WO2019119368A1 - 一种声驱动的流式细胞检测装置 - Google Patents

一种声驱动的流式细胞检测装置 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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radiation force
cell
particles
cells
acoustically driven
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PCT/CN2017/117833
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French (fr)
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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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    • 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

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  • 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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Abstract

一种声驱动的流式细胞检测装置，包括细胞操控模块（10）、成像模块（20）和图像处理模块（30），细胞操控模块（10）包括流动室和超声辐射力发生系统，超声辐射力发生系统用于对细胞颗粒产生声辐射力，操控细胞颗粒排列成平行线，实现多行聚焦，使细胞颗粒悬浮，同时驱动细胞颗粒定向输运至检测区域实现多通道并行检测。该检测装置利用声辐射力操控细胞颗粒实现输运和聚焦，因此无需复杂的泵系统驱动控制流体，也不用引入鞘液；该检测装置利用声辐射力构成的虚拟通道排列细胞，避免了腔道堵塞的问题；声辐射力可排列多行细胞，因此实现了多行聚焦的多通道并行处理；流动室成本低廉，可每次更换，因此避免了流道内残留物对新样本污染的问题。

Description

一种声驱动的流式细胞检测装置 技术领域
本申请涉及流式细胞检测装置，尤其涉及一种声驱动的流式细胞检测装置。
背景技术
流式细胞仪(Flow cytometer)是对细胞进行自动分析和分选的装置。流动室和液流驱动系统是流式细胞仪的关键部件。流动室由样品管、鞘液管和喷嘴等组成，常用光学玻璃、石英等透明、稳定的材料制作，设计和制作均很精细，是液流系统的核心。样品管贮放样品，单个细胞悬液在液流压力作用下从样品管射出；鞘液由鞘液管从四周流向喷孔，包围1在样品外周后从喷嘴射出。由于鞘液的作用，待检测细胞被限制在液流的轴线上。
因此，当前流式细胞仪主要是利用水流动力来实现细胞颗粒在微流体腔道内的输运和流动聚焦，该技术方案主要存在下述问题：悬浮颗粒易堵塞微腔道，更换腔道成本较高；流动聚焦的实验需要耗费昂贵的鞘液约束细胞排列成单列；需要利用复杂昂贵的微流体泵系统来驱动流体流动；当前流式细胞仪主要为单通道处理方式，而非多通道并行处理方式；不易清洗，流道内的残留样品会对新样品造成污染。
当前流式细胞仪主要是利用水流动力来实现细胞等待检测颗粒在微流体腔道内的输运和流动聚焦。为使流体流动，通常采用空气压缩泵压缩空气为流体流动提供必要的压力，并通过压力调节器稳定液流压力，从而形成定常流动，使得样品颗粒得以匀速流入流动室中。但是这样的泵系统所涉及的气路和控制方法很复杂，所需部件很多，系统组装体积庞大。
流动聚焦是流式细胞仪的关键模块，通常利用鞘液流聚焦的方式，对样品流形成包裹挤压作用，把样品流聚焦在通道中央处，将样品流中的细胞颗粒包夹成线性排列，从而实现细胞颗粒逐个通过检测区。这种方法引进鞘液流增加了整体液体的引入量，提高了整个液流控制系统的复杂度，另外鞘液也是一种昂贵的耗材。
