TWI252312B - Realtime microparticle sorting/manipulation system - Google Patents

Abstract

This invention proposes a novel microfluidic system for realtime cell/microparticle sorting and counting utilizing imaging controlled optical tweezer. The system is composed of a substrate, a power supply, a digital image identification program and an optical tweezer device. Digital image processing technique is used to detect and recognize the size and the number of the cells/particles. During operation, microparticles are electrokinetically focused to flow in the center of the channel. The focused particles then flow pass through an image recognition area. An integrated optical tweezer system is used to switch the flow direction of the target particles at the downstream. Particle detection and sorting can be achieved using this invention. The proposed system is capable of catching, sorting and counting specific microparticles/cells within a mixed sample and results in a simple solution for cell/microparticle manipulation in the field of micro-total-analysis-systems.

Description

1252312 IX. Description of the invention: [Technical field of the invention] The present invention relates to an instant particle classification and/or operating system, in particular to a non-contact and instant combination of digital image processing technology and optical clamp operation. Classification of particles and / or operating system. [Prior Art] In the process of biochemical reaction, it is necessary to control the biological sample in the micro-channel by some external force, so that it can be sampled in a micro-scale space for transmission, mixing, micro-valve switch or Classification and other work. At present, in most researches on cell analysis, it is necessary to separate and count specific cells or biomolecules constituting cells. Traditionally, centrifugation has been used to separate cell samples. This method is not only time-consuming and difficult to separate cells of similar volume, and the amount of samples required to separate cells by centrifugation is extremely large, which does not meet the development direction of future biochemical tests. The more advanced cell classification method uses flow cytometry. Although the system can automatically count cells, its system is huge and expensive, and the accuracy of using it to classify fine cells needs to be improved. [MR Melamed, T. Lindm . And M. L. Mendelsohn, (editors), Flow Cytometry and Sorting, 2nd. Edition, John Wiley & Sons (1991), and U.S. Patent No. 5,824,269. In addition, in order to classify cells by flow cytometry, different cells must be dyed before the operation through complex pre-processing procedures to meet the needs of the internal fluorescence excitation of the machine, M[j Krtiger, K· Smgh, A. O' Neill, c· Jackson, A· Morrison Ρ· 〇, Brien, "Development of a microfluidic device for fluorescence 98154.doc 1252312 activated cell sorting," J. Micromech. Microeng·, Vol. 12, pp. 486-494 (2002) and [Katsuragi, T. and Tani, Y·, ''Review:

Screening for Microorganism with Specific Characteristics by Flow Cytometry and Single-Cell Sorting/1 Journal of Bioscience and Bioengineering, Vol. 89, No. 3, pp. 217-222 (2000)], which is not only time consuming, but also Light dyes may be cytotoxic and impair cell activity.

With the development of MEMS technology, so far many scholars have proposed a variety of methods to classify and count cells and biomolecules. In terms of classification: some scholars use the design of tiny electrode groups in the micro-pipeline to reduce the distance between the electrodes, which can generate a non-uniform electric field at high frequency and low voltage, and use the cells to induce different dielectrophoresis in the electric field. Effects, and the principle of adsorption or repulsion to classify cells, such as [Pethig, R·, ” Dielectrophoresis: Using Inhomogeneous AC Electrical Fields to Separate and Manipulate Cells, M Crit· Rev· 扪okc/z (4)/·, 16, pp. 33 1-348 (1996)]. Or make some microstructures in the micro-pipeline with piezoelectric materials, using the effects of ultrasound to manipulate the particles, such as [Dougherty, G. M· and Pisano, Α· P·, “Ultrasonic Particle Manipulation in Microchannels Using Phased Co-planar Transducers,'' IEEE Proc·solid-state sensors and actuators workshop, Vol.l, pp. 670-673 (2003)] and [Jagannathan, H., Yaralioglu , G. G·, Ergun, AS? Khuri-

