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WO2019119368A1 - Appareil de détection de cellules de type à écoulement à entraînement acoustique - Google Patents

Appareil de détection de cellules de type à écoulement à entraînement acoustique Download PDF

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Publication number
WO2019119368A1
WO2019119368A1 PCT/CN2017/117833 CN2017117833W WO2019119368A1 WO 2019119368 A1 WO2019119368 A1 WO 2019119368A1 CN 2017117833 W CN2017117833 W CN 2017117833W WO 2019119368 A1 WO2019119368 A1 WO 2019119368A1
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WIPO (PCT)
Prior art keywords
radiation force
cell
particles
cells
acoustically driven
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/117833
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English (en)
Chinese (zh)
Inventor
郑海荣
李飞
蔡飞燕
许迪
夏向向
林勤
孟龙
肖杨
邱维宝
李永川
苏敏
黄继卿
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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Priority to PCT/CN2017/117833 priority Critical patent/WO2019119368A1/fr
Publication of WO2019119368A1 publication Critical patent/WO2019119368A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry

Definitions

  • the present application relates to a flow cytometry device, and more particularly to an acoustically driven flow cytometry device.
  • the current flow cytometry mainly uses water flow force to realize the transport and flow focusing of cell particles in the microfluidic channel.
  • the technical solution mainly has the following problems: the suspended particles are easy to block the microcavity, and the cost of replacing the channel is relatively high. High; flow-focusing experiments require expensive sheath-constrained cells to be arranged in a single column; complex and expensive microfluidic pump systems are required to drive fluid flow; current flow cytometry is primarily single-channel processing rather than multi-channel parallel processing Way; difficult to clean, residual samples in the flow channel will pollute new samples.
  • the other method does not require the use of a sheath fluid, and the cells are focused by externally applied field forces or fluid forces in the channel, allowing the cells to pass through the detection zone one by one.
  • the patent "a microfluidic chip and its preparation method and application” (application No. 201611146452.6) and “a particle sorting method and its apparatus and use” (application number 201710016125.7) use the external standing wave sound field to arrange the cell particles into one Or multiple lines to achieve single-column or multi-column focusing so that cells pass through the detection area one by one.
  • the transport, focusing and detection of the particles need to be carried out in single or multiple microchannels.
  • the suspended cell particles in the flow channel are easy to block the channel, the system is ineffective, and the replacement cost is high;
  • the technical problem to be solved by the present application is to provide an acoustically driven flow cytometry apparatus for the deficiencies of the prior art.
  • An acoustically driven flow cytometry apparatus comprising a cell manipulation module for manipulating cell particles in a sample solution, and an image processing module for smearing the stained cell particles Fluorescence imaging is performed, the image processing module is for processing and analyzing a fluorescent image, counting cell particles and estimating cell particle size, the cell manipulation module comprising a flow chamber and an ultrasonic radiation force generating system, the flow chamber for a sample solution containing cell particles; the ultrasonic radiation force generating system is used to generate acoustic radiation force on the cell particles, and the control cell particles are arranged in parallel lines to achieve multi-line focusing, suspending the cell particles, and driving the cell particles to be oriented and transported. Multi-channel parallel detection is achieved to the detection area.
  • the flow cell includes a substrate, a PDMS sidewall and a glass cap, the PDMS sidewalls being bonded to the substrate and the glass cap, respectively, the substrate being made of quartz glass, plexiglass or silicon.
  • the radiation force generating mechanism includes an ultrasonic transducer and an artificial structure placed in the flow chamber, and an electrical signal amplified by the power amplifier excites the ultrasonic transducer to generate ultrasonic waves and thereby generate a radiation force to the particles.
  • the artificial structure includes an artificial periodic structure or an artificial aperiodic structure.
  • the artificial periodic structure includes a substrate and a plurality of ridges disposed on a lower surface of the substrate, the ridges being disposed in parallel and equally spaced.
  • the ultrasonic transducer is disposed outside the flow chamber, and the ultrasonic transducer does not coincide with a geometric center of the artificial periodic structure.
  • the plurality of transducers includes at least one ultrasonic transducer and interdigitated transducers disposed in pairs and arranged in parallel, the interdigital transducers being used to synthesize a standing wave field aligning cells to achieve focusing, the ultrasonic transduction