另一种方法无需使用鞘液，通过外界施加的场力或者通道内流体作用力对细胞产生聚焦作用，使细胞一个个通过检测区。专利《一种微流 控芯片及其制备方法、应用》(申请号201611146452.6)和《一种粒子排序方法及其装置和用途》(申请号201710016125.7)利用外加的驻波声场将细胞颗粒排列成一条或者多条线，实现单列或者多列聚焦，从而使细胞一个个通过检测区域。文献中也报道了分别利用流体分离后回流作用力、流体惯性升力和涡流效用作用力、惯性迪恩流升力等流体作用力将细胞颗粒聚焦。尽管该方法不需要引入外加力场，却需要对液体进行精确控制，提高了系统的复杂度。专利公开号US2009/0042241A1的专利中采用了缩口型无鞘液流细胞颗粒聚焦方式，但是其流体通道极易被大颗粒堵塞。另外，这些方法仍然需要复杂的泵系统为液体输运细胞提供动力。
当前多数流式细胞仪是单通道聚焦处理方式，为了提高通量，多通道并行处理方式的流式细胞仪也在专利文献中有了报道。专利《一种微流控芯片及其制备方法、应用》(申请号201611146452.6)利用多个换能器产生的驻波场将细胞排列成多条线，实现了多列聚焦和并行处理。专利《用于在流式细胞仪系统中测量来自多个并行流动通道的多个发射的系统和方法》(申请号201080027480.0)公开了一种多个并行流动通道的流式细胞仪系统，以提高系统每秒检测细胞的数量。
现有技术共有缺点主要有以下三点：
均需使用复杂的泵系统驱动流体，液流驱动系统成本较高；
对颗粒的输运、聚焦和检测均需在单个或者多个微流道内进行，流道内悬浮的细胞颗粒容易堵塞通道，使系统失效，且更换成本较高；
不易清洗，流动室和液路系统中的残留样本会污染新样本。
发明内容
本申请要解决的技术问题是针对现有技术的不足，提供一种声驱动的流式细胞检测装置。
本申请要解决的技术问题通过以下技术方案加以解决：
一种声驱动的流式细胞检测装置，包括细胞操控模块、成像模块和图像处理模块，所述细胞操控模块用于对样本溶液中的细胞颗粒进行操控，所述成像模块用于对染色细胞颗粒进行荧光成像，所述图像处理模块用于处理和分析荧光图像，对细胞颗粒计数和估计细胞颗粒大小，所述细胞操控模块包括流动室和超声辐射力发生系统，所述流动室，用于盛放含有细胞颗粒的样本溶液；所述超声辐射力发生系统用于对细胞颗 粒产生声辐射力，操控细胞颗粒排列成平行线，实现多行聚焦，使细胞颗粒悬浮，同时驱动细胞颗粒定向输运至检测区域实现多通道并行检测。
所述超声辐射力发生系统包括辐射力产生机构、信号发生器和功率放大器，所述信号发生器产生的电信号经所述功率放大器放大后，激励辐射力产生机构产生超声波并进而对颗粒产生辐射力以实现对细胞的输运和操控。
所述流动室包括基底、PDMS侧壁和玻璃顶盖，所述PDMS侧壁分别与所述基底及所述玻璃顶盖键合，所述基底由石英玻璃、有机玻璃或硅制成。
所述辐射力产生机构包括超声换能器和置于流动室内的人工结构，经所述功率放大器放大后的电信号，激励所述超声换能器产生超声波并进而对颗粒产生辐射力。
所述人工结构包括人工周期结构或人工非周期结构。
所述人工周期结构包括基板和设置在所述基板下表面的多个凸条，所述凸条平行设置且间隔相等。
所述超声换能器设置在所述流动室外部，且所述超声换能器与所述人工周期结构的几何中心不重合。
所述辐射力产生机构包括置于流动室外用于合成声场的多个换能
器。
所述多个换能器包括至少一个超声换能器和成对出现且平行设置的叉指换能器，所述叉指换能器用于合成驻波场排列细胞实现聚焦，所述超声换能器用于产生偏置高斯束对细胞进行输运。
由于采用了以上技术方案，使本申请具备的有益效果在于：