Yakub, B. T·,, fAn Implementation of a Microfluidic Mixer and Switch Using Micromachined Acoustic Transducers, M 98154.doc 1252312 Some scholars have begun to use the characteristics of Optical Tweezer to make non-contact cells in microchannels. For the purpose of cell sorting, the basic principle of the optical clamp is to focus a laser beam through a high numerical aperture (NA) objective lens to form a stable position at the focus of the laser focus. Energy trap. When the tiny particles flow around the focal point, the non-mechanical contact light gradient force attracts the tiny particles to the center of the focus, thereby achieving the purpose of three-dimensional capture and manipulation. Since the size of the microcapsules that can be captured by the optical forceps is about the order of nm~μιη, which coincides with the scale of biological micro-objects, it is often used in biological research, such as [Jonas Enger, Mattias Goks6r, Kerstin Ramser , Petter Hagberg and Dag Hanstorp, "Optical tweezers applied to a microfluidic system," Lab on a Chip, Vol. 4, pp. 196_200, 2004], U.S. Patents 6,734,436, 6,744,038 and 6,797,942, and the Republic of China Patent Publication No. 593683, etc., but the efficiency of its manipulation method is rather low. In terms of identification and counting, some scholars use the micro-pipes carved out on the wafer to extend the fiber into its pipeline for transmission and reception. Laser light components, in order to detect biological samples, the process of making the wafer is also quite complicated and can not meet the needs of mass production 'such as [Lin, CH, Lee, G. Β · ''Micromachined Flow Cytometers With Embedded Etched Optic Fibers for Optical Detection, 11 J. M/cromecA. Vol. 13, pp. 447-453 (2003)] and the Republic of China Patent Disclosure Rev. 164476. There are also scholars who use expensive plating 98154.doc 1252312

The film process 'produces electrodes on the wafer, and when the particles flow, the counting and identification are generated by the impedance change signal, such as [S(m, Sanguk, Ch〇i, γ. I ee' S· S·, Fabrication of Micro cell counter with boron diffused resistor, ^ pr〇c, IEEE MEMS, pp. 311-314 (2003)], but its process is complicated and time consuming, so it is not suitable for making disposable wafers. Part of the design needs to be provided - complex wafer structures 'hard to integrate into a reliable microfluidic system, and the efficiency of cell separation is quite low. In addition, part of the design process is very expensive, not suitable: a large number of Disposable wafers. Therefore, it is necessary to provide an innovative and progressive classification and/or operating system of real-time particles to solve the above problems. [Summary of the Invention] The main purpose of the present invention is to provide a kind of Instant particle classification and/or operating system, which can be realized by using a 日 铕 w w w w w w w 早 早 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , The other purpose of this lx is to provide a sort of real-time particle classification and/or operation system, which can be used for real-time 2 Μ \ , ^ 士子子 / knife or operation, which can shorten the classification or operation, improve The classification or operation is effective and the force is applied to the particle to change the manipulation position in real time. Another object of the present invention is to say that _ 么 狄 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 楗仏 即时 即时 即时 即时 即时 即时 即时 即时 即时 即时 即时 即时 即时 即时σ丁+, ^ .. , ^ , 7 mouth is not complicated before the treatment, the operation procedure and the tumor, and it is non-m 4 + Im uf-type action, can reduce the biochemical reaction

The resulting interference and errors. U 98154.doc 1252312 For purposes of the present invention, the present invention provides an instant particle classification and/or operation system, pure, including: - a substrate, a power supply, a digital image processing device, and an optical clamp device. The substrate has a microfluidic channel for supplying a fluid therein. The fluid has a plurality of particles therein, and the microchannel has a recognizable region. The power supply is electrically connected to the substrate ' to provide the power required to drive the fluid. The digital image processing device is configured to instantly identify particles flowing through the identification region in the microchannel.

