CN216309741U

CN216309741U - Fluorescence activated cell sorting system based on moving focusing surface acoustic beam

Info

Publication number: CN216309741U
Application number: CN202122791447.3U
Authority: CN
Inventors: 褚正康
Original assignee: Sichuan Chengdian Medical Technology Consulting Co ltd
Current assignee: Sichuan Chengdian Medical Technology Consulting Co ltd
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-04-15
Anticipated expiration: 2031-11-15

Abstract

The utility model provides a fluorescence activated cell sorting system based on a moving focusing surface acoustic beam. And the laser source is used for emitting a laser beam of 1.2 mm. And a dichroic beam splitter a for reflecting the laser beam emitted from the laser source to the lower side. And the convex lens is positioned below the dichroic beam splitter a, and focuses the reflected laser beam into a tiny laser spot with the diameter of 70 mu m, so that the laser spot is aligned with the fluorescence interrogation zone of the acoustic sorting chip. When passing through the laser spot, the fluorescent particles or fluorescently labeled cells will be excited and emit light longer than the excitation wavelength. The microfluidic technology provided by the utility model has the capability of precise cell manipulation, and has great potential in the aspects of modification and miniaturization of the traditional FACS system. Fluorescence interrogation at kilohertz rates with a highly focused surface acoustic beam is used to classify micron-sized particles and biological cells.

Description

Fluorescence activated cell sorting system based on moving focusing surface acoustic beam

Technical Field

The utility model belongs to the technical field of cell sorting, and particularly relates to a fluorescence activated cell sorting system based on a mobile focusing surface acoustic beam.

Background

Fluorescence Activated Cell Sorting (FACS), a precise microscale manipulation technique capable of high-throughput cell analysis and sorting at the single-cell level, has become the fundamental technology for biological research and clinical diagnostics. In conventional FACS systems, a cell suspension containing a fluorochrome-labeled target cell type is also introduced continuously through a capillary tube and is hydrodynamically restricted by a faster sheath flow technique. The ordered individual cells are excited by the laser beam to a fluorescent label in the target biological cell. These fluorescent signals represent unique cell surface biomarkers that enable highly specific classification of individual cells downstream. A vibration mechanism then breaks the continuous flow of liquid into individual micron-sized droplets. Upon fluorescent interrogation, the droplets containing the target cells are selectively charged and electrostatically deflected into a collection vessel. Compared with batch cell sorting methods such as centrifugation and magnetic activated cell sorting, the FACS system separates target cells on a single cell basis, and therefore has higher sorting accuracy.

While very efficient and accurate, the complex sorting mechanism makes current FACS systems expensive and cumbersome, and aerosol generated in the open environment prior to the sorting process can cause potential sample contamination to the environment and a biosafety risk to machine operators by inhaling harmful aerosols. Current FACS systems are also typically deployed in a core facility shared by multiple research laboratories, resulting in scheduling conflicts and potential cross-contamination of test samples. Furthermore, the viability of sorted cells may be compromised due to the strong electric field required for sorting.

Microfluidic technology is a promising approach to overcome the disadvantages of conventional FACS systems, and micron-sized particles can be precisely manipulated in miniaturized devices. FACS equipment based on microfluidic technology has demonstrated the ability to avoid sample contamination and minimize user health risks with fully integrated actuators and aerosol-free sorting designs. Furthermore, flexible integration of additional microfluidic functions, including characterization, culture, mixing, and polymerase chain reaction, can provide more sophisticated pre-processing and analysis capabilities for microfluidic technology-based FACS systems. Microfluidic sorting mechanisms based on dielectrophoresis, acoustophoresis, MEMS valves and bubble jet were validated compared to traditional electrostatic based FACS sorting mechanisms. Although these studies have demonstrated accurate cell sorting in continuous flow by advanced microfluidic methods, many still suffer from inherent limitations, preventing their feasibility for practical applications. For example, dielectrophoresis-based cell sorting requires careful maintenance of the electrical properties of the buffer, in particular the conductivity, which can vary significantly between different biological samples. In addition, the application of strong electric fields can induce electroporation and thermal damage of cells. Bubble jet actuation relies on the instantaneous change in flow profile created by the micro-bubble generation and expansion of the side chambers of the main microfluidic channel. When the microbubble collapses after each action, it causes backflow into the cavity, pulling the cells in the opposite direction to the target exit, increasing the risk of misclassification.

