CN113967487B - A nozzle, droplet photothermal control system and its application - Google Patents
A nozzle, droplet photothermal control system and its applicationDownload PDFInfo
- Publication number
- CN113967487B CN113967487BCN202111180616.8ACN202111180616ACN113967487BCN 113967487 BCN113967487 BCN 113967487BCN 202111180616 ACN202111180616 ACN 202111180616ACN 113967487 BCN113967487 BCN 113967487B
- Authority
- CN
- China
- Prior art keywords
- outlet
- inlet
- nozzle
- reversing valve
- reagent
- 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.)
- Active
Links
Images
Classifications
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B1/00—Nozzles, spray heads or other outlets, with or without auxiliary devices such as valves, heating means
- B05B1/14—Nozzles, spray heads or other outlets, with or without auxiliary devices such as valves, heating means with multiple outlet openings; with strainers in or outside the outlet opening
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B1/00—Nozzles, spray heads or other outlets, with or without auxiliary devices such as valves, heating means
- B05B1/24—Nozzles, spray heads or other outlets, with or without auxiliary devices such as valves, heating means incorporating means for heating the liquid or other fluent material, e.g. electrically
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/02—Drop detachment mechanisms of single droplets from nozzles or pins
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Translated fromChinese技术领域technical field
本发明属于核酸检测相关技术领域，更具体地，涉及一种喷嘴、液滴光热操控系统及其应用。The invention belongs to the technical field related to nucleic acid detection, and more specifically relates to a nozzle, a liquid droplet photothermal manipulation system and applications thereof.
背景技术Background technique
核酸检测具有高灵敏度和高有效性，是使用最为广泛的病原体感染检测手段。Nucleic acid detection has high sensitivity and high effectiveness, and is the most widely used means of pathogen infection detection.
BEAMing(beads,emulsion,amplification,and magnetics)是一项将数字PCR技术和液滴技术相结合的核酸检测技术，由Devin Dressman等人在2003年提出。该技术通过向PCR反应试剂中加入油和乳化剂，形成以反应试剂为分散性相的乳状液，每个液滴内含有至多1条DNA分子和1个磁珠。然后，对乳状液进行温度循环，使得每个同时含有DNA分子和磁珠的液滴内的DNA分子单独扩增并吸附于磁珠上(乳化液扩增)。最后，去除体系中的油并加入荧光探针，并使用流式细胞仪对具有荧光效应的磁珠进行计数得到不同种类初始DNA的绝对浓度和浓度比。BEAMing技术与未经过乳化分散的核酸检测相比，由于以单个核酸分子为单位进行扩增具有较高的灵敏度和可靠性，可以检测出较低含量的突变基因。此外，BEAMing技术与未经乳化分散的核酸检测相比，由于以单个核酸分子为单位进行扩增具有较高的灵敏度和可靠性，可以检测处较低含量的突变基因。此外，BEAMing还具有筛选分离出特定种类的核酸分子的能力。BEAMing (beads, emulsion, amplification, and magnetics) is a nucleic acid detection technology that combines digital PCR technology and droplet technology. It was proposed by Devin Dressman et al. in 2003. This technology adds oil and emulsifier to the PCR reaction reagent to form an emulsion with the reaction reagent as the dispersed phase, and each droplet contains at most 1 DNA molecule and 1 magnetic bead. The emulsion is then temperature cycled so that the DNA molecules within each droplet containing both DNA molecules and magnetic beads are individually amplified and adsorbed to the magnetic beads (emulsion amplification). Finally, remove the oil in the system and add a fluorescent probe, and use a flow cytometer to count the magnetic beads with fluorescent effects to obtain the absolute concentration and concentration ratio of different types of initial DNA. Compared with nucleic acid detection without emulsification and dispersion, BEAMing technology can detect lower levels of mutant genes due to its higher sensitivity and reliability in amplification of a single nucleic acid molecule. In addition, compared with non-emulsified and dispersed nucleic acid detection, BEAMing technology can detect a lower level of mutant genes due to its higher sensitivity and reliability in the amplification of a single nucleic acid molecule. In addition, BEAMing also has the ability to screen and isolate specific types of nucleic acid molecules.
然而，由于需要在扩增前生成液滴并在扩增反应后将油相去除，BEAMing检测过程中需要经历多次搅拌、洗涤、重悬过程，不仅耗费大量时间、人力与试剂，而且大大增加了试剂泄露和交叉污染的风险。However, due to the need to generate droplets before amplification and remove the oil phase after the amplification reaction, the BEAMing detection process requires multiple stirring, washing, and resuspension processes, which not only consumes a lot of time, manpower, and reagents, but also greatly increases The risk of reagent leakage and cross-contamination is reduced.
得益于微纳制造技术的发展，微流控技术在生物检测中的优势逐渐凸显。利用微流控技术实现微流控芯片上的BEAMing检测过程，主要具有以下优势：Thanks to the development of micro-nano manufacturing technology, the advantages of microfluidic technology in biological detection have gradually become prominent. Using microfluidic technology to realize the BEAMing detection process on a microfluidic chip has the following advantages:
1)微流控芯片易于实现检测过程的自动化控制，显著减少了人力和时间成本。1) The microfluidic chip is easy to realize the automatic control of the detection process, which significantly reduces the cost of manpower and time.
2)微流控芯片免去了在不同设备间转移试剂的过程，避免了试样的交叉污染和泄露问题。2) The microfluidic chip eliminates the process of transferring reagents between different devices, and avoids cross-contamination and leakage of samples.
3)微流控结构的面积-体积比更大，因而具备更强的换热性能，适用于需要快速吸/放热或升/降温的反应。3) The area-to-volume ratio of the microfluidic structure is larger, so it has stronger heat transfer performance, and is suitable for reactions that require rapid heat absorption/release or temperature rise/fall.