在本申请的具体实施方式中，由于包括细胞操控模块、成像模块和图像处理模块，细胞操控模块包括流动室和超声辐射力发生系统，流动室用于盛放含有细胞颗粒的样本溶液；超声辐射力发生系统用于对细胞颗粒产生声辐射力，操控细胞颗粒排列成平行线，实现多行聚焦，使细胞颗粒悬浮，同时驱动细胞颗粒定向输运至检测区域实现多通道并行检测。由于本申请利用声辐射力操控细胞颗粒实现输运和聚焦，因此无需复杂的泵系统驱动控制流体，也不用引入鞘液；由于本申请利用声辐射力构成的虚拟通道排列细胞，因此不需要现有技术用到的微流道，因此避免了腔道堵塞的问题；由于声辐射力可排列多行细胞，因此实现了多行聚焦的多通道并行处理；由于本申请中的细胞操控模块中的流动室等 部件成本低廉、工艺简单，每次测量新样本时都可以更换为新器件，因此避免了现有技术中流道内残留物对新样本污染的问题。综上，本申请提供了一种无微流腔道、无微流泵、无鞘液、可丢弃、可并行处理、低廉的流式细胞检测方案。
附图说明
图1为本申请的装置在一种实施方式中的功能模块示意图；
图2为本申请的人工周期结构在一种实施方式中的结构示意图；
图3为图2所示人工周期结构的透射谱；
图4为人工周期结构调制声场沿x方向的分布；
图5为细胞在人工结构声场中受到x，y，z三个方向的声辐射力；
图6为yz平面内人工周期结构共振时的声压场分布及辐射力分量Fy和Fz的方向；
图7为yz平面内人工周期结构共振时的辐射力分量Fy和Fz沿y方向的分布；
图8为yz平面内人工周期结构非共振时的声压场分布及辐射力分量Fy和Fz的方向；
图9为yz平面内人工周期结构非共振时的声压场分布及辐射力分量Fy和Fz沿y方向的分布；
图10为声辐射力分量Fx和Fz沿输运方向x的分布；
图11为利用本申请的声驱动的流式细胞检测装置采集的图像；
图12为本申请的辐射力产生机构在一种实施方式中的结构示意图。
具体实施方式
下面通过具体实施方式结合附图对本申请作进一步详细说明。
实施例一：
如图1所示，本申请的声驱动的流式细胞检测装置，其一种实施方式，包括细胞操控模块10、成像模块20和图像处理模块30。细胞操控模块10用于对样本溶液中的细胞颗粒进行操控，成像模块20用于对染色细胞颗粒进行荧光成像，图像处理模块30用于处理和分析荧光图像，对细胞颗粒计数和估计细胞颗粒大小。细胞操控模块10可以包括流动室和超声辐射力发生系统，流动室可以是一个微腔，用于盛放含有细胞颗粒的样本溶液。超声辐射力发生系统，用于对细胞颗粒产生声辐射力， 操控细胞颗粒排列成平行线，实现多行聚焦，使细胞颗粒悬浮，同时驱动细胞颗粒定向输运至检测区域实现多通道并行检测。
成像模块20可以包括荧光激发源、光学透镜、高灵敏荧光相机。图像处理模块30可以包括计算机、高速数据采集卡、硬件控制软件和图像采集分析处理软件，图像处理模块用于处理和分析荧光图像，对细胞计数和估计细胞大小。主要利用超声辐射力发生系统产生的辐射力实现细胞的聚焦和输运。
在一种实施方式中，超声辐射力发生系统包括辐射力产生机构、信号发生器和功率放大器。信号发生器产生的电信号经功率放大器放大后，激励辐射力产生机构产生超生波，并进而对颗粒产生辐射力以实现对细胞的输运和操控。
本申请的流动室，可以包括基底、PDMS侧壁和玻璃顶盖，PDMS侧壁与基底键合，同时PDMS侧壁也与玻璃顶盖键合。在一种实施方式中，基底可由石英玻璃、有机玻璃或硅制成。
在一种实施方式中，辐射力产生机构可以包括超声换能器和人工结构。超声换能器置于流动室外，人工结构置于流动室内，经功率放大器放大后的电信号，激励超声换能器产生超声波并进而对颗粒产生辐射力。人工结构具体可以是人工周期结构或是人工非周期结构。如图2所示，人工周期结构包括基板11和设置在基板11下表面的多个凸条12，凸条12平行设置且间隔相等。图2为声辐射力发生系统中所采用的一种人工周期结构的示意图。t为板厚，p为结构的周期，w为栅格的宽度，h为栅格的高度。材料为不锈钢，参数为t＝20-100μm，h＝10-50μm,w＝20-100μm,p＝50-300μm。图3为图2所示人工周期结构的透射谱，其共振频率为5.94MHz。
本申请的超声辐射力发生系统，其中，超声换能器设置在流动室外部，超声换能器与人工周期结构的几何中心不重合。在一种实施方式中，超声换能器可以粘贴在流动室外部，具体可以粘贴在基底的下表面。超声换能器可以是偏置的高斯束声源。人工结构调制超声换能器发射声场产生了具有输运和排列等操控功能的声场，从而实现细胞的聚焦和输运。
