Measuring chip, automatic measuring system and measuring method for elastic modulus of flexible biological object

Abstract

The invention discloses a measuring chip, an automatic measuring system and a measuring method for the elastic modulus of a flexible biological object, belongs to the field of measurement of mechanical properties of biological materials, and particularly relates to the measuring chip, the automatic measuring system and the measuring method for the elastic modulus of the flexible biological object. The measuring chip comprises a glass substrate, a patterned polydimethylsiloxane (PDMS) substrate and a thin film sensor, the automatic measuring system comprises a measuring chip, a three-axis micromanipulator, an electric oil pump, a focusing manual lifting table, an inclination angle adjusting table, an image acquisition module, an LED light source and an XY electric translation table. An agarose array-film sensor composite structure is used as a flexible plane, and the flexible plane and a solid glass rigid plane form a test system. Therefore, the upper surface is a hard surface, the lower film sensor gradually deforms, and the deformation of the film is far smaller than that of the flexible sample.

Description

Technical Field

[0001] This application belongs to the field of biomaterial mechanical property measurement, specifically relating to a chip for measuring the elastic modulus of flexible biological objects, an automated measurement system and a measurement method thereof. Background Technology

[0002] In the fields of life sciences and biomedical research, elastic modulus, as a core parameter characterizing the mechanical properties of biomaterials, plays an irreplaceable and crucial role in accurately measuring it for revealing cellular behavior mechanisms, understanding tissue development patterns, and exploring the processes of disease occurrence and development. As the basic structural and functional unit of living organisms, changes in the mechanical properties of cells are often closely related to cell proliferation, differentiation, migration, and the occurrence and development of diseases. For example, the elastic modulus of cancer cells changes significantly compared to normal cells; accurate measurement of elastic modulus can provide important evidence for the early diagnosis and treatment of cancer. In the field of tissue engineering, understanding the elastic modulus of tissues helps in designing artificial tissues and organs that better conform to physiological characteristics.

[0003] Flexible biological objects encompass a variety of types, including single cells, soft blastocysts, cell clusters, and tissue sections. They not only exhibit vastly different mechanical properties but are also highly susceptible to external stimuli. Taking single cells as an example, their mechanical properties are highly heterogeneous; the elastic modulus of cells of different species and physiological states can differ by several times or even tens of times. Furthermore, the mechanical properties of cells dynamically change under external mechanical forces, temperature, and chemical stimuli. This complexity presents unique technical challenges to the accurate measurement of the elastic modulus of flexible biological objects, necessitating high-precision and high-sensitivity measurement techniques and methods.

[0004] In recent years, with the rapid development of biomedical engineering and micro-nano technology, mechanical measurement technology for flexible biological objects has ushered in an unprecedented opportunity for innovation, and a series of high-precision and high-sensitivity measurement methods and integrated platforms have emerged. At present, elastic modulus measurement technology can be mainly divided into two categories: contact and non-contact.

[0005] Among contact measurement techniques, atomic force microscopy (AFM) is the most representative tool. AFM applies controllable minute forces to the sample surface using sharp microprobes and sensitive microcantilever, and monitors the deformation of the microcantilever in real time. It then calculates the elastic modulus of the sample based on Hooke's Law. This technique, with its nanometer-level spatial resolution and piconewton-level force resolution, is particularly suitable for analyzing the local mechanical properties of biological samples, accurately acquiring mechanical information from different regions of the cell surface. Parallel plate compression technology uses two parallel plates to compress flexible biological objects. By measuring the applied force and the deformation of the sample during compression, combined with appropriate mechanical models, it analyzes the overall mechanical properties of the flexible biological object. It is often used to study the mechanical properties of larger samples such as tissue sections. Micropipette negative pressure suction technology deforms the cell membrane by adjusting the negative pressure within a micropipette. Fluid dynamics and elasticity models are used to analyze the deformation process, thereby calculating the cell's elastic modulus. This technique has been widely used in the field of cell mechanics research.

[0006] Non-contact measurement techniques, such as optical stretching and magnetic bead torsion, utilize non-contact methods like optical tweezers and magnetic fields to achieve non-destructive measurements of biological samples. These techniques are particularly suitable for biological samples that are sensitive to mechanical stimulation and easily damaged. For example, optical stretching uses gradient forces generated by optical tweezers to stretch cells, calculating the elastic modulus by measuring the cell's deformation and stress during the stretching process. Magnetic bead torsion involves labeling magnetic nanoparticles onto the cell surface, applying a rotating magnetic field to torsion the beads, causing minute deformations in the cells, and calculating the cell's elastic modulus based on the torsion angle and the applied magnetic field strength.

