Single cell dynamic monitoring method and system based on DMF and SPR imaging

Abstract

The invention discloses a single cell dynamic monitoring method and system based on DMF (Dimethyl Formamide) and SPR (Surface Plasmon Resonance) imaging, and belongs to the technical field of cell monitoring. The system comprises a DMF automatic control module which is configured to be capable of accurately controlling micro-droplets so as to be fused with an SPR optical phase sensing module and realize dynamic imaging monitoring of a single cell level; the SPR optical phase sensing module is used for carrying out SPR phase imaging on the liquid drops wrapped with the detection contents; the DMF automatic control module comprises a substrate, a ground electrode, an SPR sensing array, a hydrophobic layer, a dielectric layer and a microelectrode array; a ground electrode and a hydrophobic layer are sequentially arranged below the top-layer substrate, and an SPR sensing array is embedded in the hydrophobic layer; a dielectric layer and a hydrophobic layer are arranged above the bottom substrate; a microelectrode array is embedded in the dielectric layer; a liquid drop wrapped with a detection content is arranged between the two hydrophobic layers. According to the invention, DMF and SPR imaging can be combined to realize dynamic monitoring of single cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell monitoring, and particularly relates to a single-cell dynamic monitoring method and system based on DMF and SPR imaging. Background Art

[0002] Surface plasmon resonance (SPR) sensing technology has been widely used in fields such as biomolecular interaction analysis, disease diagnosis, and environmental detection due to its advantages of label-free and real-time monitoring of molecular binding kinetics. However, traditional SPR technology has the following key problems: (1) Large sample consumption: relying on a macroscopic flow cell (such as the Biacore system), 10 - 100 μL of sample is required for a single detection, making it difficult to adapt to the high-efficiency detection requirements of trace samples (such as single-cell secretions and rare antibodies). (2) Limited sensitivity: Traditional intensity-based SPR detects refractive index differences through changes in the intensity of reflected light, which is easily interfered by light source fluctuations, temperature drift, and background noise. The detection limit usually only reaches 1 - 10 ng / mL, making it difficult to meet the high-sensitivity requirements of trace molecules (such as early cancer markers). (3) Lack of flexibility: Most existing SPR systems use fixed detection sites, making it difficult to achieve multi-target parallel detection or dynamically adjust the detection process.

[0003] Digital microfluidics (DMF) technology precisely manipulates droplets (with a volume as low as 0.1 μL) through an electrode array, showing unique advantages in scenarios such as drug screening and single-cell analysis. However, its deep integration with SPR still faces the following challenges: (1) High integration complexity: It is necessary to construct a high-sensitivity SPR sensing layer on the surface of the microfluidic chip while avoiding interference from electrodes to optical signals and interference from the optical sensing layer to DMF manipulation. (2) Low detection throughput: Most existing DMF-SPR integration schemes are based on a single detection site, making it difficult to achieve high-throughput analysis.

[0004] Currently, the commonly used implementation mainly involves integrating a common microfluidic channel into an SPR system, and driving the sample to continuously flow through the detection site by a fluid pump. Although continuous flow detection can also be achieved and is suitable for kinetic analysis, the sample consumption is large, and the sample needs to be continuously injected (volume > 10 μL), making it impossible to handle trace samples. In addition, it has poor flexibility, the channel structure is fixed, and it is impossible to dynamically adjust the detection path or perform multi-target parallel detection. There is also a scheme that integrates local surface plasmon resonance technology (LSPR) with DMF, but it requires specially processed periodic metal structures or nanoparticles, and the preparation scheme is complex; and it still uses the traditional intensity detection method and does not fully utilize the high-sensitivity characteristics of phase imaging technology. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the above related technologies to a certain extent.

[0006] To this end, the object of the present invention is to provide a single-cell dynamic monitoring method and system based on DMF and SPR imaging, which can combine DMF and SPR imaging to achieve dynamic monitoring of single cells.