数值仿真研究了细胞在人工周期结构调制高斯声束得到的声场中所受声辐射力，揭示了人工结构声场定向输运微纳颗粒的机制。图4为人工周期结构调制声场沿x方向的分布，可以看出该声压分布服从高斯分布，其中由虚线部分为理论计算值，实线部分为实验测量值。如图5所 示，细胞在人工结构声场中受到x,y,z三个方向的声辐射力，其中x方向声辐射力Fx引起微纳颗粒朝声场最强方向的定向运动，实现输运；y方向的声辐射力Fy引起微纳颗粒的排列，实现聚焦，其对颗粒的禁闭作用，限制了微纳颗粒的侧向运动区间，构成了虚拟微腔道；z方向的声辐射力Fz负责微纳颗粒的悬浮和捕获。这三个方向声辐射力的联合作用最终导致了细胞的输运和聚焦。
图6为yz平面内人工周期结构共振时(共振频率为5.94MHz)的声压场分布及辐射力分量Fy和Fz的方向。图7为yz平面内人工周期结构共振时(共振频率为5.94MHz)的辐射力分量Fy和Fz沿y方向的分布。可以看出在圆圈所示平衡位置，辐射力分量Fz为负值，即竖直向下，因此会将细胞稳定捕获在结构表面。
图8为数值仿真计算得到的非共振时的声压场(驱动频率为5.92MHz)及辐射力分量Fy和Fz的方向。图9为数值仿真计算得到的非共振时的声压场(驱动频率为5.92MHz)及辐射力分量Fy和Fz沿y方向的空间分布。图8为yz平面内声场分布及受力示意图，可以看到非共振时的声场形态与图6共振时的声压场分布有很大的不同。圆圈所示位置为颗粒在声场中的平衡位置。在该位置，z方向，竖直向上的辐射力分量Fz刚好抵消重力作用，使颗粒悬浮；y方向，在该位置y方向声辐射力分量Fy＝0，而偏离该位置Fy不为零，方向是指向该位置的，因此该位置是颗粒在y方向的平衡位置，并沿输运方向一个个排列，从而实现聚焦。
图10为数值仿真计算得到的非共振时(驱动频率5.92MHz)辐射力分量Fx和Fz沿输运方向x的空间分布。x＝0的位置为声源的中心位置，该处声压最大。可以看出，辐射力分量Fz始终是正值，意味着该力的方向与重力方向相反，因而可以使颗粒悬浮。辐射力分量Fx在声源中心(x＝0)处为零，在偏离声源中心的位置则始终为正值。这意味着辐射力分量Fx方向是指向声源中心的，因此辐射力分量Fx会驱动颗粒朝声源定向输运。
由于本申请利用声辐射力操控细胞颗粒实现输运和聚焦，因此无需复杂的泵系统驱动控制流体，也不用引入鞘液；由于本申请利用声辐射力构成的虚拟通道排列细胞，因此不需要现有技术用到的微流道，因此避免了腔道堵塞的问题；由于声辐射力可排列多行细胞，因此实现了多行聚焦的多通道并行处理；由于本申请中的细胞操控模块中的流动室等 部件成本低廉、工艺简单，每次测量新样本时都可以更换为新器件，因此避免了现有技术中流道内残留物对新样本污染的问题。综上，本申请提供了一种无微流腔道、无微流泵、无鞘液、可丢弃、可并行处理、低廉的流式细胞检测方案。
实施例二：
实施例二为本申请的声驱动的流式细胞检测装置的具体应用例。采用C304不锈钢，基于标准化学刻蚀工艺，制备了图2所示的人工周期结构，其参数为t＝30μm，h＝20μm，w＝50μm，p＝200μm。流动室由石英玻璃基底、PDMS(聚二甲基硅氧烷)壁和玻璃顶盖构成。PDMS壁可与基底和顶盖键合。超声换能器为中心频率6MHz的PZT4压电陶瓷片，并通过环氧树脂与玻璃基底粘接在一起。超声换能器的几何中心与人工周期结构的几何中心不重合，即超声换能器需偏置放置，其目的是产生偏置的声源，使得偏离声源中心的细胞颗粒受到如图10所示的辐射力Fx的作用，从而向声源发生定向输运。控制软件控制信号发生器(AFG3102,Tektronix,Beaverton,OR,USA)产生频率5.90-5.94MHz的Chirp信号，并经由功率放大器(150A100B,Amplifier Research,Souderton,PA,USA)放大后，激励压电陶瓷片PZT4产生超声波。超声波激励人工周期结构在其表面产生图8所示局域场，并对细胞颗粒产生图9-10所示声辐射力。细胞输运至检测区域时，荧光激发光源为100W的高压汞灯激发染色细胞发出荧光，高灵敏荧光相机(QIMAGING optiMOS)记录荧光图像，并将数据传输至计算机。图像分析处理软件提取图像的荧光强度分布，并进一步计算细胞的大小和数量。