然而，现有专利中涉及的变温PCR微流控核酸检测芯片大多采用基于电流热效应(电阻丝、帕尔帖元件)的加热方式。但该方式属于外源接触式加热，主要缺陷有：However, most of the temperature-variable PCR microfluidic nucleic acid detection chips involved in existing patents use heating methods based on current thermal effects (resistance wires, Peltier elements). However, this method belongs to external contact heating, and the main defects are:
1)芯片与加热元件的空隙会影响接触热阻，对芯片加热部分的装配精度要求较高；1) The gap between the chip and the heating element will affect the contact thermal resistance, and the assembly accuracy of the chip heating part is required to be high;
2)热传导导热功率有限，难以实现快速升温；2) The heat conduction power is limited, so it is difficult to achieve rapid temperature rise;
3)变性温度区(约95℃)大部分热量向环境耗散，能量转化效率较低。3) In the denaturation temperature zone (about 95°C), most of the heat is dissipated to the environment, and the energy conversion efficiency is low.
因此急需设计一种新的液滴加热和扩增方式。Therefore, it is urgent to design a new way of heating and amplifying droplets.
发明内容Contents of the invention
针对现有技术的以上缺陷或改进需求，本发明提供了一种喷嘴、液滴光热操控系统及其应用，采用激光加热升温迅速且可控程度高，在核酸检测可以通过激光加热区实现DNA加热裂解并且提高了油包单个纳米颗粒的概率，便于后期处理，免去了在不同设备间转移试剂的过程，避免了试样的交叉污染和泄露问题。In view of the above defects or improvement needs of the prior art, the present invention provides a nozzle, a liquid droplet photothermal control system and its application, using laser heating to heat up quickly and with a high degree of controllability, and DNA can be detected through the laser heating area in nucleic acid detection. Heating and cracking increases the probability of single nanoparticles in the oil, which is convenient for post-processing, avoids the process of transferring reagents between different equipment, and avoids the problems of cross-contamination and leakage of samples.
为实现上述目的，按照本发明的一个方面，提供了一种喷嘴，所述喷嘴包括：第一出口以及多个第二出口，所述多个第二出口设于所述第一出口的出口处并相互连通，所述第一出口的出口处设置有筛孔板，所述筛孔板上设有多个筛孔，所述筛孔板的四周与所述第一出口的内壁密封可拆卸连接，所述筛孔板的一端与第二出口连通，所述筛孔板的另一端与激光照射区连通；激光，用于对所述激光照射区进行照射以对第一出口内的流体进行加热。To achieve the above object, according to one aspect of the present invention, a nozzle is provided, the nozzle includes: a first outlet and a plurality of second outlets, the plurality of second outlets are arranged at the outlet of the first outlet and communicate with each other, the outlet of the first outlet is provided with a sieve plate, and the sieve plate is provided with a plurality of sieve holes, and the periphery of the sieve plate is sealed and detachably connected with the inner wall of the first outlet , one end of the sieve plate communicates with the second outlet, and the other end of the sieve plate communicates with the laser irradiation area; the laser is used to irradiate the laser irradiation area to heat the fluid in the first outlet .
优选地，所述第一出口与多个第二出口的角度为60～90°。Preferably, the angle between the first outlet and the plurality of second outlets is 60-90°.
优选地，所述第一出口和多个第二出口的入口处均设置有单向阀。Preferably, one-way valves are provided at the entrances of the first outlet and the plurality of second outlets.
优选地，所述筛孔为一层或多层，每一筛孔的截面形状为渐缩形；所述激光照射区包括透镜，用于对所述激光进行聚焦。Preferably, the meshes are one or more layers, and the cross-sectional shape of each mesh is tapered; the laser irradiation area includes a lens for focusing the laser light.
按照本发明的另一个方面，提供了一种喷嘴的液滴光热操控系统，所述系统包括：入口换向阀和出口换向阀，所述入口换向阀设于所述第一出口的入口管道上，所述出口换向阀设于所述喷嘴的出口管道上；恒温管，所述恒温管用于维持流体的温度，所述恒温管的入口与所述出口换向阀的出口连接，所述恒温管的出口与所述入口换向阀的入口连接；所述入口换向阀的入口还与第一试剂入口连接；所述第二出口的入口管道与第二试剂入口连接，第一试剂和第二试剂在喷嘴的出口处混合。According to another aspect of the present invention, a liquid droplet photothermal control system of a nozzle is provided, the system includes: an inlet reversing valve and an outlet reversing valve, the inlet reversing valve is set at the first outlet On the inlet pipeline, the outlet reversing valve is arranged on the outlet pipeline of the nozzle; a thermostatic tube, the thermostatic tube is used to maintain the temperature of the fluid, and the inlet of the thermostatic tube is connected to the outlet of the outlet reversing valve, The outlet of the thermostatic tube is connected to the inlet of the inlet reversing valve; the inlet of the inlet reversing valve is also connected to the first reagent inlet; the inlet pipe of the second outlet is connected to the second reagent inlet, and the first The reagent and the second reagent mix at the outlet of the nozzle.
优选地，所述喷嘴和所述出口换向阀之间还设置有微泵，所述出口换向阀的出口处还设置有出口通道。Preferably, a micropump is provided between the nozzle and the outlet reversing valve, and an outlet passage is provided at the outlet of the outlet reversing valve.
