Real-time monitoring method for signal molecule release in single cell deformation process by optical tweezer manipulation combined with microelectrode sensing

Abstract

The present application relates to a kind of real-time monitoring method for realizing signal molecule release in single cell deformation process by optical tweezers manipulation combined microelectrode sensing, comprising the following steps: obtaining ITO film microelectrode of electrically active area that can only accommodate one cell size;Preparation of working electrode that can specifically respond to signal molecule concentration change;With working electrode as bottom, build electrochemical chamber of three electrode system;With biomolecule modification microsphere carrier, and place it in electrochemical chamber;Two microsphere carriers are captured respectively by double optical trap optical tweezers and contacted with cell, and cell is stretched or extruded in working electrode area to deform, and the force spectrum information and deformation variable in cell deformation process are determined;Signal molecule concentration change released in cell deformation process is monitored in real time using electrochemical workstation.The present application can be used to study the relationship between the change of single cell release chemical information and cell mechanical information under mechanical force stimulation.

Description

Technical Field

[0001] This invention belongs to the field of optical tweezers manipulation technology and also to the field of bioelectrochemical sensing. It includes interdisciplinary technical fields such as biophysics, molecular biology, nanobiotechnology and microelectrode electrochemistry. Specifically, it relates to a method for real-time monitoring of signal molecule release during single-cell deformation by combining optical tweezers manipulation with microelectrode sensing. Background Technology

[0002] Optical tweezers, first invented by Ashkin in 1986, are also known as single-beam gradient force optical traps. They utilize the gradient force created by the spatial variation of the intensity of a focused Gaussian laser beam to stably capture microparticles at the point of strongest light field, i.e., the focal point of the beam. When the laser beam moves, it can move the microparticles along with it, achieving precise manipulation of the microparticles. Compared to macroscopic methods of stretching or compressing cells, optical tweezers, using a highly focused laser to capture microspheres as handles, can apply forces of varying magnitudes and directions at the single-cell level for mechanical stimulation. Moreover, optical tweezers manipulate cell deformation non-invasively, not only achieving precise control of mechanical forces at the microscopic level while maintaining normal physiological activity, but also achieving high mechanical resolution at the pN level and high temporal resolution at the millisecond level. Therefore, optical tweezers are a universal tool for studying the mechanical changes during cell deformation at the single-cell level.

[0003] Microelectrodes are electrodes smaller than 25 μm in at least one dimension. Compared with traditional electrodes, they offer numerous advantages, such as small size, rapid response, high current density, high signal-to-noise ratio, low IR drop, easy attainment of steady state, and fast mass transfer, enabling high spatiotemporal resolution and high sensitivity detection. The emergence and application of planar electrode arrays within microelectrodes have made high-throughput cell detection possible. Cells directly cover the electrode surface, eliminating the need for target labeling and micromanipulation systems, significantly improving detection efficiency. Furthermore, microfabrication technology allows for batch fabrication with controllable electrode materials, morphology, and size. After surface modification, microelectrodes can be used to achieve highly selective and sensitive detection of neurotransmitters and key signaling molecules such as NO and CO in cells and living organisms. Therefore, microelectrode electrochemical sensing technology can be widely applied in various fields such as in vivo analysis, biosensing, and biological cell detection for real-time, high-resolution, and high-specificity detection.

[0004] As the basic structural and functional unit of life, cells constantly release various signaling molecules for intercellular communication and signal transduction, thereby coordinating overall life activities. Deformation of cells under mechanical stress affects their signal transduction and molecule release processes, thus regulating important physiological activities such as cell proliferation, differentiation, metabolism, stress response, defense, and apoptosis. The signaling molecules released by cells are mainly classified into four categories: endocrine hormones, paracrine and autocrine factors, neurotransmitters, and small gaseous signaling molecules. Except for endocrine hormones, which have long-range and prolonged effects, other signaling molecules typically act on the cell itself or nearby target cells; therefore, real-time monitoring of the release of these signaling molecules is of significant physiological importance.

