergoes a red shift; the change in the reflection peak wavelength... With changes in film thickness The following relationship must be satisfied: in, peak L0 is the initial reflection peak wavelength of the thin film, and L0 is the initial thickness of the thin film. The change in film thickness after being subjected to force is represented by compression (positive) and stretching (negative). Based on this, the local height of the hydrogel film corresponding to each pixel in the image is obtained, and the two-dimensional deformation field of the entire film is reconstructed.

9. The method for dynamic monitoring of cell interface behavior micromechanical dynamics and self-reporting of elastic modulus based on dynamic interface according to claim 6, characterized in that, Based on the gravity of the microspheres and the local deformation field they generate on the film surface, the elastic modulus E of the film is calculated using an elasticity model: Let the mass of the microsphere be m and the gravity be F=mg. The indentation depth δ generated in the contact area between the microsphere and the film is directly read from the deformation field, using the Hertzian contact model: ； ； Where R is the radius of the microsphere, E* is the reduced elastic modulus, and ν is the Poisson's ratio of the hydrogel.

10. The method for dynamic monitoring of cell interface behavior micromechanical dynamics and self-reporting of elastic modulus based on dynamic interface according to claim 6, characterized in that, Cells are seeded onto the biodynamic interface layer, and the cells undergo interface behaviors such as adhesion, spreading, migration, division and desorption. The structural color change image or reflectance spectrum signal of the photonic crystal hydrogel film sensor surface is acquired throughout the process by an optical imaging system. Based on the calibrated elastic modulus E and the film deformation field, the cell-interface interaction forces and stress distribution are further obtained: The strain-stress relationship of the material follows Hooke's law, and the strain ε is calculated from the change in reflected wavelength. ； Where, λ peak Let Δλ be the initial reflection peak wavelength of PCHFs. peak This represents the difference between the reflected peak wavelength of PCHFs after being subjected to force and its initial value. The initial lattice spacing of PCHFs; ΔL represents the difference between the lattice spacing of PCHFs after being subjected to force and its initial value; L0 represents the initial height of PCHFs; ΔL represents the height change of PCHFs after being subjected to force; E represents the elastic modulus of PCHFs; and ε represents the strain of PCHFs. A stress distribution map is obtained by calculating each pixel in the deformation field. Based on the deformation field, the interface stress distribution and interface forces are calculated using a hydrogel material mechanical model. According to the change of interface stress over time, the interface mechanical changes during cell adhesion, migration, division and detachment are obtained, realizing dynamic monitoring of the interface micromechanics throughout the entire process of cell interface behavior.

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