按照本发明的再一个方面，提供了一种液滴光热操控系统的应用，所述系统用于核酸检测，所述第一试剂为PCR反应试剂，所述PCR反应试剂包括表面包覆有DNA分子的纳米颗粒，所述纳米颗粒为具有局部表面等离激元共振性质的金属、半导体、合金、金属氧化物或以上几种的复合材料；所述第二试剂为分散剂；所述纳米颗粒在喷嘴的激光照射区进行照射，温度升高，其表面的DNA分子解旋为单链，同时PCR反应试剂内部产生微小气泡，从而体积膨胀，产生压力溢出筛孔与所述分散剂混合获得由油包覆纳米颗粒的液滴，所述液滴在所述恒温管中进行DNA单链的扩增。According to another aspect of the present invention, an application of a liquid droplet photothermal manipulation system is provided, the system is used for nucleic acid detection, the first reagent is a PCR reaction reagent, and the PCR reaction reagent includes DNA coated on the surface Molecular nanoparticles, the nanoparticles are metals, semiconductors, alloys, metal oxides or composite materials of the above with local surface plasmon resonance properties; the second reagent is a dispersant; the nanoparticles When the laser irradiation area of the nozzle is irradiated, the temperature rises, and the DNA molecules on the surface are unwound into single strands. At the same time, tiny bubbles are generated inside the PCR reaction reagent, so that the volume expands, and the pressure overflows the sieve hole and mixes with the dispersant. Droplets of oil-coated nanoparticles that undergo amplification of DNA single strands in the thermostated tube.
优选地，所述筛孔的直径为所述纳米颗粒直径的5～10倍；进一步优选，所述纳米颗粒的粒径为0.5～5μm。Preferably, the diameter of the sieve is 5-10 times the diameter of the nanoparticles; more preferably, the diameter of the nanoparticles is 0.5-5 μm.
优选地，所述多个第二出口的过流截面积与所述第一出口的过流截面积的关系为：Preferably, the relationship between the flow cross-sectional area of the plurality of second outlets and the flow cross-sectional area of the first outlet is:
其中，Sw为多个第二出口的过流截面积，So为第一出口的过流截面积，n为筛孔数，f为激光的频率，v为液滴的体积，Qp为所述微泵的体积流量，k为补偿系数，其取值为1.6～2.0。Among them, Sw is the flow cross-sectional area of multiple second outlets, So is the flow cross-sectional area of the first outlet, n is the number of sieve holes, f is the frequency of the laser, v is the volume of the droplet, andQp is The volumetric flow rate of the micropump, k is a compensation coefficient, and its value is 1.6-2.0.
优选地，所述分散剂的表面张力小于所述分散剂与流经管道避免之间的表面张力，所述分散剂包括表面活性剂和乳化剂，以防止所述分散剂吸附在油-液滴界面与流经通道壁面。Preferably, the surface tension of the dispersant is smaller than the surface tension between the dispersant and the flow-through pipeline, and the dispersant includes a surfactant and an emulsifier to prevent the dispersant from being adsorbed on oil-droplets interface and flow-through channel walls.
总体而言，通过本发明所构思的以上技术方案与现有技术相比，本发明提供的一种喷嘴、液滴光热操控系统及其应用具有如下有益效果：Generally speaking, compared with the prior art through the above technical solutions conceived by the present invention, a nozzle, a liquid droplet photothermal control system and their applications provided by the present invention have the following beneficial effects:
1.本申请中的喷嘴中多个第二出口设于所述第一出口的出口处并相互连通，所述第一出口的出口处设置有筛孔板，可以实现第一出口和第二出口的出口处，可以实现第一出口中流出的液体与第二出口处流出的液体均匀混合。1. In the nozzle of the present application, multiple second outlets are arranged at the outlet of the first outlet and communicate with each other. The outlet of the first outlet is provided with a sieve plate, which can realize the first outlet and the second outlet. At the outlet of the outlet, the liquid flowing out of the first outlet can be uniformly mixed with the liquid flowing out of the second outlet.
2.第一出口内设置有激光照射区可以实现对第一出口内的流体进行定点、定时按需无接触加热，克服现有技术中加热慢，芯片与加热元件有空隙加热效率低的问题。2. A laser irradiation area is set in the first outlet to realize fixed-point, timing and on-demand non-contact heating of the fluid in the first outlet, which overcomes the problems of slow heating and low heating efficiency in the gap between the chip and the heating element in the prior art.
3.本申请中的液滴光热操控系统通过入口换向阀和出口换向阀、喷嘴和恒温管等装置可以实现DNA分子解旋和扩增一体化设计，避免了频繁转移带来的交叉污染风险。3. The liquid droplet photothermal control system in this application can realize the integrated design of DNA molecule detwisting and amplification through the inlet reversing valve and outlet reversing valve, nozzle and thermostatic tube, avoiding the crossover caused by frequent transfer pollution risk.
4.可以通过控制筛孔和纳米颗粒的比例可以实现单个纳米颗粒的溢出，进而可以提高油包单个纳米颗粒的概率，得到的液滴更加均匀更利于后期的检测观察。4. The overflow of a single nanoparticle can be realized by controlling the ratio of the sieve hole to the nanoparticle, which in turn can increase the probability of a single nanoparticle in the oil, and the obtained droplets are more uniform and more conducive to later detection and observation.
5.本申请提供的喷嘴仅具有机械结构，简单可靠，易于加工。喷嘴与微流控芯片是可拆卸连接，不仅便于清洗和更换，而且通过更换不同筛孔大小的喷嘴可以改变生成液滴的尺寸。5. The nozzle provided by this application only has a mechanical structure, which is simple, reliable and easy to process. The nozzle and the microfluidic chip are detachably connected, which is not only convenient for cleaning and replacement, but also the size of the generated droplets can be changed by changing nozzles with different mesh sizes.