[0007] Despite continuous development and improvement in existing measurement technologies, most current measurement systems still suffer from significant limitations. On the one hand, measurement processes are not yet highly automated. From sample preparation and measurement operations to data acquisition and analysis, researchers often need to perform a large amount of manual work, which is not only time-consuming and labor-intensive but also prone to introducing human error, making it difficult to meet the demands of high-throughput and high-efficiency measurements. On the other hand, transferring flexible biological objects presents numerous challenges. Because flexible biological objects are inherently fragile and easily damaged, traditional transfer methods may cause mechanical damage, affecting the accuracy of measurement results. Simultaneously, precisely controlling the position and orientation of the sample during the measurement process is difficult, further limiting the accuracy and reliability of the measurement. Therefore, developing an elastic modulus measurement platform capable of automating the measurement process and effectively solving the challenges of transferring flexible biological objects is of significant practical importance for advancing research on the mechanical properties of flexible biological objects. Summary of the Invention

[0008] This application addresses the aforementioned problems by providing a chip for measuring the elastic modulus of flexible biological objects, an automated measurement system, and a measurement method thereof.

[0009] The first aspect of this application provides a chip for measuring the elastic modulus of flexible biological objects, including a glass substrate, a patterned polydimethylsiloxane (PDMS) substrate, and a thin-film sensor;

[0010] The thin-film sensor includes an upper PDMS thin film, an intermediate functional layer, and a lower PDMS thin film, with an overall thickness of 130~140μm. The lower PDMS thin film has a non-porous structure. The upper PDMS thin film has a first through-hole at a predetermined electrode position to provide an electrical path for external wire connection.

[0011] The measurement chip has one or more measurement slots.

[0012] A second aspect of this application provides an automated measurement system for the elastic modulus of flexible biological objects, including a measurement chip, a triaxial micromanipulator, an electric oil pump, a manual focusing lifting stage, a tilt angle adjustment stage, an image acquisition module, an LED light source, and an XY electric translation stage. The electric oil pump is connected to a micropipette via a sealed pipe, and the position of the lead screw pushing / suction is controlled by a motor to control the suction and discharge of liquid in the micropipette. The triaxial micromanipulator and the XY electric translation stage are both height-adjustable via the manual focusing lifting stage. A bracket is provided on the XY electric translation stage to support the measurement chip. There are two image acquisition modules, one directly above the measurement chip and the other to the side above it. The LED light source is located at the front end of the image acquisition module directly above the measurement chip, and the tilt angle adjustment stage is located at the bottom of the image acquisition module to the side above the measurement chip.

[0013] A third aspect of this application provides a measurement method for the aforementioned automated measurement system for the elastic modulus of flexible biological objects, comprising:

[0014] Equipment calibration and positioning: The ground micropipette is mounted on a high-precision micromanipulation arm, and the tip is manually adjusted to enter the optical microscope's field of view. Using an automated focusing system, three-dimensional image data is constructed to determine the confocal plane between the tip and the sample. A contour extraction algorithm is used to process the microscopic image, identifying the micropipette region by comparing contour geometric features, and determining the tip coordinates using the outermost contour point. For irregularly shaped biological objects, the centroid is calculated based on the contour pixel distribution to obtain its center image coordinates.

[0015] Sample selection and adsorption: The target biological object is specified in the microscopic image through the graphical user interface. The system is based on visual servo control technology to drive the microscopic manipulator arm to bring the needle tip close to the sample. It stops when the pixel distance between the two reaches a preset threshold. The electric oil pump adsorbs the biological object to the needle tip.

[0016] Sample transfer and target localization: After the micropipette adsorbs the sample, it is raised to a safe height along the Z-axis; the biaxial translation stage moves the agarose array on the thin-film sensor into the microscope field of view according to the pre-calibrated coordinates;

[0017] Sample release: The system calculates the pixel deviation between the micropipette and the target agarose array position based on machine vision, and maps it into three-dimensional motion commands for the micromanipulator after coordinate transformation model; after the manipulator moves to the target position and descends a specified height along the Z-axis, the electric oil pump releases pressure, allowing the biological object to fall smoothly into the groove of the agarose array. This process is repeated until all specified samples are transferred.

[0018] Preparation of measuring equipment: After the sample transfer is completed, adjust the position and angle of the side-view microscope to obtain a clear side view image of the sample; replace the micropipette used for adsorption, install the ground glass platform, adjust its verticality for measurement, and use a digital microscope to measure and record the initial diameter of the sample.

[0019] Measurement platform positioning: The microscopic operating table moves to the target position according to the sample coordinates recorded in step one, and then decreases its speed after descending to the specified height;

[0020] Contact and loading: The measurement chip comes into contact with the biological object at a constant speed. In the initial stage of contact, only a small elastic deformation occurs and no detectable force signal is generated.

[0021] Data acquisition and measurement termination: Continue loading until the thin-film sensor acquires a complete force-deformation curve, and simultaneously record the current diameter of the sample and the change in sensor resistance; repeat this process until all sample measurements are completed.