  • the device is used to generate a biased Gaussian beam to transport cells.
  • the cell manipulation module includes a flow chamber and an ultrasonic radiation force generation system for containing a cell manipulation module, an imaging module, and an image processing module, and the flow chamber is for holding a sample solution containing cell particles;
  • the force generation system is used to generate acoustic radiation force on the cell particles, and the control cell particles are arranged in parallel lines to achieve multi-line focusing, suspending the cell particles, and driving the cell particles to be transported to the detection area to achieve multi-channel parallel detection.
  • the present application utilizes acoustic radiation force to manipulate cell particles for transport and focusing, there is no need for a complicated pump system to drive the control fluid, nor to introduce a sheath fluid; since the present application utilizes a virtual channel composed of acoustic radiation forces to align cells, it does not need to be present.
  • microchannels used in the technology, thus avoiding the problem of clogging of the channel; multi-row parallel processing is realized by multi-line focusing due to the arrangement of multiple rows of cells due to acoustic radiation force; due to the cell manipulation module in the present application
  • the components such as the flow chamber are low in cost and simple in process, and can be replaced with new devices each time a new sample is measured, thereby avoiding the problem of contamination of the new sample by the residue in the flow channel in the prior art.
  • the present application provides a flow-free cell detection solution without microfluidic channels, no microfluidic pumps, no sheath fluid, disposable, parallel processing, and low cost.
  • FIG. 1 is a schematic diagram of functional modules of an apparatus of the present application in an embodiment
  • FIG. 2 is a schematic structural view of an artificial cycle structure of the present application in an embodiment
  • Figure 3 is a transmission spectrum of the artificial periodic structure shown in Figure 2;
  • Figure 4 is a distribution of the artificial sound structure modulating the sound field along the x direction
  • Figure 5 shows the acoustic radiation force of the cells in the three directions of x, y, and z in the artificial structure sound field
  • Figure 7 is a distribution of the radiation force components Fy and Fz in the y direction when the artificial periodic structure resonates in the yz plane;
  • Figure 8 is a view showing the distribution of the sound pressure field and the directions of the radiation force components Fy and Fz when the artificial periodic structure in the yz plane is non-resonant;
  • Figure 9 is a distribution of sound pressure field and distribution of radiation force components Fy and Fz along the y direction when the artificial periodic structure in the yz plane is non-resonant;
  • Figure 10 is a distribution of acoustic radiation force components Fx and Fz along the transport direction x;
  • Figure 11 is an image acquired using the acoustically driven flow cytometry apparatus of the present application.
  • FIG. 12 is a schematic structural view of a radiation force generating mechanism of the present application in an embodiment.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • an embodiment of the acoustically driven flow cytometry apparatus of the present application includes a cell manipulation module 10, an imaging module 20, and an image processing module 30.
  • the cell manipulation module 10 is for manipulating the cell particles in the sample solution
  • the imaging module 20 is for fluorescence imaging of the stained cell particles
  • the image processing module 30 is for processing and analyzing the fluorescence image, counting the cell particles and estimating the cell particle size.
  • the cell manipulation module 10 can include a flow chamber and an ultrasonic radiation force generating system, and the flow chamber can be a microcavity for holding a sample solution containing cell particles.
  • Ultrasonic radiation force generation system is used to generate acoustic radiation force on cell particles, manipulate cell particles to be arranged in parallel lines, achieve multi-line focusing, suspend cell particles, and drive cell particles to be transported to the detection area to achieve multi-channel parallel detection.
  • Imaging module 20 can include a fluorescent excitation source, an optical lens, a highly sensitive fluorescent camera.
  • the image processing module 30 can include a computer, a high speed data acquisition card, hardware control software, and image acquisition analysis processing software for processing and analyzing the fluorescent image, counting the cells, and estimating the cell size.
  • the focus and transport of the cells are mainly achieved by the radiation force generated by the ultrasonic radiation force generating system.
  • the ultrasonic radiation force generating system includes a radiation force generating mechanism, a signal generator, and a power amplifier.
  • the electrical signal generated by the signal generator is amplified by the power amplifier, and the excitation radiation generating mechanism generates an ultrasonic wave, and then generates a radiation force to the particle to realize the transportation and manipulation of the cell.