6.本申请提供的液滴光热操控系统采用周期性光信号和筛孔结构控制液滴的生成与循环，确保液滴在每个循环中至少被光源加热一次，从而保证每个液滴内DNA的充分扩增。6. The droplet photothermal control system provided by this application uses periodic light signals and sieve structure to control the generation and circulation of droplets, ensuring that the droplets are heated by the light source at least once in each cycle, so as to ensure that each droplet Adequate amplification of DNA.
附图说明Description of drawings
图1示意性示出了本实施例弯喷嘴的结构示意图；Fig. 1 schematically shows the structural representation of the curved nozzle of this embodiment;
图2示意性示出了本实施例直喷嘴的结构示意图；Fig. 2 schematically shows the structural representation of the straight nozzle of the present embodiment;
图3A示意性示出了本实施例单层筛孔的筛孔板示意图；Fig. 3A schematically shows a schematic diagram of a sieve plate of a single-layer sieve in this embodiment;
图3B示意性示出了本实施例多层筛孔的筛孔板示意图；Fig. 3B schematically shows a schematic diagram of a sieve plate with multi-layer sieves in this embodiment;
图4示意性示出了本实施例筛孔的截面示意图；Fig. 4 schematically shows a schematic cross-sectional view of a sieve hole in this embodiment;
图5示意性示出了本实施例液滴光热操控系统的结构示意图。FIG. 5 schematically shows a schematic structural diagram of the liquid droplet photothermal manipulation system of this embodiment.
在所有附图中，相同的附图标记用来表示相同的元件或结构，其中：Throughout the drawings, the same reference numerals are used to designate the same elements or structures, wherein:
100-喷嘴：110-第一出口；120-第二出口；130-筛孔板；140-激光照射区；100-nozzle: 110-first outlet; 120-second outlet; 130-sieve plate; 140-laser irradiation area;
200-入口换向阀；300-出口换向阀；400-恒温管；500-第一试剂入口；600-第二试剂入口；700-微泵；800-出口通道；900-单向阀。200-inlet reversing valve; 300-outlet reversing valve; 400-thermostatic tube; 500-first reagent inlet; 600-second reagent inlet; 700-micro pump; 800-outlet channel; 900-one-way valve.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.
请参阅图1及图2，本发明第一方面提供了一种喷嘴100，所述喷嘴100可以为直喷嘴或弯喷嘴，所述喷嘴100包括第一出口110以及多个第二出口120，所述多个第二出口120设于所述第一出口110的出口处并相互连通，所述第一出口的出口处设置有筛孔板130，所述筛孔板130上设有多个筛孔，所述筛孔板130的四周与所述第一出口120的内壁密封可拆卸连接，所述筛孔板130的一端与第二出口120连通，所述筛孔板120的另一端与激光照射区140连通；所述激光照射区120用于接收激光照射以对第一出口110内的流体进行加热。Referring to Fig. 1 and Fig. 2, the first aspect of the present invention provides a
所述第一出口110与多个第二出口120的角度优选为60～90°，进一步优选为90°，即第一出口110和第二出口120形成一个T形。The angle between the
所述第一出口110和多个第二出口120的入口处均设置有单向阀。One-way valves are provided at the inlets of the
所述筛孔为一层或多层(如图3A和图3B所示)，每一筛孔的截面形状为渐缩形(如图4所示)，多个筛孔可以均匀布置与筛孔板也可以根据需要按一定的规律布置。例如，内环较疏外环较密的形式布置。Described sieve hole is one or more layers (as shown in Figure 3A and Figure 3B), and the cross-sectional shape of each sieve hole is taper (as shown in Figure 4), and a plurality of sieve holes can be arranged evenly with sieve hole The boards can also be arranged according to certain rules as required. For example, the inner ring is sparser and the outer ring is denser.
所述激光照射区包括透镜，用于对所述激光进行聚焦，激光的输出波长范围优选为740～830nm。The laser irradiation area includes a lens for focusing the laser, and the output wavelength range of the laser is preferably 740-830nm.
所述第一出口由耐高温和高压冲击的透明材料制造，优选为石英玻璃，采用光刻蚀技术加工出筛孔，筛孔板可拆卸，便于清洗和更换。The first outlet is made of a transparent material resistant to high temperature and high pressure impact, preferably quartz glass, and the sieve holes are processed by photoetching technology. The sieve hole plate is detachable and easy to clean and replace.
本申请另一方面提供了一种包括上述喷嘴的液滴光热操控系统，如图5所示，所述系统包括喷嘴100、入口换向阀200、出口换向阀300和恒温管400，其中：Another aspect of the present application provides a liquid droplet photothermal control system including the above-mentioned nozzle, as shown in FIG. :
入口换向阀200和出口换向阀300，所述入口换向阀200设于所述第一出口110的入口管道上，所述出口换向阀300设于所述喷嘴100的出口管道上；An
恒温管400，所述恒温管400用于维持流体的温度，所述恒温管400的入口与所述出口换向阀300的出口连接，所述恒温管400的出口与所述入口换向阀200的入口连接；所述入口换向阀200的入口还与第一试剂入口500连接；
所述第二出口120的入口管道与第二试剂入口600连接，所述第一试剂和第二试剂在喷嘴100的出口处混合。The inlet pipe of the
所述喷嘴100和所述出口换向阀300之间还设置有微泵700作为驱动系统，所述出口换向阀300的出口处还设置有出口通道800。微泵700包括但不限于使用S型气动式微泵、压电微泵驱动、电磁驱动。作为优选，使用S型气动微泵作为驱动系统。A
工作时可以通过控制入口换向阀200和出口换向阀300的阀门转向实现流体流向的改变，进而可以实现流体在恒温管内循环运动。During operation, the fluid flow direction can be changed by controlling the valve turning of the
在入口换向阀200和第一试剂入口500之间、在第二试剂入口600与第二出口120之间、以及喷嘴100与入口换向阀200之间均设置有单向阀900。A one-
本申请再一方面提供了一种液滴光热操控系统的应用，所述系统尤其适用于核酸检测。Another aspect of the present application provides an application of a liquid droplet photothermal manipulation system, which is especially suitable for nucleic acid detection.