[0022] The beneficial effects of this application are:

[0023] This application uses a flexible sensing film and a customized rigid glass platform to replace the traditional parallel plate extrusion method, thus adapting to automated operation. Micromanipulation techniques are employed to automate the transfer and measurement of the elastic modulus of flexible biological objects, improving the success rate and speed of biological object transfer, reducing waste of biological samples, and increasing the scale and efficiency of measurements, thereby achieving high-throughput testing. Attached Figure Description

[0024] Figure 1 A schematic diagram of an automated system for measuring the elastic modulus of flexible biological objects;

[0025] Figure 2 This is a schematic diagram of the support structure on the XY electric translation platform;

[0026] Figure 3 This diagram illustrates the aspiration and transfer of flexible biological samples from the culture medium; the procedure follows steps a, b, c, and d.

[0027] Figure 4 A schematic diagram for automatically completing the measurement of the elastic modulus; the operation procedure follows abcd;

[0028] Figure 5 Electron micrograph of molten, ground hard capillary glass under a 4X magnification microscope;

[0029] Figure 6 This is a schematic diagram of the mechanical model in the embodiment;

[0030] Figure 7 This is a schematic diagram of the measuring slot in the fully connected state of Example 2. Detailed Implementation

[0031] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0032] Specific Implementation Method 1: This implementation method provides a chip for measuring the elastic modulus of flexible biological objects, comprising a glass substrate, a patterned polydimethylsiloxane (PDMS) substrate, and a thin-film sensor.

[0033] The thin-film sensor includes an upper PDMS thin film, an intermediate functional layer, and a lower PDMS thin film, with an overall thickness of 130~140μm. The lower PDMS thin film has a non-porous structure. The upper PDMS thin film has a first through-hole at a predetermined electrode position to provide an electrical path for external wire connection.

[0034] The measurement chip has one or more measurement slots.

[0035] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that one or more second through-holes are formed on the patterned PDMS substrate. The number of second through-holes is the same as the number of measurement grooves. The positions of the second through-holes correspond one-to-one with the positions of the measurement grooves. The second through-holes and the area covered by the thin-film sensor on the second through-holes constitute the measurement grooves. Everything else is the same as in Specific Implementation Method One.

[0036] Specific Implementation Method 3: The difference between this implementation method and Specific Implementation Method 1 is that the number of the second through holes is two or more, and the connection relationship of the two or more second through holes is either completely non-connected, locally connected, or completely connected.

[0037] The term "globally disconnected" means that all second vias are independent and there are no interconnected paths between them.

[0038] The local connectivity is a subset of second vias forming a connected set, which has no connecting path with other second vias; wherein, the number of such connected subsets can be one or more;

[0039] The full connectivity means that all the second through holes form a single connected system, and there is a connection path between any two second through holes;

[0040] When the connectivity is local or global, a gas insertion port is placed in the middle of the connectivity path. Everything else is the same as in Specific Implementation Method 1.

[0041] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method One in that the glass substrate, the patterned PDMS substrate, and the thin-film sensor are permanently bonded together as a whole through ion bonding. Everything else is the same as in Specific Implementation Method One.

[0042] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method One in that it also includes an agarose array, which is non-fixedly placed within the measuring tank. Everything else is the same as in Specific Implementation Method One.

[0043] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method One in that the thickness of both the upper and lower PDMS films is 50 μm, and the thickness of the intermediate functional layer is 35 μm. Everything else is the same as in Specific Implementation Method One.

[0044] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method One in that the measurement chip is prepared in the following manner:

[0045] Hard optical glass is selected as the glass substrate;

[0046] The unpatterned surface of the patterned PDMS substrate is connected to the glass substrate by ion bonding;

[0047] The patterned surface of the patterned PDMS substrate is bonded to the underlying PDMS film;

[0048] A metal mask is tightly bonded to the upper surface of the lower PDMS film, and the cutout area of ​​the metal mask corresponds to the pattern design of the intermediate functional layer. A special conductive ink is uniformly sprayed onto the surface of the metal mask in a mist using an aerosol spraying system. Under the action of gravity and surface tension, the special conductive ink penetrates into the cutout area of ​​the metal mask, forming the intermediate functional layer with the pattern corresponding to the pattern design on the surface of the lower PDMS film.

[0049] The upper PDMS film is bonded to the upper surface of the lower PDMS film to form the thin-film sensor. Everything else is the same as in Specific Embodiment 1.

[0050] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method Seven in that the patterned PDMS substrate is prepared in the following manner:

[0051] The PDMS prepolymer and curing agent are mixed evenly at a mass ratio of 1:10 to form a mixture, which is then poured into a photocurable mold.

[0052] Place the mixture in a vacuum chamber for 1 hour to remove air bubbles.

[0053] Transfer the mixture to a constant temperature chamber and cure it at 70°C for 3 hours;

[0054] The patterned polydimethylsiloxane PDMS substrate is obtained by demolding the cured mixture. Everything else is the same as in Specific Embodiment Seven.

[0055] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method Five in that the agarose array is prepared in the following manner:

[0056] Molds with predetermined patterns are printed using photopolymerization technology;

[0057] The agarose is heated to a molten state by microwave and then poured into a container;

[0058] A photocurable stamp mold is used to imprint agarose colloid in a molten state. After the agarose cools and solidifies, it is demolded to obtain an agarose array with a predetermined microstructure. The prepared agarose array is arranged in a 4×3 matrix, forming a circular array structure with a diameter of 300 μm. Other aspects are the same as in Specific Embodiment Five.