[0007] In order to solve the above technical problems, the present invention is implemented as follows: An embodiment of the present invention provides a single-cell dynamic monitoring system based on DMF and SPR imaging, and the system includes: A DMF automatic control module, configured to be able to precisely control micro-droplets so as to be integrated with the SPR optical phase sensing module to achieve dynamic imaging monitoring at the single-cell level; An SPR optical phase sensing module, used for performing SPR phase imaging on droplets wrapped with detection contents.

[0008] In addition, according to the single-cell dynamic monitoring system based on DMF and SPR imaging of the present invention, it may also have the following additional technical features: In some embodiments, the DMF automatic control module includes a substrate, a ground electrode, an SPR sensing array, a hydrophobic layer, a dielectric layer, and a microelectrode array; The substrate includes two layers, which are respectively arranged on the top layer and the bottom layer; The ground electrode and the first hydrophobic layer are sequentially arranged below the top-layer substrate; the SPR sensing array is embedded in the first hydrophobic layer; The dielectric layer and the second hydrophobic layer are sequentially arranged above the bottom-layer substrate; the microelectrode array is embedded in the dielectric layer; A number of droplets wrapped with detection contents are arranged between the first hydrophobic layer and the second hydrophobic layer.

[0009] In some embodiments, the microelectrode array is a metal or indium tin oxide electrode array; The microelectrode array is connected to the peripheral circuit system through a flexible circuit board flexible cable.

[0010] In some embodiments, the SPR sensing array is arranged in a region facing the prism in the SPR optical phase sensing module.

[0011] In some embodiments, the SPR sensing array has the same thickness as the first hydrophobic layer.

[0012] In some embodiments, the microelectrode array is embedded at the bottom of the dielectric layer.

[0013] In some embodiments, the preparation method of the SPR sensing array is: magnetron sputtering a gold, silver or copper metal film, and then obtaining it through photolithography and stripping; a two-dimensional material is compounded on the metal film to enhance the sensitivity.

[0014] In some of these embodiments, the SPR optical phase sensing module includes a light source, a collimator, an acousto-optic tunable filter, a polarizer, a liquid crystal phase retarder, a cylindrical lens group, a prism, an objective lens, an analyzer, a lens, and a camera; the prism is disposed on the upper surface of the DMF automatic control module; The light path sequentially passes through the light source, the collimator, the acousto-optic tunable filter, the polarizer, the liquid crystal phase retarder, the cylindrical lens group, the prism, the DMF automatic control module, the prism, the objective lens, the analyzer, the lens, and the camera.

[0015] An embodiment of the present invention also provides a single-cell dynamic monitoring method based on DMF and SPR imaging, which is implemented by using the single-cell dynamic monitoring system based on DMF and SPR imaging described in any one of the above; the content of the method includes: Droplet loading and calibration: Inject PBS droplets and transport them to the SPR sensing area, adjust the incident light angle of the SPR optical phase sensing module, calibrate the SPR resonance angle, and establish a phase-refractive index standard curve through PBS solutions with different concentrations; Trace sample detection: Drive a certain amount of single-cell droplets to the imaging sensing area through the DMF automatic control module; the driving is multi-droplet parallel driving to multiplex the sensing area in a time-sharing manner; collect SPR phase signals in real time through the SPR optical phase sensing module; monitor the cell adhesion process, then load drug droplets to merge with the single-cell droplets, and capture the dynamic changes of cell secretions through SPR phase imaging; the SPR optical phase sensing module or the intelligent control unit identifies the number of cells in the droplets, eliminates multi-cell or empty droplets, and retains single-cell droplets.