[0005] Currently, there is a lack of a method for real-time monitoring of the stress on cell deformation and the dynamic release response of signal molecules at the single-cell level. This invention combines optical tweezers manipulation technology and microelectrode electrochemical sensing technology. Polystyrene microspheres are used as optical tweezers handles to induce cell deformation. Then, by modifying the surface of the microelectrode with electrode materials that can respond to changes in signal molecules, the release of signal molecules can be monitored in real time. This invention contributes to the study of the effects of mechanical stress on endothelial cells, macrophages, and nerve cells at the single-cell level. Summary of the Invention

[0006] The purpose of this invention is to provide a method for real-time monitoring of signal molecule release during single-cell deformation through optical tweezers manipulation combined with microelectrode sensing. Addressing the limitations of existing research in mechanically stretching / compressing and accurately measuring force on single normal cells without damaging them, and the resulting low spatiotemporal resolution, this invention combines microelectrode electrochemical sensing technology with optical tweezers technology. For the first time, it achieves the stretching or compression of unlabeled normal cells using dual-optical-tweezers, and monitors the changes in the concentration of signal molecules released by the cells under optical tweezers in real time. This allows for the study of the relationship between deformation, force spectrum information, and signal molecule release during cell deformation. Therefore, this invention provides a strategy that combines optical tweezers with target-free electrochemical sensing technology to achieve the stretching or compression of single cells and monitor the release of signal molecules and cell deformation in real time with high spatiotemporal resolution. This offers a universal approach to mechanically stimulating changes in the morphology and physiology of single cells.

[0007] The solution adopted by this invention to achieve its objective is: a method for real-time monitoring of signal molecule release during single-cell deformation through optical tweezers manipulation combined with microelectrode sensing, characterized by comprising the following steps:

[0008] (1) Fabricate ITO thin film microelectrodes that can only accommodate an electroactive region the size of a single cell;

[0009] (2) Based on the characteristics of signal molecules, ITO thin film microelectrodes with exposed electrochemically active regions are prepared using chemical modification methods to create working electrodes that can specifically respond to changes in the concentration of signal molecules.

[0010] (3) An electrochemical chamber with a three-electrode system is constructed with the working electrode as the bottom to contain cells and cell culture medium;

[0011] (4) Modify the microsphere carrier with biomolecules and place it in the electrochemical chamber;

[0012] (5) Two microsphere carriers were captured by dual optical trap optical tweezers and brought into contact with cells. The cells were stretched or squeezed in the working electrode area to cause deformation. The force spectrum information and deformation amount during the cell deformation process were measured.

[0013] (6) Use an electrochemical workstation to monitor the changes in the concentration of signal molecules released during cell deformation in real time.

[0014] Preferably, in step (1), an ITO thin film is sputtered by magnetron sputtering, and then photolithography is performed using a negative photoresist to obtain an ITO thin film microelectrode that can only accommodate an electroactive region the size of a single cell. The negative photoresist is at least one of SU-8 2000.5, NRD6015, and HD 4100. The ITO thin film electrode of the non-electroactive part is repeatedly photolithographically lithographically lithographically and passivated multiple times to obtain an ITO thin film microelectrode that can only accommodate an electroactive region the size of a single cell.

[0015] Multiple photolithography passivation processes on the ITO glass slides enable the electroactive sites of the microelectrodes to monitor the release of signal molecules from a single cell.

[0016] The cells referred to are all cell lines capable of being mechanically stretched using dual-light trap optical tweezers, including but not limited to human cardiomyocytes (AC16 cells), human umbilical vein endothelial cells (HUVEC cells), fusion cells of mouse neurocytoma and rat glioma (NG108-15 cells), mouse mononuclear macrophage leukemia cells (RAW264.7 cells), and mouse hippocampal neurons (HT22 cells).