所述第一试剂为PCR反应试剂，所述PCR反应试剂包括表面包覆有DNA分子的纳米颗粒，所述纳米颗粒为具有局部表面等离激元共振性质的金属、半导体、合金、金属氧化物或以上几种的复合材料；所述第二试剂为分散剂；所述纳米颗粒在喷嘴的激光照射区进行照射，温度升高，其表面的DNA分子解旋为单链，同时PCR反应试剂内部产生微小气泡，从而体积膨胀，产生压力溢出筛孔与所述分散剂混合获得由油包覆纳米颗粒的液滴，所述液滴在所述恒温管中进行DNA单链的扩增。The first reagent is a PCR reaction reagent, and the PCR reaction reagent includes nanoparticles coated with DNA molecules on the surface, and the nanoparticles are metals, semiconductors, alloys, and metal oxides with local surface plasmon resonance properties. or the composite materials of the above several kinds; the second reagent is a dispersant; the nano-particles are irradiated in the laser irradiation area of the nozzle, and the temperature rises, and the DNA molecules on the surface unwind into single strands, and at the same time, the inside of the PCR reaction reagent Micro-bubbles are generated, so that the volume expands, and the pressure is generated to overflow the sieve and mix with the dispersant to obtain liquid droplets coated with oil nanoparticles, and the liquid droplets undergo DNA single-strand amplification in the thermostat tube.
本实施例中，PCR反应试剂包括但不限于DNA模版、游离核苷酸、DNA聚合酶、PCR缓冲液。In this embodiment, the PCR reaction reagents include but not limited to DNA template, free nucleotides, DNA polymerase, and PCR buffer.
纳米颗粒为具有局部表面等离激元共振性质的金属、半导体、合金、金属氧化物及上述材料组成的复合材料,其粒径在0.5～5μm之间。作为优选，使用超顺磁四氧化三铁纳米粒子(以下简称磁珠)，粒径为1.05μm±0.1μm。Nanoparticles are metals, semiconductors, alloys, metal oxides and composite materials composed of the above materials with local surface plasmon resonance properties, and their particle diameters are between 0.5 and 5 μm. Preferably, superparamagnetic ferroferric oxide nanoparticles (hereinafter referred to as magnetic beads) are used, with a particle size of 1.05 μm±0.1 μm.
所述纳米颗粒表面修饰有DNA的至少一种初始DNA的PCR引物。作为优选，使用生物素-链霉亲和素结合的方式进行修饰，PCR引物包括初始DNA的正向引物或反向引物中的一种。进一步地，引物与纳米颗粒表面的距离通过在生物素和插入一定长度的连接序列进行调控，使单个光信号激发纳米颗粒的时变温度场中，与引物杂交的目标核酸所在区域的最高温度在核酸变性温度(约为94℃，与DNA中G-C含量有关)附近。The nanoparticle is surface-modified with at least one PCR primer for the original DNA. Preferably, the biotin-streptavidin combination method is used for modification, and the PCR primer includes one of the forward primer or the reverse primer of the original DNA. Further, the distance between the primer and the surface of the nanoparticle is regulated by inserting a certain length of linking sequence in the biotin, so that in the time-varying temperature field in which a single optical signal excites the nanoparticle, the maximum temperature of the region where the target nucleic acid hybridized with the primer is at Nucleic acid denaturation temperature (about 94 ° C, related to the G-C content in DNA) around.
所述光源可以以恒定的频率发出具有一定总能量的光信号，并通过透镜等方式聚焦于试剂喷嘴。作为优选，光源采用激光器。进一步地，作为优选，光源的输出波长范围为740～830nm。作为优选，聚焦镜头采用线状聚焦，其聚焦位置位于喷嘴接近出口位置。The light source can emit a light signal with a certain total energy at a constant frequency, and focus it on the reagent nozzle through a lens or the like. Preferably, the light source is a laser. Further, preferably, the output wavelength range of the light source is 740-830 nm. Preferably, the focusing lens adopts linear focusing, and its focusing position is located near the outlet of the nozzle.
所述筛孔的直径为所述纳米颗粒直径的5～10倍；进一步优选，所述纳米颗粒的粒径为0.5～5μm。The diameter of the sieve hole is 5-10 times of the diameter of the nanoparticles; more preferably, the particle diameter of the nanoparticles is 0.5-5 μm.
所述多个第二出口的过流截面积与所述第一出口的过流截面积的关系为：The relationship between the flow cross-sectional area of the plurality of second outlets and the flow cross-sectional area of the first outlet is:
其中，Sw为多个第二出口的过流截面积，So为第一出口的过流截面积，n为筛孔数，f为激光的频率，v为液滴的体积，Qp为所述微泵的体积流量，k为补偿系数，其取值为1.6～2.0。Among them, Sw is the flow cross-sectional area of multiple second outlets, So is the flow cross-sectional area of the first outlet, n is the number of sieve holes, f is the frequency of the laser, v is the volume of the droplet, andQp is The volumetric flow rate of the micropump, k is a compensation coefficient, and its value is 1.6-2.0.