[0016] In addition, according to the single-cell dynamic monitoring system based on DMF and SPR imaging of the present invention, the following additional technical features may also be provided: In some of these embodiments, after the detection is completed, drive the cleaning liquid to the sensing area through the DMF automatic control module to remove residues.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: In the embodiment of the present invention, the provided single-cell dynamic monitoring system based on DMF and SPR imaging uses an SPR sensing surface that does not require specially processed periodic metal structures or nanoparticles, reducing processing costs; at the same time, combined with the new active digital microfluidics technology, the response time is reduced, higher throughput can be achieved, sample contamination can be effectively reduced, and label-free real-time dynamic analysis of single cells can be performed; The single-cell dynamic monitoring method based on DMF and SPR imaging of the present invention is implemented by using the single-cell dynamic monitoring system based on DMF and SPR imaging, and thus has at least all the features and advantages of the single-cell dynamic monitoring system based on DMF and SPR imaging, which will not be elaborated here. Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 FIG. is a schematic structural diagram of a DMF-SPR manipulation-detection integrated system disclosed in an embodiment of the present invention; Figure 2 FIG. is a schematic cross-sectional structural diagram of a DMF automatic control module disclosed in an embodiment of the present invention.

[0019] DESCRIPTION OF THE REFERENCE NUMERALS: 1 - mercury lamp light source; 2 - collimator; 3 - acousto-optic tunable filter; 4 - polarizer; 5 - liquid crystal phase retarder; 6 - concave cylindrical lens; 7 - convex cylindrical lens; 8 - prism; 9 - objective lens; 10 - analyzer; 11 - lens; 12 - camera; 13 - DMF automatic control module; 131 - substrate; 132 - ground electrode; 133 - SPR sensing array; 134 - hydrophobic layer; 135 - droplet; 136 - dielectric layer; 137 - microelectrode array. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings through specific embodiments and their application scenarios.

[0022] Please refer to Figure 1 As shown in the figure, in some embodiments of the present invention, a DMF-SPR manipulation-detection integrated system is provided, which includes two major parts: an SPR optical phase sensing module and a DMF automatic control module. The system schematic diagram is as shown in Figure 1As shown in the figure, it mainly includes a mercury lamp light source 1, a collimator 2, an acousto-optic tunable filter 3 (AOTF), a polarizer 4, a liquid crystal phase retarder 5 (LCVR), a concave cylindrical lens 6, a convex cylindrical lens 7, a prism 8, an objective lens 9, an analyzer 10, a lens 11, a camera 12 and a DMF automatic control module 13. The prism 8 is disposed on the upper surface of the DMF automatic control module 13. The mercury lamp light source 1, the collimator 2, the acousto-optic tunable filter 3 (AOTF), the polarizer 4, the liquid crystal phase retarder 5 (LCVR), the concave cylindrical lens 6, the convex cylindrical lens 7, the prism 8, the DMF automatic control module 13, the prism 8, the objective lens 9, the analyzer 10, the lens 11 and the camera 12 are optically connected in sequence.

[0023] In some embodiments of the present invention, the cross-sectional structure of the DMF automatic control module 13 is as Figure 2 shown, and mainly includes a substrate 131, a ground electrode 132, an SPR sensing array 133, a hydrophobic layer 134, a droplet 135 containing a detection content, a dielectric layer 136, and a microelectrode array 137 of metal or indium tin oxide (ITO). The substrate 131 has two layers, which are respectively arranged on the top layer and the bottom layer, and a ground electrode 132 is arranged below the top substrate; two hydrophobic layers 134 are arranged below the ground electrode 132, and a plurality of droplets 135 containing detection contents are arranged between the two hydrophobic layers 134; an SPR sensing array 133 is embedded in the hydrophobic layer above the droplet 135, and the SPR sensing array 133 is arranged in the area facing the prism 8; a dielectric layer 136 is arranged below the hydrophobic layer 134 below the droplet 135, and the lower surface of the dielectric layer 136 is connected to the upper surface of the bottom substrate; a microelectrode array 137 is embedded at the bottom of the dielectric layer 136. The microelectrode array 137 is connected to the peripheral circuit system through a flexible printed circuit (FPC) flexible cable.