Health - identification results. The optical caliper device is actuated based on the identification result to process the identified particles. [Embodiment] A schematic diagram showing a preferred embodiment of the classification and/or operating system of the instant particles of the present invention is shown with reference to FIG. It is to be noted that the particles to which the present invention is applied include, but are not limited to, cells, bacteria, fine particles, blood cells, and the like. The instant particle classification and/or operating system i includes a substrate 10, a power supply 20, a digital image processing device 3A, and an optical clamp device. Referring also to Figure 2, there is shown a top plan view of the substrate 10 in the classification and/or operation system of the instant particles of the present invention. The substrate 1 (e.g., a wafer) has a microchannel that is used to supply a fluid therein. In this embodiment, the microchannel comprises a main flow channel, the same feed channel 12, a first buffer flow channel 133, a first buffer feed channel 32, and a second buffer. The flow channel 133, a second buffer feed tank 134, a first splitter channel 141, a first storage tank 142, a second branch channel 143 and a second storage tank 144. One end of the main flow channel is connected to the sample feeding trough 12, and the other end is connected to the 98154.doc -9-1252312 first knives 141 and the second shunt 143, and is connected at about its middle portion. The first buffer flow channel 133 and the second buffer flow channel 13 3 . The main flow path 11 has an identification area Π 1 and a focus area 112. In this embodiment, the length of the main flow channel 11 is 2.5 cm, and the lengths of the first shunt channel 141, the second shunt channel 143, the first buffer flow channel 131, and the second buffer flow channel 133 are both For 〇5cm, all runners have a width of i〇〇^m and a depth of 40μηι. The sample feed tank 12 is configured to inject a fluid into the main flow channel, and the flow direction of the flow is from the left side in the figure to the right side in the figure, and the fluid has a plurality of particles in the present embodiment. It is a 1 mM side sodium solution (Na2B4 〇 7.l 〇 H2 〇) (pH = 9.2) and contains a concentration of a1〇-3Mi Rhodamine B dye. The fluid is mixed with a plurality of particles, which are polymer beads of polystyrene (p〇lyStyrene, ps), have a refractive index of l58, and have a density of 1.05 g/cm3. It is equivalent to cells, so it is suitable for replacing biological samples for testing. Wherein "□" represents the first particle 5 1 (diameter of 1 〇 array),,, 〇, and represents the second ruthenium particle 52 (diameter 12 μιη). The two ends of the first buffer flow channel 13 1 are respectively connected to the main flow channel 丨i and the first buffer liquid feeding groove 132 for injecting a first buffer liquid into the main flow channel 11. The two ends of the second buffer flow path 133 are respectively connected to the main flow channel 11 and the second buffer liquid feed channel 13 4 for injecting a second buffer to the main flow channel 11. Due to the injection of the first buffer and the second buffer, the flow of the fluid can be compressed to a width of a single particle, so that the particles can be sequentially flowed in the fluid, and there is no 98154 at the same time. .doc -10- 1252312 Two or more particles pass through the identification zone ,1, causing the optical clamp device 4 to mishandle. In this embodiment, the first buffer and the second buffer are the same, and each is a 1 mM sodium borate solution. In this embodiment, the first buffer flow channel 131 and the second buffer flow channel 133 are perpendicular to the main flow channel 11, respectively. However, in other applications, the first buffer flow channel 13 1 and the first The two buffer flow channels 13 3 may be obliquely intersected with the main flow path 11, respectively. The two ends of the first branching channel 141 are respectively connected to the main flow channel 11 and the first storage channel 142 for introducing the classified first particles 51 into the first storage slot 142. The two ends of the second branching channel 143 are respectively connected to the main flow channel n and the second storage groove 144 for introducing the classified second particles 52 into the second storage tank 144. In this embodiment, the first shunt 141 and the second shunt 143 are respectively oblique to the main channel u. However, in other applications, the first shunt 141 and the second shunt 143 may respectively It is perpendicular to the main channel 11.

The power supply 20 is electrically connected to the sample feeding tank 12, the first buffer feeding tank 32, the second buffer feeding tank 134, the first storage tank 142 and the second storage tank, respectively. 144. The power supply is coupled to a high voltage power supply to provide the power required to drive the fluid on the substrate 1 to drive the fluids by electroosmotic flow drive. Referring to FIG. 1 ', the digital image processing device 30 is configured to instantly recognize particles flowing through the identification region (1) in the main flow channel U to generate a recognition result. The digital image processing apparatus includes a first light source, a first lens 32, a beam splitter 33, a mirror 34, a first objective lens, a (10) camera 36, and a processing single opening 17, " Take ^ (example 'one computer'). The first light source 3 is configured to generate a first light beam, and is projected to the substrate 10 via the first lens 32, the beam splitter 33, the data mirror 34, and the first objective lens 35, the cCD camera The image is taken near the recognition area 111 of the substrate 10, and the image is transmitted to the processing unit 37. In the present embodiment, the first light source 31 is an incoherent light source. The first objective lens 35 is 20 times multiple and the NA value is 0.4. in

Figure. The processing unit 37 has an image processing software, and the image processing software includes a user interface 38, and the user interface 38 includes a classifier 381, a counter 382, and a display module 383. Referring to FIG. 3, there is shown a schematic diagram of a user interface of the instant particle classification and/or real-time image recognition in the operating system processing unit of the present invention for designating the particle type to be classified according to the particle size and The difference in color is used for identification. This counter 382 is the result of the calculation. The display module 383 displays an instant image near the identification area U1, and the size of the identification area 111 is adjustable. Digital image processing technology used in the present invention