Due to low power consumption and good biocompatibility, acoustic electrophoresis has proven to be a promising microfluidic technology, allowing the classification of micro-scale cells and particles according to size, density and compressibility. Some recent studies have utilized acoustic electrophoresis for fluorescence-activated screening of particles, cells and droplets. Johansson et al [1] and Jokobssson et al [2] demonstrate fluorescence-activated cell and particle sorting using standing waves, respectively. These devices are built based on Bulk Acoustic Wave (BAW) transducers, resulting in a wide sorting area (on the order of a few millimeters) where large numbers of non-target cells can be sorted to the collection outlet of the target cells. Nawaz et al [3] demonstrated a standing field Surface Acoustic Wave (SAW) driven FACS system utilizing a focused transducer to produce sorting region widths on the order of the acoustic wavelength (120 μm). However, the use of standing waves inherently limits the maximum translational displacement of the target cell to less than a quarter of the acoustic wavelength. Too small a separation distance between target and non-target cells may affect the accuracy of cell sorting, particularly for larger cells. In addition, the moving acoustic field for particle manipulation was transmitted and localized to a 100 μm wide region and demonstrated the feasibility of sorting droplets and cells in a fluorescence-activated system. However, the additional polymer layer gray between the fluid and the piezoelectric substrate attenuates the acoustic waves and reduces the effectiveness of the acoustic propagation effect increasing the acoustic power may cause a significant temperature rise, may damage the cells, and compromise their viability after sorting. Another approach to creating local acoustic forces is proposed by using concentrated acoustic traveling waves.

Technical scheme of prior art I

A method for high throughput cell sorting based on Standing Surface Acoustic Waves (SSAWs). A pair of focused inter-digital transducers (FIDTs) is used to produce a high resolution and energy efficient SSAW. Therefore, sorting throughput is significantly improved compared to conventional acoustic cell sorting methods. And the 10 μm polystyrene particles were successfully sorted, the minimum drive time was 72 μ s, which means potential sorting rates of over 13800 times/sec. Without the use of a detection unit, the actual sequencing throughput of 3300 specimens per second can be demonstrated.

Disadvantages of the prior art

These devices are built based on Bulk Acoustic Wave (BAW) transducers, resulting in a wide sorting area (on the order of a few millimeters) where large numbers of non-target cells can be sorted to the collection outlet of the target cells. And current FACS systems are rather complex, expensive, bulky, and have potential sample contamination and bio-safety issues due to aerosol generation in the open environment.

Technical scheme of prior art II

An acoustic flowbore Fluorescence Activated Cell Sorting (FACS) device that can simultaneously perform on-demand, high-throughput, high-resolution cell detection and sorting, integrated on a single chip. The technique was applied to an acoustic flow-port FACS instrument using "microfluidic drift" technique to accurately focus the cells/particles in three dimensions and achieve flow of single file particles/cells through the laser interrogation zone. The fluorescently labeled particles/cells are then sorted using short standing field surface acoustic wave (SSAW) pulses (150 μ s) triggered by an electronic feedback system.

The second prior art has the defects

Standing wave use inherently limits the maximum translational displacement of the target cell to less than a quarter of an acoustic wavelength. Too small a separation distance between target and non-target cells may affect the accuracy of cell sorting, particularly for larger cells.

SUMMERY OF THE UTILITY MODEL

The utility model aims to solve the defects of the prior art, provides the fluorescence activated cell sorting system based on the moving focusing surface acoustic beam, can highly accurately sort single particles and cells, improves the purity and the cell activity of the sorted cells, and has good biocompatibility.