[0017] Preferably, in step (2), the ITO thin film microelectrode can be chemically modified with different electrode materials using irreversible adsorption, covalent linkage or self-assembly techniques according to the signal molecules to be specifically detected; preferably, the signal molecules are molecules that act on the cell itself or nearby target cells and can be detected by electrochemical methods; preferably, the signal molecules include at least one of gaseous signal molecules, paracrine or autocrine factors and neurotransmitters.

[0018] The gaseous signaling molecule is at least one of NO, CO, and H2S; the paracrine or autocrine factor is at least one of growth factor and cytokine; and the neurotransmitter is at least one of acetylcholine and glutamate.

[0019] Preferably, the electrode material includes nanomaterials (nanoparticles, nanotubes, and nanowires), membrane materials (Nafion), electroactive polymers (PEDOT), metalloporphyrin molecules, enzymes, etc., which are modified into microelectrodes through irreversible adsorption, covalent linkage, or self-assembly techniques.

[0020] Preferably, the specific application includes at least one of the following: modifying an ITO electrode with noble metal nanoparticles to respond to small molecules with redox properties, and modifying an ITO electrode with glutamate oxidase to respond to neurotransmitters that do not have electrochemical activity.

[0021] Preferably, in step (3), the working electrode is used as the bottom, and the PDMS film with holes on the top is used as the chamber to contain the cell culture medium. The counter electrode and the reference electrode are inserted through the holes to form an electrochemical chamber with a three-electrode system.

[0022] Preferably, in step (4), the microsphere carrier is a polystyrene microsphere or a silica microsphere, and the biomolecule includes at least one of concanavalin A, transferrin-biotin, integrin, and folic acid.

[0023] Preferably, in step (5), the cells are in at least one of the following states: adherent, semi-adherent, and suspended.

[0024] Preferably, in step (5), the cell deformation is the membrane deformation in the initial state and after squeezing or stretching the cell; the force spectrum information is the mechanical information converted from the relative position of the two microspheres captured by the dual-light trap optical tweezers as they deviate from the center of the light trap due to the cell membrane tension.

[0025] Preferably, in step (6), the electrochemical workstation applies a working voltage according to the requirements of the working electrode for the specific detection of signal molecules, so as to monitor the change in the concentration of signal molecules based on the real-time change of the current.

[0026] This invention provides a method for real-time monitoring of signal molecule release during single-cell deformation through optical tweezers manipulation combined with microelectrode sensing. The invention utilizes microfabrication techniques such as photolithography to fabricate planar microelectrodes that respond to changes in signal molecule concentration. Real-time changes in electrode current reflect changes in the content of signal molecules released by a single cell during mechanical stretching or compression based on optical tweezers. Using dual-light-tweezers manipulation, microspheres that can connect to the cell are captured, and the cell is stretched or compressed to induce deformation. Force spectrum information and deformation during the cell's stress process are measured, while changes in microelectrode current are monitored on an electrochemical workstation, thus reflecting changes in the content of signal molecules released by the cell under mechanical stimulation. This method can be widely applied in the fields of biomechanics and biochemistry to study the relationship between changes in chemical information released by single cells under mechanical stimulation and cellular mechanical information.

[0027] The present invention has the following advantages and beneficial effects:

[0028] 1. The method provided by this invention combines optical tweezers and microelectrode electrochemical sensing, which enables real-time monitoring of the relationship between the force spectrum information and the released signal molecules on cells while they are being stretched or compressed by polystyrene microspheres.

[0029] 2. This invention uses dual-light trap optical tweezers to manipulate microspheres to squeeze or stretch individual cells, which avoids the problem that the average value of population deformation measured by macroscopic stretching cell platforms cannot reflect the individual cells.

[0030] 3. This invention uses optical tweezers to deform cells, which can precisely and non-damagingly compress or stretch cells and measure pN-level forces under microscopic deformation while ensuring cell viability.