图11为利用声驱动的流式细胞检测系统采集的图像。实验所用细胞为直径15μm的MCF-7肿瘤细胞。细胞经钙黄绿素荧光染料染色后注入流动室。超声辐射力发生系统产生的辐射力对这些细胞进行排列、并将这些细胞输运至检测区域。当这些细胞到达检测区域时，荧光激发光源激发染色细胞发出绿色荧光，荧光图像由高灵敏荧光相机记录，经由高速采集卡传入计算机。计算机的图像分析处理软件自动提取荧光强度曲线，并根据峰值大小和峰的数量分别计算细胞的尺寸和细胞个数。图11提取了虚线框中单列细胞的荧光强度曲线。
实施例三：
实施例三与实施例一的区别在于辐射力产生机构的结构不同。如图12所示，辐射力产生机构可以包括多个换能器，多个换能器置于流动室外且用于合成声场。
在一种实施方式中，多个换能器包括至少一个超声换能器和成对出现且平行设置的叉指换能器，叉指换能器用于合成驻波场排列细胞实现聚焦，超声换能器用于产生偏置高斯束对细胞进行输运。叉指换能器可以有一对，也可以有多队，图12中包括一对叉指换能器，即第一叉指换能器13和第二叉指换能器14，其中超声换能器15选用压电换能器PZT，40为细胞。叉指换能器1和叉指换能器2用于合成驻波场排列细胞实现聚焦，压电换能器PZT用于产生偏置高斯束对细胞进行输运。
超声辐射力发生系统可以包括多个超声换能器、信号发生器和功率放大器组成。一部分超声换能器用于操控细胞排列聚焦；另一部分换能器用作偏置的高斯束声源驱动颗粒输运。多个超声换能器发射声波合成的声场产生了具有输运和排列等操控功能的声场，从而实现细胞的聚焦和输运。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换。

Claims (10)

  1. 一种声驱动的流式细胞检测装置，包括细胞操控模块、成像模块和图像处理模块，所述细胞操控模块用于对样本溶液中的细胞颗粒进行操控，所述成像模块用于对染色细胞颗粒进行荧光成像，所述图像处理模块用于处理和分析荧光图像，对细胞颗粒计数和估计细胞颗粒大小，其特征在于，所述细胞操控模块包括流动室和超声辐射力发生系统，所述流动室，用于盛放含有细胞颗粒的样本溶液；所述超声辐射力发生系统用于对细胞颗粒产生声辐射力，操控细胞颗粒排列成平行线，实现多行聚焦，使细胞颗粒悬浮，同时驱动细胞颗粒定向输运至检测区域实现多通道并行检测。
  2. 如权利要求1所述的声驱动的流式细胞检测装置，其特征在于，所述超声辐射力发生系统包括辐射力产生机构、信号发生器和功率放大器，所述信号发生器产生的电信号经所述功率放大器放大后，激励辐射力产生机构产生超声波并进而对颗粒产生辐射力以实现对细胞的输运和操控。
  3. 如权利要求2所述的声驱动的流式细胞检测装置，其特征在于，所述流动室包括基底、PDMS侧壁和玻璃顶盖，所述PDMS侧壁分别与所述基底及所述玻璃顶盖键合，所述基底由石英玻璃、有机玻璃或硅制成。
  4. 如权利要求3所述的声驱动的流式细胞检测装置，其特征在于，所述辐射力产生机构包括超声换能器和置于流动室内的人工结构，经所述功率放大器放大后的电信号，激励所述超声换能器产生超声波并进而对颗粒产生辐射力。
  5. 如权利要求4所述的声驱动的流式细胞检测装置，其特征在于，所述人工结构包括人工周期结构或人工非周期结构。
  6. 如权利要求5所述的声驱动的流式细胞检测装置，其特征在于，所述人工周期结构包括基板和设置在所述基板下表面的多个凸条，所述凸条平行设置且间隔相等。
  7. 如权利要求4所述的声驱动的流式细胞检测装置，其特征在于，所述人工结构采用不锈钢材料制成。
  8. 如权利要求6所述的声驱动的流式细胞检测装置，其特征在于，所述超声换能器设置在所述流动室外部，且所述超声换能器与所述 人工周期结构的几何中心不重合。
  9. 如权利要求3所述的声驱动的流式细胞检测装置，其特征在于，所述辐射力产生机构包括置于流动室外用于合成声场的多个换能器。
  10. 如权利要求9所述的声驱动的流式细胞检测装置，其特征在于，所述多个换能器包括至少一个超声换能器和成对出现且平行设置的叉指换能器，所述叉指换能器用于合成驻波场排列细胞实现聚焦，所述超声换能器用于产生偏置高斯束对细胞进行输运。
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