The utility model adopts the following technical scheme:

the fluorescence activated cell sorting system based on the moving focusing surface acoustic beam comprises a laser source, a dichroic beam splitter, a convex lens, an acoustic sorting chip, a photomultiplier and a band-pass filter;

the laser source is used for emitting a laser beam of 1.2 mm;

a dichroic beam splitter a for reflecting the laser beam emitted from the laser light source to the lower side;

a convex lens; the laser beam splitter is positioned below the dichroic beam splitter a, and focuses the reflected laser beam into a tiny laser spot with the diameter of 70 mu m, so that the laser spot is aligned with a fluorescence interrogation zone of the acoustic sorting chip;

a dichroic beam splitter b located at an upper side of the dichroic beam splitter a, reflecting the fluorescence passing through the convex lens and the dichroic beam splitter a;

the band-pass filter is positioned at one side of the dichroic beam splitter b and is used for filtering low-frequency and high-frequency interference signals and receiving a target wavelength signal to the photomultiplier;

the photomultiplier is positioned at one side of the band-pass filter and is used for identifying fluorescent particles or fluorescent cells which are excited to emit longer-wavelength fluorescence;

once the photomultiplier detects the fluorescence emission of a single target cell, the peak value of the output voltage triggers a function generator to generate a pulse alternating current signal;

and the acoustic sorting chip is positioned on the lower side of the convex lens, applies the amplified pulse alternating current signal to the focused digital-to-digital transducer, generates a focused surface acoustic wave beam, couples the surface acoustic wave beam into adjacent fluid, and enables the detected fluorescent particles or fluorescent cells to be transversely converted into a streamline which is in the same direction as a target outlet, thereby realizing the screening of the fluorescent cells or fluorescent particles.

Further, the system also comprises a red LED lamp, an objective lens, a conventional mirror surface, a long-pass filter and a high-speed camera;

a red LED lamp positioned on the upper side of the dichroic beam splitter b to illuminate the acoustic sorting chip;

an objective lens located at the lower side of the acoustic sorting chip for amplifying the light;

a conventional mirror surface located at a lower side of the objective lens, reflecting the light;

the long-pass filter is positioned on one side of the conventional mirror surface and used for blocking strong light of laser wavelength;

and the high-speed camera is positioned on one side of the long-pass filter and is used for monitoring the whole sorting process.

Further, the acoustic sorting chip is mounted on the 3D printing support.

Furthermore, the microfluidic channel of the acoustic sorting chip is connected with the injector pump through a pipeline, and the focusing digital transducer of the acoustic sorting chip is connected with the output of the power amplifier.

Further, the laser source was a 35mW 473nm wavelength solid state laser source.

Further, on the focusing inter-digital transducer, the width of the surface acoustic wave beam generating the focusing is 50 um.

The utility model has the beneficial effects that:

1. the acoustic sorting chip is used for sorting cells or particles, aerosol cannot be generated, so that potential sample pollution cannot be caused to the environment, and biological safety risks can be caused to system operators by sucking harmful aerosol.

2. The sorting precision of the cells or the particles and the survival rate of the cells are improved. The system is applied to separate the fluorescent-labeled MCF-7 breast cancer cells from the diluted whole blood sample, and the purity of the sorted MCF-7 cells is higher than 86%. The cell viability before and after acoustic sorting is higher than 95%, which shows that the cell viability is good in biocompatibility.

3. The system volume is greatly reduced by utilizing the microfluidic technology.

Drawings

FIG. 1 is a schematic structural view of the present invention;

in the figure: the device comprises a 1-laser source, a 2-dichroic beam splitter a, a 3-convex lens, a 4-acoustic sorting chip, a 5-dichroic beam splitter b, a 6-photomultiplier, a 7-band-pass filter, an 8-objective lens, a 9-conventional mirror surface, a 10-high-speed camera, an 11-red LED lamp and a 12-long-pass filter.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described below clearly and completely, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in FIG. 1, the fluorescence activated cell sorting system based on moving focused surface acoustic beam of the utility model comprises a laser source 1, a dichroic beam splitter, a convex lens 3, an acoustic sorting chip 4, a photomultiplier 6 and a band-pass filter 7.