  • the flow chamber of the present application may include a substrate, a PDMS sidewall, and a glass cap, the PDMS sidewalls being bonded to the substrate while the PDMS sidewalls are also bonded to the glass cap.
  • the substrate can be made of quartz glass, plexiglass or silicon.
  • the radiation force generating mechanism can include an ultrasonic transducer and an artificial structure.
  • the ultrasonic transducer is placed outside the flow chamber, and the artificial structure is placed in the flow chamber, and the electrical signal amplified by the power amplifier excites the ultrasonic transducer to generate ultrasonic waves and thereby generate radiation force to the particles.
  • the artificial structure may specifically be an artificial periodic structure or an artificial aperiodic structure. As shown in FIG. 2, the artificial periodic structure includes a substrate 11 and a plurality of ribs 12 disposed on a lower surface of the substrate 11, and the ridges 12 are disposed in parallel and at equal intervals. 2 is a schematic diagram of an artificial periodic structure employed in an acoustic radiation force generating system.
  • FIG. 1 is a transmission spectrum of the artificial periodic structure shown in Figure 2, with a resonant frequency of 5.94 MHz.
  • the ultrasonic radiation force generating system of the present application wherein the ultrasonic transducer is disposed outside the flow chamber, and the ultrasonic transducer does not coincide with the geometric center of the artificial periodic structure.
  • the ultrasonic transducer can be affixed to the exterior of the flow chamber, specifically to the lower surface of the substrate.
  • the ultrasonic transducer can be a biased Gaussian beam source.
  • the artificial structure modulating the ultrasonic transducer emits a sound field to produce a sound field with manipulation functions such as transport and alignment, thereby achieving cell focusing and transport.
  • Figure 4 shows the distribution of the sound field in the x-direction of the artificial periodic structure. It can be seen that the sound pressure distribution obeys the Gaussian distribution, where the dotted line is the theoretical calculation value and the solid line part is the experimental measurement value. As shown in Fig.
  • the cells are subjected to acoustic radiation forces in the three directions of x, y, and z in the artificial structure sound field, wherein the x-direction acoustic radiation force Fx causes the orientation movement of the micro-nano particles toward the strongest direction of the sound field to achieve transport;
  • the acoustic radiation force Fy in the y direction causes the arrangement of the micro/nano particles to achieve focusing, and the confinement effect on the particles limits the lateral motion interval of the micro/nano particles, forming a virtual microcavity;
  • the acoustic radiation force Fz in the z direction is responsible for Suspension and capture of micro-nano particles.
  • the combined action of the acoustic radiation forces in these three directions ultimately leads to the transport and focusing of the cells.
  • Fig. 6 is a view showing the sound pressure field distribution and the directions of the radiation force components Fy and Fz at the time of resonance of the artificial periodic structure in the yz plane (resonance frequency is 5.94 MHz).
  • Fig. 7 is a view showing the distribution of the radiation force components Fy and Fz in the y direction when the artificial periodic structure resonates in the yz plane (resonance frequency is 5.94 MHz). It can be seen that in the equilibrium position shown by the circle, the radiation force component Fz is negative, that is, vertically downward, so that the cells are stably captured on the surface of the structure.
  • Fig. 8 is a diagram showing the sound pressure field (driving frequency of 5.92 MHz) and the directions of the radiating force components Fy and Fz at the time of non-resonance calculation by numerical simulation.
  • Fig. 9 is a spatial distribution of the sound pressure field (driving frequency of 5.92 MHz) and the radiation force components Fy and Fz along the y direction obtained by numerical simulation.
  • Figure 8 is a schematic diagram of the sound field distribution and force in the yz plane. It can be seen that the sound field morphology at the time of non-resonance is very different from the sound pressure field distribution at the time of resonance in Figure 6. The position shown by the circle is the equilibrium position of the particles in the sound field.
  • Fig. 10 is a spatial distribution of the non-resonant (driving frequency 5.92 MHz) radiation force components Fx and Fz along the transport direction x obtained by numerical simulation.
  • the radiation force component Fz is always a positive value, meaning that the direction of the force is opposite to the direction of gravity, so that the particles can be suspended.
  • the present application utilizes acoustic radiation force to manipulate cell particles for transport and focusing, there is no need for a complicated pump system to drive the control fluid, nor to introduce a sheath fluid; since the present application utilizes a virtual channel composed of acoustic radiation forces to align cells, it does not need to be present.