[0031] 4. The optical tweezers device and electrochemical sensing device used in this invention both have high time resolution at the millisecond level, so as to more realistically and quickly reflect the actual situation of signal molecule release under microscopic deformation of single cells.

[0032] 5. The present invention employs a microelectrode electrochemical sensing method that eliminates the need for probe-labeled targets, thereby minimizing disturbances to cellular mechanisms and improving the signal-to-background ratio of signal molecule detection. Attached Figure Description

[0033] Figure 1 This is a flowchart of the microelectrode fabrication process;

[0034] Figure 2 This is a schematic diagram of the electrochemical chamber structure;

[0035] Figure 3 A schematic diagram illustrating the detection of signaling molecules released by cells using microelectrodes under the stretching or squeezing of optical tweezers;

[0036] Figure 4This is a schematic diagram illustrating the manipulation of microspheres by dual optical trap optical tweezers on cells.

[0037] Figure 5 This relates the force spectrum and the change in current generated by the microelectrode when optical tweezers manipulate microspheres on cells. Detailed Implementation

[0038] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[0039] like Figure 1 The above is a flowchart of the fabrication process of the microelectrode of the present invention. Figure 2 This is a schematic diagram of the electrochemical chamber structure of the present invention. Figure 3 This is a schematic diagram illustrating the detection of signaling molecules released by cells using microelectrodes under the stretching or compression of optical tweezers. Figure 4 This is a schematic diagram illustrating the manipulation of microspheres by dual optical trap optical tweezers in cells according to the present invention.

[0040] Figure 5 This diagram illustrates the relationship between the force spectrum of the optically manipulated microspheres and the current generated by the microelectrode when applied to cells. The diagram shows a direct correlation between the mechanical changes experienced by the cells and the current generated on the microelectrode when the microspheres are manipulated. By converting the standard curve of signal molecule concentration versus current magnitude, the release of signal molecules under different levels of mechanical force can be obtained. These signal molecules include, but are not limited to, those described in Examples 1-3.

[0041] Example 1

[0042] A method for real-time monitoring of NO signaling molecule release during the deformation of a single endothelial cell (HUVEC) via optical tweezers manipulation combined with microelectrode sensing includes the following steps:

[0043] (1) The desired ITO pattern is obtained by photolithography on the cover glass using positive photoresist (AZ 5214IR), and then an ITO thin film is deposited on the ITO pattern by magnetron sputtering.

[0044] (2) A large area of ​​SU-8 passivation layer was covered on a coverslip with deposited ITO and the operation was repeated twice to obtain an ITO microelectrode with an exposed electroactive region the size of a cell.

[0045] (3) Au-PEDOT conductive polymer was obtained by polymerization reaction of HAuCl4 and EDOT and chemically modified onto ITO microelectrode by drop coating. A working voltage of 0.7V (vs. Ag / AgCl) was applied to specifically respond to the concentration change of NO signal molecules.

[0046] (4) Using the microelectrode mentioned above as the bottom, and a PDMS film with holes as the chamber to contain the cell culture medium, a top glass is added to close the chamber and leave enough holes to insert the counter electrode and reference electrode, and then connected to an electrochemical workstation to form an electrochemical chamber with a three-electrode system.

[0047] (5) Add cultured HUVEC cells and polystyrene microspheres modified with concanavalin A to the electrochemical chamber and place the electrochemical chamber on the sample stage of the optical tweezers device.

[0048] (6) Two polystyrene microspheres were simultaneously captured by dual optical trap optical tweezers and brought into contact with the cell membrane of HUVEC. After the two were combined, the microspheres were manipulated to place the cell in the electroactive region of the microelectrode, and the deformation of the cell membrane and force spectrum information were monitored in real time while the cell was stretched or squeezed to deform.