In which a laser beam having a diameter of 1.2mm is emitted from the laser light source 1 and then reflected by the dichroic beam splitter a 2. The laser beam is focused by a convex lens 3 into a tiny laser spot of 70 μm diameter, aligned with the fluorescence interrogation zone of the acoustic sorting chip 4. Once the fluorescent particles or fluorescently labeled cells enter the laser spot, the fluorescent species are excited and emit light longer than the excitation wavelength. The fluorescent emission through the convex lens 3 and the dichroic beam splitter a2 is reflected by another dichroic beam splitter b5 to a photomultiplier tube (PMT)6, which photomultiplier tube (PMT)6 may convert the received light intensity into a voltage signal. The band pass filter 7 is placed in front of the photomultiplier tube (PMT)6, the PMT 6 being transparent only to the fluorescence emission wavelength.

The suspended particles or cells are hydrodynamically restricted by two sheath flows, so the non-fluorescent subpopulations follow the original flow line, and by default flow into the waste outlet. Once the photomultiplier tube 6(PMT) detects the fluorescence emission of a single target cell, its output voltage peak triggers a function generator to generate a pulsed AC signal. After signal amplification, the pulsed ac signal is applied to a focusing digital transducer (FIDT) to produce a focused surface acoustic beam that couples into the adjacent fluid and laterally converts the detected particles or cells into a streamline co-directed to the target outlet.

In summary, fluorescence detection focuses an activation pulse on a line acoustic surface acoustic wave beam (FTSAW) to screen individual target particles or cells from a heterogeneous population.

The red light emitting LED lamp 11 illuminates the acoustic sorting chip from top to bottom. After light amplification by the objective 8 and reflection by the conventional mirror 9, the entire sorting process is monitored by a high-speed camera 10, imaging from the bottom of the apparatus. The long pass filter 12 blocks the intense light at the laser wavelength to avoid overexposure of the high speed camera 10.

The acoustic separation chip 4 of the utility model is a micro-fluidic channel layer made of Polydimethylsiloxane (PDMS) and lithium niobate (LiNbO)₃) A focused digital transducer (FIDT) fabricated on a substrate. In which a 40 μm deep microfluidic channel was fabricated by a widely used soft lithography process by casting Polydimethylsiloxane (PDMS) on a prefabricated master.

X-propagation LiNbO is cut in a y-cutting mode at 128 degrees by adopting a standard lift-off process₃A Cr/Al layer (7nm/200nm) was deposited on the surface of the substrate to prepare a focused digital transducer (FIDT). The focusing digital transducer (FIDT) consists of 36 pairs of coaxial arcuate electrodes, both of which have a width and spacing of 7.5 μm corresponding to an acoustic substrate wavelength of 30 μm and a resonance frequency of 132 MHz.

The aperture of the proximal end of the focusing digital transducer (FIDT) is 160 μm, and the included angle with the distal end is 26 degrees. Subsequently 300nm thick SiO was deposited using electron beam excitation₂Layer to prevent corrosion and promote bonding with Polydimethylsiloxane (PDMS). Followed by Polydimethylsiloxane (PDMS) and lithium niobate (LiNbO)₃) The pads were aligned and bonded together after 150 seconds of air plasma treatment to form the final acoustic sorting chip 4.

Polydimethylsiloxane (PDMS) channels and thin Polydimethylsiloxane (PDMS) layers with micro-pillars and supports were fabricated separately using standard soft lithography techniques. And bonding the Polydimethylsiloxane (PDMS) channel with the micro-column layer after the plasma treatment to form the disposable PDMS channel device. The disposable channeling device was placed by hand on a SAW transducer consisting of a piezoelectric substrate with inter-digital transducers (IDTs). The channel device can be removed and discarded without difficulty and the surface acoustic transducer can be reused.