所述分散剂可以采用满足如下条件的液体：Described dispersant can adopt the liquid that meets following condition:
1)与水不互溶；1) immiscible with water;
2)不影响DNA聚合酶的正常工作；2) It does not affect the normal work of DNA polymerase;
3)与分散剂的表面张力小于分散剂与通道壁之间的表面张力。3) The surface tension with the dispersant is smaller than the surface tension between the dispersant and the channel wall.
作为优选，使用碳酸双(2-乙基己基)酯钠盐和矿物油的混合物作为分散剂。此外，所述分散剂内添加有表面活性剂与乳化剂，用于阻止PCR反应试剂成分吸附在油-液滴界面与微通道壁面。Preferably, a mixture of bis(2-ethylhexyl) carbonate sodium salt and mineral oil is used as dispersant. In addition, surfactants and emulsifiers are added in the dispersant to prevent PCR reagent components from being adsorbed on the oil-droplet interface and the microchannel wall.
所述恒温管为环状，可以为由上下两片带环状凹槽的薄片封接而成。其材质选用疏水亲油材质，使得所述分散剂与微通道的接触角大于试剂与微通道的接触角，作为优选，选用PDMS(聚二甲基硅氧烷)。恒温管沿流体的流动方向依次设置有弯管(冷却区)，恒温发热区(恒温区)，圆柱形空腔(缓冲区)，微换向阀，微单向阀，最后回到试剂喷嘴。The thermostatic tube is ring-shaped, and may be formed by sealing two upper and lower sheets with annular grooves. The material is hydrophobic and lipophilic, so that the contact angle between the dispersant and the microchannel is greater than that between the reagent and the microchannel. Preferably, PDMS (polydimethylsiloxane) is selected. The thermostatic tube is provided with an elbow (cooling area), a constant temperature heating area (constant temperature area), a cylindrical cavity (buffer zone), a micro-reversing valve, a micro-check valve, and finally a reagent nozzle along the flow direction of the fluid.
恒温发热区通过ITO(氧化铟锡)溅射到玻璃基片上，配合光刻电路形成加热器和温度传感器，再通过芯片控制实现恒温发热。The constant temperature heating area is sputtered onto the glass substrate through ITO (indium tin oxide), and the heater and temperature sensor are formed with the photolithography circuit, and then the constant temperature heating is realized through chip control.
圆柱形空腔的顶部也包覆有PCR封板膜与外界空气连通，用于排出热发泡生成的气体，维持通道内的压力平衡。The top of the cylindrical cavity is also covered with a PCR sealing film to communicate with the outside air, which is used to discharge the gas generated by thermal foaming and maintain the pressure balance in the channel.
本申请中的系统可以实现如下目的：The system in this application can achieve the following objectives:
1)基于热发泡的原理，生成具有一定大小和尺寸，包含至多1个所述纳米颗粒的油包水液滴。1) Based on the principle of thermal foaming, water-in-oil droplets with a certain size and size containing at most one nanoparticle are generated.
2)使用周期性光信号操控液滴的生成(或通过)速率，同时将附着于所述纳米颗粒的双链DNA片段快速加热至变性温度，使其双链解旋为两条单链，在冷却后均与纳米颗粒结合。2) Use periodic light signals to control the generation (or passage) rate of droplets, and at the same time, quickly heat the double-stranded DNA fragments attached to the nanoparticles to the denaturation temperature, so that the double strands can be unwound into two single strands. Both combine with nanoparticles after cooling.
3)令液滴通过一恒温区域，使DNA聚合酶在最佳活性温度下把与纳米颗粒结合的单链DNA分子扩增为双链。3) Let the liquid droplet pass through a constant temperature area, so that the DNA polymerase can amplify the single-stranded DNA molecules bound to the nanoparticles into double strands at the optimal activity temperature.
4)重复2)与3)，直到每个纳米颗粒上附着的DNA片段数量足够进行荧光探针检测为止。4) Repeat 2) and 3) until the number of DNA fragments attached to each nanoparticle is sufficient for fluorescent probe detection.
本申请中系统的使用方法具体如下：The method of using the system in this application is as follows:
1)使光源周期性地发出光信号，聚焦于喷嘴。1) Make the light source periodically emit light signals and focus on the nozzle.
2)切换入口换向阀至左位，PCR反应试剂经单向阀注入第一出口，分散剂经单向阀注入第二出口。2) Switch the inlet reversing valve to the left position, the PCR reagent is injected into the first outlet through the one-way valve, and the dispersant is injected into the second outlet through the one-way valve.
3)每次当第一出口接收到光信号时，由于纳米颗粒的局部等离激元共振效应，将光能转化为热能，在纳米颗粒周围形成温度场，与纳米颗粒结合的DNA温度在很短的时间内急剧上升至沸点附近。此时，PCR反应试剂中附着于所述纳米颗粒的DNA达到变性温度，双链解旋为单链(其中一条固定在磁珠上，另一条为游离单链)；同时，PCR反应试剂内部产生微小气泡，从而体积膨胀，产生压力，反应试剂溢出筛孔，与分散剂混合形成尺寸略大于筛孔的液滴。3) Every time when the first outlet receives an optical signal, due to the local plasmon resonance effect of the nanoparticle, the light energy is converted into heat energy, and a temperature field is formed around the nanoparticle, and the temperature of the DNA combined with the nanoparticle is very high. In a short period of time, it rises sharply to near the boiling point. At this time, the DNA attached to the nanoparticles in the PCR reaction reagent reaches the denaturation temperature, and the double strands unwind into single strands (one of which is fixed on the magnetic beads, and the other is a free single strand); at the same time, the DNA generated inside the PCR reaction reagent The tiny bubbles expand in volume and generate pressure, and the reaction reagent overflows the sieve and mixes with the dispersant to form droplets slightly larger than the sieve.