  • microchannels used in the technology, thus avoiding the problem of clogging of the channel; multi-row parallel processing is realized by multi-line focusing due to the arrangement of multiple rows of cells due to acoustic radiation force; due to the cell manipulation module in the present application
  • the components such as the flow chamber are low in cost and simple in process, and can be replaced with new devices each time a new sample is measured, thereby avoiding the problem of contamination of the new sample by the residue in the flow channel in the prior art.
  • the present application provides a flow-free cell detection solution without microfluidic channels, no microfluidic pumps, no sheath fluid, disposable, parallel processing, and low cost.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • Embodiment 2 is a specific application example of the acoustically driven flow cytometry apparatus of the present application.
  • the flow cell consists of a quartz glass substrate, a PDMS (polydimethylsiloxane) wall and a glass top cover.
  • the PDMS wall can be bonded to the substrate and the top cover.
  • the ultrasonic transducer is a PZT4 piezoelectric ceramic piece with a center frequency of 6 MHz and is bonded to the glass substrate by an epoxy resin.
  • the geometric center of the ultrasonic transducer does not coincide with the geometric center of the artificial periodic structure, that is, the ultrasonic transducer needs to be biased, and the purpose is to generate a biased sound source, so that the cell particles deviating from the center of the sound source are subjected to the image as shown in FIG.
  • the illustrated radiation force Fx acts to direct the transport to the sound source.
  • the control software control signal generator (AFG3102, Tektronix, Beaverton, OR, USA) generates Chirp signals with a frequency of 5.90-5.94 MHz and is amplified by a power amplifier (150A100B, Amplifier Research, Souderton, PA, USA) to excite the piezoelectric ceramics.
  • the sheet PZT4 produces ultrasonic waves.
  • the ultrasonically excited artificial periodic structure produces a localized field as shown in Figure 8 on its surface and produces the acoustic radiation force shown in Figures 9-10 for the cellular particles.
  • the fluorescent excitation source is a 100W high-pressure mercury lamp that excites the stained cells to fluoresce, and a highly sensitive fluorescent camera (QIMAGING optiMOS) records the fluorescent images and transmits the data to a computer.
  • the image analysis processing software extracts the fluorescence intensity distribution of the image and further calculates the size and number of cells.
  • Figure 11 is an image acquired using an acoustically driven flow cytometry system.
  • the cells used in the experiment were MCF-7 tumor cells having a diameter of 15 ⁇ m.
  • the cells were stained with calcein fluorescent dye and injected into the flow cell.
  • the radiation generated by the ultrasonic radiation force generating system aligns these cells and transports these cells to the detection area.
  • the fluorescent excitation source excites the stained cells to emit green fluorescence
  • the fluorescence image is recorded by a highly sensitive fluorescent camera and transmitted to the computer via a high-speed acquisition card.
  • the image analysis processing software of the computer automatically extracts the fluorescence intensity curve, and calculates the cell size and the number of cells according to the peak size and the number of peaks, respectively.
  • Figure 11 extracts the fluorescence intensity curves of a single column of cells in the dashed box.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • the plurality of transducers includes at least one ultrasonic transducer and interdigitated transducers arranged in pairs and arranged in parallel, the interdigital transducers for synthesizing standing wave field alignment cells for focusing, ultrasonic transposition
  • the energy device is used to generate a biased Gaussian beam to transport the cells.
  • the interdigital transducers may have a pair or a plurality of teams, and FIG. 12 includes a pair of interdigital transducers, namely a first interdigital transducer 13 and a second interdigital transducer 14, wherein the ultrasonic transducer is transposed
  • the device 15 uses a piezoelectric transducer PZT, 40 is a cell.
  • the interdigital transducer 1 and the interdigital transducer 2 are used to synthesize a standing wave field array of cells for focusing, and the piezoelectric transducer PZT is used to generate a biased Gaussian beam for transporting cells.
  • the ultrasonic radiation force generation system may include a plurality of ultrasonic transducers, a signal generator, and a power amplifier. A portion of the ultrasonic transducer is used to manipulate the cell alignment focus; another portion of the transducer acts as a biased Gaussian beam source to drive particle transport.
  • the sound field synthesized by a plurality of ultrasonic transducers to generate sound waves produces a sound field with manipulation functions such as transport and alignment, thereby achieving cell focusing and transport.