[0049] (7) HUVEC cells release NO during the stretching or compression process. During this process, the microelectrode can monitor the release of NO in real time, thereby obtaining the change of nitric oxide release of HUVEC cells under mechanical deformation.

[0050] Example 2

[0051] A method for real-time monitoring of glutamate (Glu) release during synaptic deformation of a single hippocampal neuron (HT22) via optical tweezers manipulation combined with microelectrode sensing includes the following steps:

[0052] (1) As described in Example 1, an ITO microelectrode with an exposed electroactive region the size of a cell is obtained by microfabrication techniques such as photolithography.

[0053] (2) H2PtCl6 was added to the ITO microelectrode for electroplating, so that a layer of Pt was electrochemically deposited on the ITO surface to sensitively respond to changes in H2O2 concentration.

[0054] (3) Add glutamate oxidase (GluOx) to the above Pt / ITO microelectrode and dry overnight to obtain GluOx / Pt / ITO microelectrode that can convert Glu into H2O2 electroactive molecules, thereby achieving a specific response to changes in the concentration of Glu signal molecules.

[0055] (4) Using the microelectrode mentioned above as the bottom, and a PDMS film with holes as the chamber to contain the cell culture medium, a top glass is added to close the chamber and leave enough holes to insert the counter electrode and reference electrode, and then connected to an electrochemical workstation to form an electrochemical chamber with a three-electrode system.

[0056] (5) Add cultured HT22 cells and polystyrene microspheres modified with concanavalin A to the electrochemical chamber and place the electrochemical chamber on the sample stage of the optical tweezers device.

[0057] (6) Two polystyrene microspheres were simultaneously captured by optical tweezers. After the two were combined, the microspheres were manipulated to place the synapse of this cell in the electroactive region of the microelectrode. The deformation and force spectrum information of the synapse were monitored in real time while the synapse was deformed by stretching or squeezing.

[0058] (7) HT22 synapses release Glu during the stretching or compression process. During this process, the microelectrode can monitor the release of Glu in real time, thereby obtaining the change of Glu release of HT22 synapses under mechanical deformation.

[0059] Example 3

[0060] A real-time monitoring method for the release of redox homeostasis-related signaling molecules (H2O2 and NO) during the deformation of a single macrophage (RAW264.7) via optical tweezers manipulation combined with microelectrode sensing includes the following steps:

[0061] (1) The desired ITO pattern is obtained by photolithography on the cover glass using positive photoresist (AZ 5214IR), and then an ITO thin film is deposited on the ITO pattern by magnetron sputtering.

[0062] (2) Add AgNWs solution to the ITO film and dry it to obtain an ITO film with a uniformly covered disordered Ag NTs conductive layer.

[0063] (3) Using Ag NTs as templates, an ITO thin film with AuNTs conductive layer was obtained by in-situ substitution reaction using gold precursor [Au(en)2]Cl3.

[0064] (4) The obtained Au NTs / ITO film is placed in a mixed aqueous solution of H2PtCl6 and HCOOH to undergo a redox reaction, so that the generated Pt NP is deposited on the Au NTs / ITO film to obtain an Au@PtNTs / ITO electrode that can electrochemically respond to H2O2 and NO.

[0065] (5) The electrode was covered with a large area of ​​SU-8 passivation layer by photolithography and the operation was repeated twice to obtain an Au@Pt NTs / ITO microelectrode with an exposed size of one cell.

[0066] (6) Using the Au@Pt NTs / ITO microelectrode as the bottom, a PDMS film with holes is used as the chamber to contain the cell culture medium. A top glass is added to close the chamber and leave enough holes to insert the counter electrode and reference electrode. The chamber is then connected to an electrochemical workstation to form an electrochemical chamber with a three-electrode system.

[0067] (7) Add cultured RAW264.7 cells and polystyrene microspheres modified with concanavalin A to the electrochemical chamber and place the electrochemical chamber on the sample stage of the optical tweezers device.