[0059] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method Nine in that the connections between the agarose array, the glass substrate, the patterned PDMS substrate, and the thin-film sensor are all formed by permanent bonding through ion bonding, connecting them into a whole; wherein, the agarose array is non-fixed. Everything else is the same as in Specific Implementation Method Nine.

[0060] Specific Implementation Method Eleven: This implementation method differs from Specific Implementation Method Seven in that the specially formulated conductive ink comprises multi-walled carbon nanotubes, carbon black, polydimethylsiloxane, and isopropanol. Everything else is the same as in Specific Implementation Method Seven.

[0061] Specific Implementation Method Twelve: This implementation method differs from Specific Implementation Method Eleven in that the specially made conductive ink is prepared in the following manner:

[0062] Multi-walled carbon nanotubes and carbon black powder, with a mass ratio of 1:1, were placed separately in isopropanol in different containers.

[0063] Two containers were placed on a magnetic stirrer and stirred continuously at room temperature for 1 hour. After the magnetic stirring was completed, the two containers were transferred to a water bath and subjected to ultrasonic treatment for 1 hour to obtain a multi-walled carbon nanotube-isopropanol dispersion and a carbon black-isopropanol dispersion.

[0064] Multi-walled carbon nanotube-isopropanol dispersion and carbon black-isopropanol dispersion were added together with PDMS prepolymer into the same clean container to form a mixed system;

[0065] The container containing the mixture was placed in a 50°C constant temperature water bath and continuously stirred for 6 hours. During the stirring process, isopropanol evaporated, ultimately yielding the specially formulated conductive ink. Other procedures are the same as in Specific Embodiment Eleven.

[0066] Specific Implementation Method Thirteen: This implementation method provides an automated measurement system for the elastic modulus of flexible biological objects, comprising a measurement chip 7, a triaxial micromanipulator 1, an electric oil pump 2, a manual focusing lifting platform 3, a tilt angle adjustment platform 4, an image acquisition module 5, an LED light source 6, and an XY electric translation stage 8. The electric oil pump 2 is connected to a micropipette via a sealed pipe, and the position of the lead screw pushing / suction is controlled by a motor to control the suction and discharge of liquid in the micropipette. The triaxial micromanipulator 1 and the XY electric translation stage 8 are both height-adjustable via the manual focusing lifting platform 3. A bracket is provided on the XY electric translation stage 8 to support the measurement chip 7. There are two image acquisition modules 5, one directly above the measurement chip 7 and the other to the side above it. The LED light source 6 is located at the front end of the image acquisition module 5 directly above the measurement chip 7, and the tilt angle adjustment platform 4 is located at the bottom of the image acquisition module 5 located to the side above the measurement chip 7.

[0067] Specific Implementation Method Fourteen: This implementation method provides a measurement method for an automated measurement system for the elastic modulus of flexible biological objects, including:

[0068] Equipment calibration and positioning: The ground micropipette is mounted on a high-precision micromanipulation arm, and the tip is manually adjusted to enter the optical microscope's field of view. Using an automated focusing system, three-dimensional image data is constructed to determine the confocal plane between the tip and the sample. A contour extraction algorithm is used to process the microscopic image, identifying the micropipette region by comparing contour geometric features, and determining the tip coordinates using the outermost contour point. For irregularly shaped biological objects, the centroid is calculated based on the contour pixel distribution to obtain its center image coordinates.

[0069] Sample selection and adsorption: The target biological object is specified in the microscopic image through the graphical user interface. The system is based on visual servo control technology to drive the microscopic manipulator arm to bring the needle tip close to the sample. It stops when the pixel distance between the two reaches a preset threshold. The electric oil pump adsorbs the biological object to the needle tip.

[0070] Sample transfer and target localization: After the micropipette adsorbs the sample, it is raised to a safe height along the Z-axis; the biaxial translation stage moves the agarose array on the thin-film sensor into the microscope field of view according to the pre-calibrated coordinates;

[0071] Sample release: The system calculates the pixel deviation between the micropipette and the target agarose array position based on machine vision, and maps it into three-dimensional motion commands for the micromanipulator after coordinate transformation model; after the manipulator moves to the target position and descends a specified height along the Z-axis, the electric oil pump releases pressure, allowing the biological object to fall smoothly into the groove of the agarose array. This process is repeated until all specified samples are transferred.

[0072] Preparation of measuring equipment: After the sample transfer is completed, adjust the position and angle of the side-view microscope to obtain a clear side view image of the sample; replace the micropipette used for adsorption, install the ground glass platform, adjust its verticality for measurement, and use a digital microscope to measure and record the initial diameter of the sample.

[0073] Measurement platform positioning: The microscopic operating table moves to the target position according to the sample coordinates recorded in step one, and then decreases its speed after descending to the specified height;

[0074] Contact and loading: The measuring chip 7 comes into contact with the biological object at a constant speed. In the initial stage of contact, only a small elastic deformation occurs and no detectable force signal is generated.