4)由于液滴流经的流道的高表面积-体积比(S表/V通道≈4/D通道)和流经弯管形成的二次流现象，乳化液在弯管区快速冷却，游离的DNA单链与纳米颗粒上修饰的引物杂交。作为优选，流经弯管后，乳化液将会依次通过：4) Due to the high surface area-volume ratio of the flow channel through which the droplets flow (Stable /Vchannel ≈ 4/Dchannel ) and the secondary flow phenomenon formed by flowing through the bend, the emulsion cools rapidly in the bend area, free Single strands of DNA hybridize to the modified primers on the nanoparticles. As a preference, after flowing through the elbow, the emulsion will pass through:
a)恒温区域，DNA聚合酶从于引物杂交DNA的3’-OH端以5’-3’方向合成DNA双链。a) In the constant temperature region, DNA polymerase synthesizes DNA double strands in the 5'-3' direction from the 3'-OH end of the primer-hybridized DNA.
b)缓冲区域，在液体初次流入恒温管时排出微通道内原有的气体；并在循环过程中排除因热发泡产生的气泡，维持液体压力。b) The buffer area, when the liquid flows into the thermostatic tube for the first time, the original gas in the microchannel is discharged; and the bubbles generated by thermal foaming are eliminated during the circulation process to maintain the liquid pressure.
当乳化液进入缓冲区域时，切换入口换向阀至右位，切断PCR反应试剂和分散剂的流入，进入内循环。When the emulsion enters the buffer area, switch the inlet reversing valve to the right position, cut off the inflow of PCR reagents and dispersants, and enter the internal circulation.
5)乳化液经单向阀回到喷嘴。液滴由于其尺寸略大于筛孔，将不会通过筛孔直接流出，而是聚集在筛孔内侧。接收到下一次光信号时，靠近筛孔的液滴受热膨胀，在互相挤压下变形通过筛孔，回到步骤4)。5) The emulsion returns to the nozzle through the one-way valve. The droplets, due to their size slightly larger than the sieve openings, will not flow directly through the sieve openings, but gather inside the sieve openings. When the next optical signal is received, the liquid droplets close to the sieve hole are heated and expanded, deformed and passed through the sieve hole under mutual extrusion, and return to step 4).
6)步骤4)和5)在光信号的控制下重复，使得液滴周期性地经历受热、冷却、恒温的过程，液滴内部的DNA实现扩增。6) Steps 4) and 5) are repeated under the control of the light signal, so that the droplet undergoes the process of heating, cooling, and constant temperature periodically, and the DNA inside the droplet is amplified.
7)经历若干循环后，当单个液滴内的DNA浓度达到可以进行荧光检测的标准时，切换出口换向阀至右位，将环状微通道中的乳化液全部导出，用于后续的荧光探针检测和计数。7) After several cycles, when the DNA concentration in a single droplet reaches the standard for fluorescence detection, switch the outlet reversing valve to the right position to export all the emulsion in the annular microchannel for subsequent fluorescence detection. Needle detection and counting.
实施例1Example 1
本实施例提供一种液滴光热操控系统的应用。具体包括以下步骤：This embodiment provides an application of a liquid droplet photothermal manipulation system. Specifically include the following steps:
1)一种液滴光热操控系统，结构如附图5所示，使用如附图2所示的直线式喷嘴，筛孔尺寸约为10±0.5μm。使用808nm，3000mW的近红外光纤激光器，以长约10mm，宽约2mm的条形光斑聚焦于喷嘴。调制激光器，使得光斑以2Hz的频率发送光信号。接通恒温发热片，直到恒温区的温度达到65℃。打开气动微泵。1) A liquid droplet photothermal manipulation system, the structure of which is shown in Figure 5, using a linear nozzle as shown in Figure 2, with a sieve size of about 10±0.5 μm. Use an 808nm, 3000mW near-infrared fiber laser to focus on the nozzle with a strip-shaped spot about 10mm long and 2mm wide. The laser is modulated so that the spot sends an optical signal at a frequency of 2 Hz. Turn on the constant temperature heating sheet until the temperature in the constant temperature zone reaches 65°C. Turn on the pneumatic micropump.
2)切换入口换向阀至左位，PCR反应试剂和分散剂以约3：1的速度比分别由针筒经单向阀注入喷嘴。2) Switch the inlet reversing valve to the left position, and the PCR reaction reagent and dispersant are respectively injected into the nozzle from the syringe through the one-way valve at a speed ratio of about 3:1.
PCR反应试剂按照以下方法调配：每150μL PCR反应试剂中含有2.5μM正向引物和400μM反向引物各3μL，模板DNA10μL(由rbm39a和rbm39b通过逆转录合成，等位基因a和b的比例约为49：1)，正向引物修饰的磁珠6μL，dNTPs预混液3μL，10×PCR缓冲液15μL，PlatinumTaq DNA聚合酶9μL(5U/μL)，水101μL。The PCR reaction reagent was prepared according to the following method: each 150 μL PCR reaction reagent contained 3 μL each of 2.5 μM forward primer and 400 μM reverse primer, and 10 μL of template DNA (synthesized by rbm39a and rbm39b through reverse transcription, the ratio of alleles a and b was approximately 49:1), 6 μL of forward primer-modified magnetic beads, 3 μL of dNTPs master mix, 15 μL of 10×PCR buffer, 9 μL of PlatinumTaq DNA polymerase (5 U/μL), and 101 μL of water.
分散剂按照以下方法调配：7％聚甘油-4异硬脂酸酯ABIL WE09乳化剂，20％矿物油，73％碳酸二乙基己酯。Tegosoft DEC软化剂。The dispersant was formulated as follows: 7% polyglyceryl-4 isostearate ABIL WE09 emulsifier, 20% mineral oil, 73% diethylhexyl carbonate. Tegosoft DEC softener.