The technique can effectively separate the dynamic object from the static background, and does not need to perform any pre-processing on the image during the analysis process, that is, it has the function of instant recognition. After the processing unit 37 recognizes, an identification result is generated, and according to the identification result, the identified particle is processed by whether the shirt drives the light sweet device 40. Referring again to FIG. 1, the optical clamp device 40 includes a second light source 41, a second lens 42, an electronic shutter (Shutte (10), a mirror 44, and a second objective lens 45. The second light source 41 is a laser. a light source for generating a laser beam' which can be a semiconductor laser (SDmL2jDS Uniphase, 98154.doc -12-1252312 USA), a mode-locked titanium sapphire () laser (Mira9〇〇, Coherent, USA), intracavity The frequency doubled semiconductor excites a solid state laser (VenH-lOW, Coherent, USA) or a gas laser. The laser beam passes through the second lens 42, the electronic shutter (Shutter) 43, the mirror 44, and the second objective lens 45, focusing to the focus area 112 of the substrate 1 (Fig. 2). In the present embodiment, the second objective lens 45 is multiplied by 1〇〇, and the value is 〇·8. The child's shutter 43 can be interrupted. The laser beam is used to control the time point at which the laser beam is turned on, and the electronic shutter 43 is electrically connected to the processing unit 37 and controlled by the processing unit 37 to determine the time of the laser conduction. Processing the identified particles at point '. Again, the operation of this embodiment is as follows. First, a fluid is provided in the sample feed tank 12, the fluid includes a plurality of first particles 51 and a plurality of first particles 52. Next, the first buffer and the second buffer are provided for the fluid The flow is compressed to a width of a single particle such that the 4th particle 5 1 , 52 can be sequentially flowed through the identification zone 111 in the fluid. If the digital image processing device 3 〇 recognizes the passage through the identification zone 4 The particle of 111 is the first particle 5丨, and the processing unit 37 causes the optical clamp device 40 to actuate, that is, the laser is focused on the focal region 112. Then, when the first particle 51 is close to the When the focus area is in focus, it is then horizontally attracted by a light gradient force, and is shifted upwards in the figure. At this time, the optical clamp device 40 is immediately turned off (the processing unit 37 controls the electronic shutter 43 to block the conduction of the laser. ), since the flow of the fluid exhibits a "laminar flow," the sheet ~: mouth, a particle 5 1 will continue to flow along the upper side of the main flow path 11, and finally enter the first branch flow path 141 and the first A storage tank 142. 98154.doc 13 1252312 The digital image processing device 3 recognizes that the particles passing through the identification area 111 are the second particles 52, and the processing unit 37 causes the light sweet device to be disabled, that is, the processing unit 37 controls the electronic shutter. 43. The second particle 52 is continuously flowed along the lower side of the main flow path 11 without affecting, and finally enters the second distribution channel 143 and the second storage tank 144. The second storage tank 142 is the second particle w in the first storage tank 142, and the classification of the particles is completed. The above is the case of particle classification, however, in other applications, the power of the laser can be enhanced to directly A-specific particles (e.g., the first particle $1), or bombardment into two halves. This can be used to destroy or delete harmful cells. 4a to 4f, there are shown schematic views of a preferred embodiment of the process of substrate 10 for the classification and/or operation of the present invention. First, a first substrate 60 is provided. In the present embodiment, the first substrate 60 is a glass slide used in a microscope. However, in other applications, the material of the first substrate 6 can be used. Molecular material. The first substrate 6 was annealed by 4 〇〇〇c, and after releasing the & residual stress, it was washed with a boiling solution (2S04(/^) · h202(%) = 3:1) for ten minutes. Then, a thickness of about 3 μηι 2 AZ4620 positive photoresist film 7 is applied on the surface of the first substrate 6〇 as an etching mask for etching the first substrate 60 in a buffered oxide etchant (BOE). Curtain, then use the tuned mask 8 to perform standard lithography procedures, as shown in Figure 4a. Thereafter, 1M hydrochloric acid (HC1) is used to remove the 98154.doc 14 1252312 swell generated during the etching process, and the surface of the substrate can be 40 μm deep on the surface of the substrate 12 over a period of 45 minutes. (including the mainstream, the younger-buffer flow channel 133, the first channel 11, the sample feed tank buffer feed tank 132, the second buffer flow channel 133 flow channel 141, the first storage The groove 142 and the storage tank 144)' are as shown in Fig. 4b. The second buffer feed slot 134, the first split second split runner 143, and the second

The photoresist film 7 is subsequently removed using a hydroxide desorption (KQH) solution, as shown in Figure 4c. Then, the sample feed tank 12, the first buffer feed tank 132, the second buffer feed tank 134, the first storage tank 142 and the The second storage tank 144 respectively drills a liquid inlet and outlet hole as shown in Fig. 4d. Then, the first substrate 60 is inverted, and a second substrate 9 is para-bonded to the upper surface of the first substrate 60. In the embodiment, the second substrate 9 is a thickness of A 170 μηη coverslip was spun and spun in a 58 〇 γ high temperature furnace for ten minutes, as shown in Figure 4e. Finally, the substrate 1 is obtained, as shown in FIG. The above embodiments are merely illustrative of the principles and functions of the present invention, and are not intended to limit the scope of the present invention. The scope of the invention should be as set forth in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a preferred embodiment of a particle and/or an operating system of the present invention; FIG. 2 is a schematic plan view showing a substrate of the present invention; 98154.doc -15- 1252312 A schematic diagram of a graphical user interface for instant image recognition in a processing unit of the present invention; and Figures 4a through 5 are schematic diagrams showing a preferred embodiment of the process of the substrate of the present invention. [Main component symbol description]