[0075] Data acquisition and measurement termination: Continue loading until the thin-film sensor acquires a complete force-deformation curve, and simultaneously record the current diameter of the sample and the change in sensor resistance; repeat this process until all sample measurements are completed.

[0076] The effectiveness of this application is verified through the following embodiments:

[0077] Example 1: A chip for measuring the elastic modulus of flexible biological objects includes a glass substrate, a patterned polydimethylsiloxane (PDMS) substrate, and a thin-film sensor;

[0078] The thin-film sensor includes an upper PDMS thin film, an intermediate functional layer, and a lower PDMS thin film, with an overall thickness of 130~140μm. The lower PDMS thin film has a non-porous structure. The upper PDMS thin film has a first through-hole at a predetermined electrode position to provide an electrical path for external wire connection.

[0079] The measurement chip has three measurement slots;

[0080] Three second through holes are formed on the patterned PDMS substrate. The positions of the second through holes correspond one-to-one with the positions of the measurement grooves. The second through holes and the area of ​​the thin film sensor covering the second through holes constitute the measurement grooves.

[0081] The number of second through holes is three, and the connection relationship of the second through holes is that they are completely disconnected.

[0082] The glass substrate, the patterned PDMS substrate, and the thin-film sensor are permanently bonded together as a whole by ion bonding.

[0083] It also includes an agarose array, which is non-fixedly placed in the measuring tank;

[0084] The thickness of the upper PDMS film and the lower PDMS film is 50 μm, and the thickness of the intermediate functional layer is 35 μm.

[0085] The measurement chip is fabricated in the following manner:

[0086] Hard optical glass is selected as the glass substrate;

[0087] The unpatterned surface of the patterned PDMS substrate is connected to the glass substrate by ion bonding;

[0088] The patterned surface of the patterned PDMS substrate is bonded to the underlying PDMS film;

[0089] A metal mask is tightly bonded to the upper surface of the lower PDMS film, and the cutout area of ​​the metal mask corresponds to the pattern design of the intermediate functional layer. A special conductive ink is uniformly sprayed onto the surface of the metal mask in a mist using an aerosol spraying system. Under the action of gravity and surface tension, the special conductive ink penetrates into the cutout area of ​​the metal mask, forming the intermediate functional layer with the pattern corresponding to the pattern design on the surface of the lower PDMS film.

[0090] The upper PDMS film is bonded to the upper surface of the lower PDMS film to form the thin-film sensor;

[0091] The patterned PDMS substrate is prepared by the following method:

[0092] The PDMS prepolymer and curing agent are mixed evenly at a mass ratio of 1:10 to form a mixture, which is then poured into a photocurable mold.

[0093] Place the mixture in a vacuum chamber for 1 hour to remove air bubbles.

[0094] Transfer the mixture to a constant temperature chamber and cure it at 70°C for 3 hours;

[0095] The patterned polydimethylsiloxane PDMS substrate was obtained by demolding the cured mixture.

[0096] The agarose array was prepared in the following manner:

[0097] Molds with predetermined patterns are printed using photopolymerization technology;

[0098] The agarose is heated to a molten state by microwave and then poured into a container;

[0099] A photocurable stamp mold is used to press and mold agarose colloid in a molten state. After the agarose cools and solidifies, it is demolded to obtain an agarose array with a predetermined microstructure. The prepared agarose array is arranged in a 4×3 matrix to form a circular array structure with a diameter of 300μm.

[0100] The connections between the agarose array, the glass substrate, the patterned PDMS substrate, and the thin-film sensor are all formed by permanent bonding through ion bonding, connecting them into a whole; wherein, the agarose array is non-fixed;

[0101] The specially formulated conductive ink comprises multi-walled carbon nanotubes, carbon black, polydimethylsiloxane, and isopropanol.

[0102] The specially formulated conductive ink is prepared in the following manner:

[0103] Multi-walled carbon nanotubes and carbon black powder, with a mass ratio of 1:1, were placed separately in isopropanol in different containers.

[0104] Two containers were placed on a magnetic stirrer and stirred continuously at room temperature for 1 hour. After the magnetic stirring was completed, the two containers were transferred to a water bath and subjected to ultrasonic treatment for 1 hour to obtain a multi-walled carbon nanotube-isopropanol dispersion and a carbon black-isopropanol dispersion.

[0105] Multi-walled carbon nanotube-isopropanol dispersion and carbon black-isopropanol dispersion were added together with PDMS prepolymer into the same clean container to form a mixed system;

[0106] The container containing the mixture was placed in a constant temperature water bath at 50°C, and the mixture was continuously stirred for 6 hours. During the stirring process, isopropanol evaporated, and the special conductive ink was finally obtained.