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  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • General Physics & Mathematics (AREA)
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  • Optical Measuring Cells (AREA)

Abstract

L'invention concerne un appareil de détection de cellules de type à écoulement à entraînement acoustique, comprenant un module de manipulation de cellules (10), un module d'imagerie (20) et un module de traitement d'image (30), le module de manipulation de cellules (10) comprenant une chambre d'écoulement et un système de génération de force de rayonnement ultrasonore, et le système de génération de force de rayonnement ultrasonore étant utilisé pour générer une force de rayonnement acoustique en direction de particules cellulaires, et pour manipuler les particules cellulaires de façon à les agencer en lignes parallèles, ce qui permet d'obtenir une focalisation multi-lignes, mettre en suspension les particules cellulaires et les amener à être transférées de manière orientée vers une zone de détection de façon à réaliser une détection parallèle à canaux multiples. L'appareil de détection utilise une force de rayonnement acoustique pour manipuler les particules cellulaires en vue de leur transfert et de leur focalisation, et, par conséquent, aucun système de pompe compliqué n'est nécessaire pour entraîner et commander un fluide, et aucun fluide de gainage n'a besoin d'être introduit. L'appareil de détection utilise un canal virtuel formé par la force de rayonnement acoustique pour agencer les cellules, ce qui permet d'éviter le problème de colmatage d'une cavité. La force de rayonnement acoustique peut agencer de multiples lignes de cellules, et, par conséquent, un traitement parallèle à canaux multiples de focalisation multi-lignes peut être réalisé. La chambre d'écoulement est peu coûteuse et peut être changée à chaque fois, et par conséquent, on évite le problème de la contamination d'un nouvel échantillon par un résidu resté dans un canal d'écoulement .
PCT/CN2017/117833 2017-12-21 2017-12-21 Appareil de détection de cellules de type à écoulement à entraînement acoustique Ceased WO2019119368A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102762990A (zh) * 2009-12-04 2012-10-31 生命技术公司 用于声学流式细胞计量术的装置、系统、方法和计算机可读介质
CN103191791A (zh) * 2013-03-01 2013-07-10 东南大学 生物微粒高通量分选和计数检测的集成芯片系统及应用
CN103203328A (zh) * 2013-03-14 2013-07-17 深圳先进技术研究院 基于结构声场操控和筛选颗粒的系统及方法
WO2014006145A1 (fr) * 2012-07-05 2014-01-09 Universität Leipzig Dispositif et procédé de caractérisation de cellules
CN105214742A (zh) * 2015-10-10 2016-01-06 中国科学院深圳先进技术研究院 基于人工结构声场的微流体系统及操控微粒的方法
CN105242004A (zh) * 2009-04-13 2016-01-13 华盛顿大学 整体决策液份分级

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105242004A (zh) * 2009-04-13 2016-01-13 华盛顿大学 整体决策液份分级
CN102762990A (zh) * 2009-12-04 2012-10-31 生命技术公司 用于声学流式细胞计量术的装置、系统、方法和计算机可读介质
WO2014006145A1 (fr) * 2012-07-05 2014-01-09 Universität Leipzig Dispositif et procédé de caractérisation de cellules
CN103191791A (zh) * 2013-03-01 2013-07-10 东南大学 生物微粒高通量分选和计数检测的集成芯片系统及应用
CN103203328A (zh) * 2013-03-14 2013-07-17 深圳先进技术研究院 基于结构声场操控和筛选颗粒的系统及方法
CN105214742A (zh) * 2015-10-10 2016-01-06 中国科学院深圳先进技术研究院 基于人工结构声场的微流体系统及操控微粒的方法

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