1 Classification and/or operation of the instant particles of the present invention 10 Substrate 11 Main channel 12 Sample feed slot 20 Power supply 30 Digital image processing device 31 First light source 32 First lens 33 Beam splitter 34 Filter 35 First objective lens 36 CCD camera 37 processing unit 38 graphic user interface 40 optical clamp device 41 second light source 42 brother 'one lens 43 electronic shutter 44 mirror 98154.doc -16- 1252312

45 51 52 60 70 80 90 111 112 131 132 133 134 141 142 143 144 381 382 383 Second objective first particle second particle first substrate positive photoresist film mask second substrate identification area focus area first buffer Flow channel first buffer feed tank second buffer flow channel second buffer feed tank first split channel first storage tank second split runner second storage tank sorter counter display module 98154.doc -17-

Claims (1)

1252313⁄4 101851 Patent Application Text Applicable Patent Renewal This month}Zhouzhou 10, Patent Application Range··1. Two kinds of instant particle classification and/or operating system, including: -Base: with a micro flow channel 'The microchannel is used to supply a fluid to the fluid and a plurality of particles in the fluid, the microchannel has an identification zone; - a power supply is electrically connected to the substrate to provide driving The power required by the fluid; a digital image processing device that instantly recognizes particles flowing through the identification zone in the microchannel to generate an identification result; and an optical clamp device that operates according to the identification result for processing The identified particles. 2. System 3 as Request 1 • System 4 as Request 1 • System as Request 1 • System 6 as Request 1 • System 7 as Request 1 • System 8 as Request 1 The system of claim 1 wherein the particles are cells. Wherein the particles are bacteria. Wherein the particles are blood cells. The material of the substrate is glass. The material of the substrate is 矽. The material of the substrate is a polymer. The micro flow channel includes: a main flow channel for the fluid to flow therein, having the identification region; a first buffer flow channel connected to the main flow channel for injecting a first buffer liquid to the main flow channel; a second buffer flow channel is connected to the main flow channel for injecting a second buffer solution into the main flow channel, and the first buffer liquid and the second buffer liquid are injected to compress the flow of the fluid to a single particle Width, such that 98154-941130.doc 1252312 the particles can be sequentially flowed in the identification zone; the first flow channel is connected to the main channel for deriving the plurality of first particles after classification; and the first runner And connecting the main channel to derive a plurality of second particles after classification. ^ θ The system of claim 8, wherein the first buffer flow path and the second buffer flow path are perpendicular to the main flow path, respectively. 10. The system of claim 8, wherein the first buffer flow path and the second buffer flow path are respectively oblique to the main flow path. U. The system according to claim 8, wherein the first branch channel and the second branch channel are respectively perpendicular to the main channel. 12. The system of claim 8, wherein the first shunt and the second shunt are respectively oblique to the main flow. The system of claim 1, wherein the digital image processing device comprises: a first light source for generating a first light beam and projected onto the identification area on the substrate; a CCD camera for capturing the An image near the substrate identification area; and a processing unit for receiving the image transmitted by the CCD camera, and processing the image to generate the identification result. 14. The system of claim 13, wherein the processing unit has a graphical user interface. The graphical user interface includes: a classifier for setting a particle type to be classified; a counter for calculating The result is a display module that displays an instant image near the identification area, and the size of the identification area 98154-941130.doc 1252312 can be adjusted. The system of claim 1, wherein the optical clamp device comprises: a first light source for generating a laser beam and focusing to a focal region on the substrate; and an electric: Shaanxi gate, which is occludable The laser beam is used to control the laser light: a pass between the point and the electronic shutter is electrically connected to the digital 16. 17. < is flattened and controlled by the digital image processing device to determine the point in time at which the laser beam is turned on. Such as the priming of claim 1 5, the jewel laser, the particle of claim 1. The laser beam is selected from the group consisting of semiconductor lightning solid lasers and gas lasers. The optical clamp device is attracted to the identified system 18. The system particles of claim 1. Wherein the optical clamp device destroys the identified 98154-941130.doc