[0068] (8) Two polystyrene microspheres were simultaneously captured by dual optical trap optical tweezers and placed in contact with the cell membrane of RAW264.7. After the two were combined, the microspheres were manipulated to place the cell in the electroactive region of the microelectrode, and the deformation of the cell membrane and force spectrum information were monitored in real time while the cell was stretched or squeezed to deform.

[0069] During stretching or compression, RAW264.7 cells release H2O2 and NO, substances that reflect cellular redox homeostasis. By applying working voltages of 0.4V and 0.7V (vs. Ag / AgCl) to specifically respond to changes in the concentration of H2O2 and NO signaling molecules, real-time monitoring of H2O2 and NO release can be achieved, thus revealing changes in redox homeostasis of RAW264.7 cells under mechanical deformation.

[0070] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for real-time monitoring of signal molecule release during single-cell deformation by combining optical tweezers manipulation with microelectrode sensing, characterized in that, Includes the following steps: (1) Fabrication of ITO thin film microelectrodes that can only accommodate an electrochemically active region the size of a single cell; (2) Based on the characteristics of signal molecules, the ITO thin film microelectrode with exposed electrochemically active region is prepared by chemical modification method to prepare a working electrode that can specifically respond to changes in the concentration of signal molecules; (3) An electrochemical chamber with a three-electrode system is constructed with the working electrode as the bottom to contain cells and cell culture medium; (4) Modify the microsphere carrier with biomolecules and place it in an electrochemical chamber, wherein the biomolecule is concanavalin A and the microsphere carrier is polystyrene microspheres; (5) Two microsphere carriers are captured by dual optical trap optical tweezers and brought into contact with cells. The cells are stretched or squeezed in the working electrode area to cause deformation. The force spectrum information and deformation amount during the cell deformation process are measured. The cell deformation amount is the membrane deformation amount in the initial state and after squeezing or stretching the cells. The force spectrum information is the mechanical information converted from the relative position of the two microspheres captured by the dual optical trap optical tweezers as they deviate from the center of the optical trap due to the cell membrane tension. (6) Use an electrochemical workstation to monitor the changes in the concentration of signal molecules released during cell deformation in real time.

2. The method according to claim 1, characterized in that: In step (1), an ITO thin film is sputtered by magnetron sputtering, and then photolithography is performed using a negative photoresist. The negative photoresist is at least one of SU-8 2000.5, NRD6015, and HD 4100. The ITO thin film electrode with non-electroactive parts is repeatedly photolithographically ...

3. The method according to claim 1, characterized in that: In step (2), different electrode materials are chemically modified on the ITO thin film microelectrode according to the signal molecules to be specifically detected by irreversible adsorption, covalent connection or self-assembly technology.

4. The method according to claim 3, characterized in that: The signaling molecules are molecules that act on the cell itself or nearby target cells and are detected using electrochemical methods.

5. The method according to claim 3, characterized in that: The signaling molecule is at least one of gaseous signaling molecules, paracrine or autocrine factors, and neurotransmitters.

6. The method according to claim 3, characterized in that: The electrode material includes at least one of the following: noble metal nanoparticles modifying ITO electrodes to respond to small molecules with redox properties, and glutamate oxidase modifying ITO electrodes to respond to neurotransmitters that do not have electrochemical activity.

7. The method according to claim 1, characterized in that: In step (3), the working electrode is used as the bottom, and the PDMS film with holes on the top is used as the chamber to contain the cell culture medium. The counter electrode and the reference electrode are inserted through the holes to form an electrochemical chamber with a three-electrode system.

8. The method according to claim 1, characterized in that: In step (5), the cells are at least one of the following states: adherent, semi-adherent, and suspended.

9. The method according to claim 1, characterized in that: In step (6), the electrochemical workstation applies a working voltage according to the requirements of the working electrode for the specific detection of signal molecules, thereby monitoring the changes in the concentration of signal molecules based on the real-time changes in the current.

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