[0107] An automated measurement system for the elastic modulus of flexible biological objects includes a measurement chip 7, a triaxial micromanipulator 1, an electric oil pump 2, a manual focusing lifting platform 3, a tilt angle adjustment platform 4, an image acquisition module 5, an LED light source 6, and an XY electric translation stage 8. The electric oil pump 2 is connected to a micropipette via a sealed pipe, and its position is controlled by a motor to control the suction / discharge of liquid in the micropipette. Both the triaxial micromanipulator 1 and the XY electric translation stage 8 are height-adjustable via the manual focusing lifting platform 3. A bracket is provided on the XY electric translation stage 8 to support the measurement chip 7. Two image acquisition modules 5 are provided, one directly above the measurement chip 7 and the other to its side. The LED light source 6 is located at the front end of the image acquisition module directly above the measurement chip 7, and the tilt angle adjustment platform 4 is located at the bottom of the image acquisition module to its side.

[0108] This embodiment uses 100 μm diameter microbeads as an example to illustrate the operation of 293T cell spheres. First, 293T cells are passaged in an agarose array and cultured for several days until they form spheres. The formed cell spheres are then transferred to culture dishes and placed into the corresponding wells on the platform for operation. This method is applicable to flexible biological samples such as single cells and cell aggregates. The specific operation procedure is as follows:

[0109] Step (a) Equipment calibration and positioning: The 80 μm diameter micropipette was mounted on a high-precision micromanipulation arm. The tip was manually adjusted to enter the field of view of the optical microscope. Using an automated focusing system, three-dimensional image data was constructed to determine the confocal plane between the tip and the sample. The microscopic image was processed using a contour extraction algorithm. The micropipette region was identified by comparing the contour geometric features, and the tip coordinates were determined by the outermost contour point. For irregularly shaped biological objects, the centroid was calculated based on the contour pixel distribution to obtain the center image coordinates.

[0110] Step (b) Sample selection and adsorption: The target biological object is specified in the microscopic image through the graphical user interface. The system, based on visual servo control technology, drives the microscopic manipulator to bring the needle tip close to the sample. It stops when the pixel distance between the two reaches a preset threshold. The electric oil pump adsorbs the biological object to the needle tip.

[0111] Step (c) Sample transfer and target localization: After the micropipette adsorbs the sample, it is raised to a safe height along the Z-axis; the biaxial translation stage moves the agarose array on the thin-film sensor into the microscope field of view according to the pre-calibrated coordinates;

[0112] Step (d) Sample Release: The system calculates the pixel deviation between the micropipettes and the target agarose array position based on machine vision, and maps it into three-dimensional motion commands for the micromanipulator through a coordinate transformation model. After the manipulator moves to the target position and descends a specified height along the Z-axis, the electric hydraulic pump releases pressure, allowing the biological object to fall smoothly into the agarose array groove. This process is repeated until all specified samples have been transferred.

[0113] 2.2 Task 2: Automatically complete the measurement of elastic modulus

[0114] Step (a) Preparation of measuring equipment: After the sample transfer is completed, adjust the position and angle of the side-view microscope to obtain a clear side view image of the sample; replace the micropipette used for adsorption, install the ground glass platform, adjust its verticality for measurement, and use a digital microscope to measure and record the initial diameter of the sample.

[0115] Step (b) Measurement platform positioning: The microscopic operating table moves to the target position according to the sample coordinates recorded in Task 1, and then decreases its speed after descending to the specified height;

[0116] Step (c) Contact and Loading: The glass platform comes into contact with the biological object at a constant speed. In the initial stage of contact, only a small elastic deformation occurs and no detectable force signal is generated.

[0117] Step (d) Data Acquisition and Measurement Termination: Continue loading until the thin-film sensor acquires a complete force-deformation curve, and simultaneously record the current diameter of the sample and the change in sensor resistance; repeat this process until all sample measurements are completed.

[0118] 3. Methods for calculating the elastic modulus

[0119] The system establishes a measurement model through a force-resistance calibration experiment: a rigid platform is extended 3-4 times and fixed to a micromanipulator arm, with its end being a molten and smoothed rigid capillary glass. The host computer controls the manipulator arm to apply displacement along the Z-axis, and a high-precision weighing device is used to measure the force acting on the thin-film sensor in real time, simultaneously recording the sensor resistance change. The least squares method is used to fit the resistance-force curve, establishing a quantitative relationship model between the two. The end shape is molten and smoothed rigid capillary glass, such as... Figure 5 The image shown is under 4X magnification, with the scale unit being 21.3 micrometers per division.

[0120] Once the applied force is calibrated, we obtain the value of F1 in the mechanical model shown in the figure above. Based on the biomechanical measurement method proposed in this application, an agarose array-thin-film sensor composite structure is used as a flexible plane, forming a test system with a rigid solid glass plane. Therefore, the upper surface is hard, while the lower thin-film sensor gradually deforms, but the deformation of the thin film will be much smaller than that of the flexible sample. The elastic model is calculated as follows:

[0121] During the experiment, the initial height X1 of the organoid was first recorded, and the height X2 after deformation was measured after the compression platform stopped applying pressure; its deformation δ=X1-X2.