[0024] In some embodiments of the present invention, a single-cell dynamic monitoring method based on DMF and SPR imaging is provided. The steps of the single-cell dynamic monitoring method include: Step 1, chip (i.e., DMF automatic control module 13) preparation: The substrate 131 of the chip is mainly quartz glass or ITO conductive glass; the SPR sensing array 133 is mainly prepared by first magnetron sputtering a metal thin film and then through processes such as photolithography and stripping. The material composition of the SPR sensing array 133 can be metals such as gold, silver, and copper, or other materials can be added on the metal, such as two-dimensional materials or phase change materials such as graphene, MoS2, and MXene, and the sensitivity can be enhanced by adjusting the thickness. The preparation and assembly of other layers of the chip can refer to the prior art, and the present invention does not make excessive limitations.

[0025] Step 2. System initialization: Place a prism on the DMF chip, inject phosphate buffered saline (PBS) through the liquid inlet, and tear it into droplets 135 of 2×2 electrodes. Transport the droplets 135 to the SPR sensing area, adjust the incident light angle, and calibrate the SPR resonance angle. The excitation beam is generated by a mercury lamp source 1. The broadband light emitted from the source is coupled into a liquid-core optical fiber through a built-in coupling lens group in the source. The light emerging from the optical fiber is collimated into parallel light by a short-focus lens collimator 2 and then incident on an AOTF (acousto-optic tunable filter 3) for modulation. The light further phase-calibrated by a polarizer (polarizer 4) and a liquid crystal phase retarder 5 passes through a cylindrical lens group (concave cylindrical lens 6, convex cylindrical lens 7) for beam expansion to form an elliptical beam to expand the observation area, and is incident after passing through a coupling lens (prism 8). The incident angle θ on the SPR sensing film is approximately 72.5° under the condition of a pure gold film (which can be calculated and adjusted accordingly according to the different materials of the SPR sensing layer). The incident light excites the surface plasmon wave on the sensing chip to produce the SPR effect. The reflected light carrying the sample information is captured by a CCD / CMOS camera system after passing through an analyzer 10 to obtain the signal carrying the phase information, and the change in the refractive index of the chip surface is characterized by demodulating the phase shift of the signal. Establish a standard curve with PBS solutions of different concentrations.

[0026] Step 3. Standard sample manipulation and detection process: The microfluidic controller drives a 0.1 μL sample droplet to the SPR sensing area, synchronously collects the SPR phase signal of the droplet. If a target molecule is detected, it automatically drives a cleaning droplet to remove the residual sample. Manipulate multiple droplets in parallel (such as antigen, antibody, control droplets), and time-division multiplex the SPR sensing area; avoid droplet collisions through dynamic path planning to improve the detection throughput.

[0027] Step 4. Cell culture and single-cell dynamic monitoring application: Inject the suspension containing target cells (such as tumor cells) (density 10 4 cells / mL) into the droplet manipulation area of the DMF chip through the liquid inlet; the CCD captures the droplet images in real time, and based on the image, identifies the number of cells in the droplet, and eliminates the droplets containing multiple cells or empty droplets. Drive the single-cell droplet to the SPR sensing area, and monitor the cell adhesion process in real time through the change in the phase signal (adhesion time 10 - 30 minutes). Load droplets containing drugs through the liquid inlet, and the microfluidic controller drives the drug droplets to merge with the target single-cell droplets to ensure uniform diffusion of the drug around the cells. Capture information such as cell secretions through SPR phase imaging.

[0028] In some embodiments of the present invention, the DMF-SPR manipulation-detection integrated system can have other system forms. For example, the light source can be replaced with a mercury lamp, LED, supercontinuum laser, or other light sources; the form of the beam expander system can also be changed, and elements such as a collimator / lens group can be additionally added; the phase detection part can be replaced with a structure such as a Mach-Zehnder interferometer.

[0029] For the parts not described in detail in the present invention, reference can be made to the prior art or they are well-known technologies to those skilled in the art. This embodiment does not make any limitations in this regard and will not be described in detail herein.

[0030] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the claims of the present invention, and all of them fall within the protection scope of the present invention.