In the utility model, an acoustic separation chip 4 is arranged on a 3D printing support, a microfluidic channel is connected with an injector pump through a pipeline, and a focusing digital transducer (FIDT) is connected with the output of a power amplifier. The 3D printing support with the sorting chip is then placed on the stage with the high speed camera 10 leading to the microscope.

As shown in fig. 1, the optical assembly and acoustic sorting chip includes a 35mW 473nm wavelength solid state laser source, a pure red LED lamp 11, a bandpass filter 7, a convex lens 3, a photomultiplier tube 6, a dichroic beamsplitter a2 and a dichroic beamsplitter b 5. These are assembled and mounted on a manual z-axis platform, integrating the system of the present invention. The system of the utility model is manually aligned with the microscope objective on the xy plane, and then adjusted along the z axis, so that the laser spot in the microfluidic channel of the acoustic sorting chip is minimum.

The distance between the laser spot and the focused acoustic beam can be adjusted by moving the XY stage of the microscope. During each fluorescence detection, the system outputs a high-level voltage signal to the function generator. In the 0.1 mus delay trigger mode, the function generator generates a pulsed ac signal of determined duration and power to the power amplifier, which is then applied to a focused digital transducer (FIDT) to generate surface acoustic waves.

The utility model demonstrates that single particle horizontal manipulation uses a highly focused propagating surface acoustic beam, demonstrating the system of the utility model, which uses fluorescence probing of highly focused surface acoustic beams at kilohertz rates to sort micron-sized particles and biological cells. The width of the sonic beam is 50 μm, and a high degree of precision in sorting single particles and cells can be achieved. We applied the acoustic system to separate fluorescently-labeled MCF-7 breast cancer cells from a diluted whole blood sample, with the purity of the sorted MCF-7 breast cancer cells being higher than 86%. The cell viability before and after acoustic sorting is higher than 95%, which shows that the cell viability has good biocompatibility and can be widely applied to biomedical research.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. The fluorescence-activated cell sorting system based on the moving focusing surface acoustic beam is characterized by comprising a laser source, a dichroic beam splitter, a convex lens, an acoustic sorting chip, a photomultiplier and a band-pass filter;

the laser source is used for emitting a laser beam of 1.2 mm;

a convex lens; the dichroic beam splitter is positioned below the dichroic beam splitter a and used for focusing the reflected laser beam into a tiny laser spot with the diameter of 70 mu m so as to align the laser spot with a fluorescence interrogation zone of the acoustic sorting chip;

a dichroic beam splitter b located at an upper side of the dichroic beam splitter a, for reflecting the fluorescence passing through the convex lens and the dichroic beam splitter a;

the band-pass filter is positioned at one side of the dichroic beam splitter b and used for filtering low-frequency and high-frequency interference signals and receiving a target wavelength signal to the photomultiplier;

the photomultiplier is positioned at one side of the band-pass filter and is used for identifying fluorescent particles or fluorescent cells which are excited to emit longer-wavelength fluorescence; once the photomultiplier detects the fluorescence emission of a single target cell, the peak value of the output voltage triggers a function generator to generate a pulse alternating current signal;

2. The fluorescence-activated cell sorting system based on moving focused surface acoustic beams according to claim 1, characterized in that the acoustic sorting chip is mounted on a 3D printing support.

3. The fluorescence-activated cell sorting system based on moving focused surface acoustic beams according to claim 1, wherein the microfluidic channel of the microfluidic channel acoustic sorting chip is connected with an injector pump through a pipeline, and the focusing digital transducer of the acoustic sorting chip is connected with the output of the power amplifier.

4. The fluorescence-activated cell sorting system based on moving focused surface acoustic beams according to claim 1, characterized in that the laser source is a 35mW 473nm wavelength solid state laser source.

5. The fluorescence activated cell sorting system based on moving focused surface acoustic beams according to claim 3, characterized in that the width of the focused surface acoustic beam generated on the focusing digital transducer is 50 um.