[0122] According to the principle of mechanical equilibrium, the vertical force F acting on the system is generated jointly by the thin-film sensor and the rigid plane. Based on the Tatara theoretical model, the equation for calculating the elastic modulus E is established:

[0123]

[0124] The formulas for calculating L and K are as follows:

[0125]

[0126] The key parameters are defined as a, which is the contact characteristic length, E, which is the equivalent stiffness coefficient, and Poisson's ratio v (taken as a typical value of 0.5 for biomaterials).

[0127] Example 2: The difference between this example and Example 1 is that the three second through holes are fully connected.

[0128] A gas insertion port is set at the middle position of the connecting path. Everything else is the same as in Example 1.

[0129] A gas-controlled input port can be set at the center of the through-hole, connecting to a small electric air pump to input air pressure into the channel, causing the thin-film sensor to deform in a central bulge shape. The maximum height of the deformation has a calibrable relationship with the sensor's resistance change and the external air pressure input, allowing users to controllably intervene externally via a host computer interface to obtain the desired degree of thin-film change. Simultaneously, the entire measurement tank is sealed with a biocompatible material, leaving the space above for sterilization before relevant biological culture can be carried out.

[0130] Applicable subjects: For example, the culture of IPSC-CM 2D cardiomyocytes. Specifically, since directly collecting cardiomyocytes from patients is often scarce, induced pluripotent stem cell-derived cardiomyocytes (IPSC-CM) are a typical in vitro human cardiomyocyte biological model, obtained by inducing differentiation of iPSCs in vitro. These cells possess electrophysiological, contractile, and gene expression characteristics similar to natural cardiomyocytes. Therefore, they have great application value in disease modeling, drug screening, regenerative medicine, and personalized medicine. Induced pluripotent stem cells (iPSCs) refer to stem cells that have been reprogrammed to possess pluripotency through gene reprogramming. These cells can differentiate into almost all cell types in vivo.

[0131] The purpose of this type of cardiomyocyte culture is that, without relevant external environmental stimuli, the differentiation and maturity of the cells are often insufficient during the culture process, which manifests as small beating amplitude and uneven beating between cells. However, certain external physical or chemical stimuli can be added during the culture process to promote the differentiation and maturity of the cells, thereby obtaining a better in vitro model.

[0132] The connection between the two:

[0133] The controllable thin film deformation proposed above can satisfy the following requirements for the cultivation of IPSC-CM:

[0134] 1. The need for a nurturing environment;

[0135] 2. In-situ external physical stimulation is required (such as long-term pulsation at a specific frequency to simulate the pulsation of the human heart, or long-term sustained pulsation to provide the necessary physical pressure to simulate the internal environment, etc.).

[0136] 3. At the same time, after the culture is completed, the differentiation maturity of IPSC-CM can be measured, such as the amplitude and frequency of jumping.

[0137] While this application has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely examples of the principles and applications of this application. Therefore, it should be understood that many modifications can be made to the exemplary embodiments, and other arrangements can be designed without departing from the spirit and scope of this application as defined by the appended claims. It should be understood that different dependent claims and features described herein can be combined in ways different from those described in the original claims. It is also understood that features described in conjunction with individual embodiments can be used in other described embodiments.

Claims

1. A chip for measuring the elastic modulus of flexible biological objects, characterized in that, Includes glass substrates, patterned polydimethylsiloxane (PDMS) substrates, and thin-film sensors; The thin-film sensor includes an upper PDMS thin film, an intermediate functional layer, and a lower PDMS thin film, with an overall thickness of 130~140μm. The lower PDMS thin film has a non-porous structure. The upper PDMS thin film has a first through-hole at a predetermined electrode position to provide an electrical path for external wire connection. The measurement chip has one or more measurement slots.

2. The chip for measuring the elastic modulus of flexible biological objects according to claim 1, characterized in that, One or more second through holes are formed on the patterned PDMS substrate. The number of second through holes is the same as the number of measurement slots. The positions of the second through holes correspond one-to-one with the positions of the measurement slots. The second through holes and the area of ​​the thin film sensor covering the second through holes constitute the measurement slots.

3. A chip for measuring the elastic modulus of flexible biological objects according to claim 2, characterized in that, The number of second through holes is two or more, and the connection relationship between the two or more second through holes is either completely non-connected, locally connected, or completely connected. The term "globally disconnected" means that all second vias are independent and there are no interconnected paths between them. The local connectivity is a subset of the second vias that form a connected set, and there is no connecting path between this subset and other second vias. The global connectivity refers to all the second through holes forming a single connected system, with a connection path existing between any two second through holes.

4. The chip for measuring the elastic modulus of flexible biological objects according to claim 1, characterized in that, The glass substrate, the patterned PDMS substrate, and the thin-film sensor are permanently bonded together as a whole through ion bonding.

5. A chip for measuring the elastic modulus of flexible biological objects according to claim 1, characterized in that, It also includes an agarose array, which is placed non-fixedly within the measuring tank.

6. A chip for measuring the elastic modulus of flexible biological objects according to claim 1, characterized in that, The thickness of the upper PDMS film and the lower PDMS film is 50 μm, and the thickness of the intermediate functional layer is 35 μm.