Claims

1. A single-cell dynamic monitoring system based on DMF and SPR imaging, characterized in that, The system includes: A DMF automatic control module, configured to accurately manipulate microdroplets so as to be integrated with the SPR optical phase sensing module to achieve dynamic imaging monitoring at the single-cell level; An SPR optical phase sensing module for performing SPR phase imaging on droplets wrapped with detection contents.

2. The single-cell dynamic monitoring system based on DMF and SPR imaging according to claim 1, characterized in that The DMF automatic control module includes a substrate, a ground electrode, an SPR sensing array, a hydrophobic layer, a dielectric layer, and a microelectrode array; The substrate includes two layers, which are respectively arranged at the top layer and the bottom layer; The ground electrode and the first hydrophobic layer are sequentially arranged below the top-layer substrate; the SPR sensing array is embedded in the first hydrophobic layer; The dielectric layer and the second hydrophobic layer are sequentially arranged above the bottom-layer substrate; the microelectrode array is embedded in the dielectric layer; A number of droplets wrapped with detection contents are arranged between the first hydrophobic layer and the second hydrophobic layer.

3. The single-cell dynamic monitoring system based on DMF and SPR imaging according to claim 2, characterized in that, The microelectrode array is a metal or indium tin oxide electrode array; The microelectrode array is connected to the peripheral circuit system through a flexible circuit board flexible cable.

4. The single-cell dynamic monitoring system based on DMF and SPR imaging according to claim 2, wherein The SPR sensing array is arranged in the area directly opposite to the prism in the SPR optical phase sensing module.

5. The single-cell dynamic monitoring system based on DMF and SPR imaging according to claim 2, characterized in that, The SPR sensing array has the same thickness as the first hydrophobic layer.

6. The single-cell dynamic monitoring system based on DMF and SPR imaging according to claim 2, wherein, The microelectrode array is embedded at the bottom of the dielectric layer.

7. The single-cell dynamic monitoring system based on DMF and SPR imaging according to claim 2, wherein The preparation method of the SPR sensing array is: magnetron sputtering a gold, silver or copper metal film, and then obtaining it through photolithography and stripping; a two-dimensional material is compounded on the metal film to enhance the sensitivity.

8. The single-cell dynamic monitoring system based on DMF and SPR imaging according to claim 1, characterized in that, The SPR optical phase sensing module includes a light source, a collimator, an acousto-optic tunable filter, a polarizer, a liquid crystal phase retarder, a cylindrical lens group, a prism, an objective lens, an analyzer, a lens, and a camera; the prism is arranged on the upper surface of the DMF automatic control module; The light path sequentially passes through the light source, the collimator, the acousto-optic tunable filter, the polarizer, the liquid crystal phase retarder, the cylindrical lens group, the prism, the DMF automatic control module, the prism, the objective lens, the analyzer, the lens, and the camera.

9. A single-cell dynamic monitoring method based on DMF and SPR imaging, characterized in that, The method is implemented by using the single-cell dynamic monitoring system based on DMF and SPR imaging according to any one of claims 1-8; the content of the method includes: Droplet loading and calibration: Inject PBS droplets and transport them to the SPR sensing area, adjust the incident light angle of the SPR optical phase sensing module, calibrate the SPR resonance angle, and establish a phase-refractive index standard curve through PBS solutions with different concentrations; Trace sample detection: Drive a certain amount of single-cell droplets to the imaging sensing area through the DMF automatic control module; the driving is multi-droplet parallel driving to multiplex the sensing area in a time-division manner; collect SPR phase signals in real time through the SPR optical phase sensing module; monitor the cell adhesion process, then load drug droplets and merge them with the single-cell droplets to capture the dynamic changes of cell secretions through SPR phase imaging; the SPR optical phase sensing module or the intelligent control unit identifies the number of cells in the droplets, eliminates multi-cell or empty droplets, and retains single-cell droplets.

10. The single-cell dynamic monitoring method based on DMF and SPR imaging according to claim 9, characterized in that, After the detection is completed, drive the cleaning liquid to the sensing area through the DMF automatic control module to remove residues.

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