7. A chip for measuring the elastic modulus of flexible biological objects according to claim 1, characterized in that, The measurement chip is fabricated in the following manner: Hard optical glass is selected as the glass substrate; The unpatterned surface of the patterned PDMS substrate is connected to the glass substrate by ion bonding; The patterned surface of the patterned PDMS substrate is bonded to the underlying PDMS film; A metal mask is tightly bonded to the upper surface of the lower PDMS film, and the cutout area of ​​the metal mask corresponds to the pattern design of the intermediate functional layer. Using an aerosol jetting system, a special conductive ink is uniformly sprayed in a mist onto the surface of the metal mask. Under the action of gravity and surface tension, the special conductive ink penetrates into the cutout area of ​​the metal mask, forming the intermediate functional layer with a pattern corresponding to the design on the surface of the underlying PDMS film. The upper PDMS film is bonded to the upper surface of the lower PDMS film to form the thin-film sensor.

8. A chip for measuring the elastic modulus of flexible biological objects according to claim 7, characterized in that, The patterned PDMS substrate is prepared by the following method: The PDMS prepolymer and curing agent are mixed evenly at a mass ratio of 1:10 to form a mixture, which is then poured into a photocurable mold. Place the mixture in a vacuum chamber for 1 hour to remove air bubbles. Transfer the mixture to a constant temperature chamber and cure it at 70°C for 3 hours; The patterned polydimethylsiloxane PDMS substrate is obtained by demolding the cured mixture.

9. An automated measurement system for the elastic modulus of flexible biological objects, characterized in that, The system includes a measurement chip (7) as described in any one of claims 1-8, a three-axis micromanipulation arm (1), an electric oil pump (2), a manual focusing lifting platform (3), a tilt angle adjustment platform (4), an image acquisition module (5), an LED light source (6), and an XY electric translation stage (8); the electric oil pump (2) is connected to a micropipette via a sealed pipe, and the position of the screw push / suction is controlled by a motor to control the suction and discharge of liquid in the micropipette; the three-axis micromanipulation arm (1) and the XY electric translation stage (8) are both height-adjusted via the manual focusing lifting platform (3); a bracket is provided on the XY electric translation stage (8) to support the measurement chip (7); there are two image acquisition modules (5), one directly above the measurement chip (7) and the other to the side above the measurement chip (7); the LED light source (6) is provided at the front end of the image acquisition module (5) directly above the measurement chip (7), and the tilt angle adjustment platform (4) is provided at the bottom of the image acquisition module (5) to the side above the measurement chip (7).

10. The measurement method of the automated measurement system for the elastic modulus of flexible biological objects as described in claim 9, characterized in that, include: Equipment calibration and positioning: The grinding micropipette is mounted on a high-precision micromanipulation arm, and the needle tip is manually adjusted to enter the field of view of the optical microscope. Using an automated focusing system, three-dimensional image data is constructed to determine the confocal plane between the needle tip and the sample. A contour extraction algorithm is used to process the microscopic image, and the micropipette region is identified by comparing the contour geometric features. The needle tip coordinates are determined by the outermost contour point. For irregular circular biological objects, the centroid is calculated based on the contour pixel distribution to obtain its center image coordinates. Sample selection and adsorption: The target biological object is specified in the microscopic image through the graphical user interface. The system is based on visual servo control technology to drive the microscopic manipulator arm to bring the needle tip close to the sample. It stops when the pixel distance between the two reaches a preset threshold. The electric oil pump adsorbs the biological object to the needle tip. Sample transfer and target localization: After the micropipette adsorbs the sample, it is raised to a safe height along the Z-axis; The biaxial translation stage moves the agarose array on the thin-film sensor into the microscope's field of view according to the pre-calibrated coordinates; Sample release: The system calculates the pixel deviation between the micropipette and the target agarose array position based on machine vision, and maps it into three-dimensional motion commands for the micromanipulator after coordinate transformation model; after the manipulator moves to the target position and descends a specified height along the Z-axis, the electric oil pump releases pressure, allowing the biological object to fall smoothly into the groove of the agarose array. This process is repeated until all specified samples are transferred. Preparation of measuring equipment: After the sample transfer is completed, adjust the position and angle of the side-view microscope to obtain a clear side view image of the sample; replace the micropipette used for adsorption, install the ground glass platform, adjust its verticality for measurement, and use a digital microscope to measure and record the initial diameter of the sample. Measurement platform positioning: The microscopic operating table moves to the target position according to the sample coordinates recorded in step one, and then decreases its speed after descending to the specified height; Contact and loading: The measuring chip (7) comes into contact with the biological object at a constant speed. In the initial stage of contact, only a small elastic deformation occurs and no detectable force signal is generated. Data acquisition and measurement termination: Continue loading until the thin-film sensor acquires a complete force-deformation curve, and simultaneously record the current diameter of the sample and the change in sensor resistance; repeat this process until all sample measurements are completed.

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