3)当乳化液进入缓冲区域时，切换入口换向阀至右位，切断试剂和分散剂的流入，进入内循环。3) When the emulsion enters the buffer area, switch the inlet reversing valve to the right position, cut off the inflow of reagents and dispersants, and enter the internal circulation.
4)30min后，切换出口换向阀至右位，将环状微通道中的乳化液全部导出，进行平均液滴直径测试和磁珠包含率测试。4) After 30 minutes, switch the outlet reversing valve to the right position, export all the emulsion in the annular microchannel, and conduct the average droplet diameter test and magnetic bead inclusion rate test.
5)向导出的乳化液中加入液滴破坏剂，离心并吸去上层油层，使用磁力架静置后除去所有水分，再加入100μL的TK缓冲液重悬，在4℃下保存样品。5) Add a droplet breaker to the derived emulsion, centrifuge and absorb the upper oil layer, use a magnetic stand to remove all water, then add 100 μL of TK buffer to resuspend, and store the sample at 4°C.
液滴破坏剂按以下方式调配：1％Trition-X表面活性剂，1％SDS(十二烷基硫酸钠)，1mM EDTA(乙二胺四乙酸)，10mM Tris-HCL(三羟甲基氨基甲烷盐酸盐)，100mM NaCl(氯化钠)。The droplet breaker was prepared in the following manner: 1% Trition-X surfactant, 1% SDS (sodium dodecyl sulfate), 1 mM EDTA (ethylenediaminetetraacetic acid), 10 mM Tris-HCL (trishydroxymethylamino Methane hydrochloride), 100 mM NaCl (sodium chloride).
TK缓冲液按以下方式调配：20mM Tris-HCL，50mM KCL(氯化钾)TK buffer is prepared in the following way: 20mM Tris-HCL, 50mM KCL (potassium chloride)
实施例2Example 2
本实施例与实施例1不同之处在于，使用附图1所示的弯曲式喷嘴，筛孔尺寸相同。The difference between this embodiment and Embodiment 1 is that the curved nozzle shown in Figure 1 is used, and the mesh size is the same.
实施例3Example 3
本实施例与实施例1不同之处在于，弯曲式喷嘴出口处使用附图3B所示的双层筛孔结构，筛孔尺寸相同。The difference between this embodiment and Embodiment 1 is that the outlet of the curved nozzle uses a double-layer mesh structure as shown in Fig. 3B, and the mesh size is the same.
实施例4Example 4
本实施例与实施例1不同之处在于，筛孔尺寸约为15±0.5μm。The difference between this embodiment and embodiment 1 is that the mesh size is about 15±0.5 μm.
实施例5Example 5
本实施例与实施例1不同之处在于，激光信号的频率为4Hz。The difference between this embodiment and Embodiment 1 is that the frequency of the laser signal is 4 Hz.
测试实施例。将实施例1-5进行测试。Test Example. Examples 1-5 were tested.
1)平均液滴直径测试，使用光学显微镜和测微尺细胞随机测量同一乳化液样品中10个含有磁珠的液滴的平均直径(Davg10)。1) The average droplet diameter test, using an optical microscope and a micrometer cell to randomly measure the average diameter (Davg10) of 10 droplets containing magnetic beads in the same emulsion sample.
2)液滴不合格率测试，使用光学显微镜随机测量同一乳化液样品50个液滴里不含有磁珠(n(a+b))和含有至少两个磁珠(n(≥2))的液滴占总液滴数(n)的比例。2) Droplet failure rate test, using an optical microscope to randomly measure 50 droplets of the same emulsion sample that do not contain magnetic beads (n(a+b)) and contain at least two magnetic beads (n(≥2)) The ratio of droplets to the total number of droplets (n).
3)扩增杂交率测试，使结合在磁珠上的DNA与荧光探针杂交后，使用流式细胞仪测量同时结合rbm39a与rbm39b(n(a+b))的磁珠占只结合rbm39a(n(a))和只结合rbm39b(n(b))的磁珠的比率。3) Amplified hybridization rate test, after hybridizing the DNA bound to the magnetic beads with the fluorescent probe, use flow cytometry to measure the percentage of magnetic beads bound to rbm39a and rbm39b (n(a+b)) at the same time only bound to rbm39a ( n(a)) and the ratio of beads bound only to rbm39b (n(b)).
4)扩增准确度测试，使用流式细胞仪测量只结合rbm39a(n(a))和只结合rbm39b(n(b))的磁珠的比率。4) Amplification accuracy test, using a flow cytometer to measure the ratio of magnetic beads that only bind to rbm39a (n(a)) and only bind to rbm39b (n(b)).
测试结构如表1所示。The test structure is shown in Table 1.
表1Table 1
通过测试结果可知：It can be seen from the test results that:
1)正常状况下，生成液滴的平均直径由筛孔直径决定，略大于筛孔直径；1) Under normal conditions, the average diameter of the generated droplets is determined by the diameter of the sieve hole, which is slightly larger than the diameter of the sieve hole;
2)双层筛孔与单层筛孔相比，由于总过流面积增大，脉冲的压力被消减，生成液滴变小；2) Compared with the single-layer sieve, the double-layer sieve has an increased total flow area, the pressure of the pulse is reduced, and the generated droplets become smaller;
3)激光频率过大会导致液泛现象而使生成液滴变小。3) Excessive laser frequency will cause liquid flooding and make the generated droplets smaller.
4)生成液滴越小，生成空液滴的概率越大，有效样本越少；4) The smaller the generated droplet, the greater the probability of generating an empty droplet, and the fewer effective samples;
5)生成液滴越大，生成空液滴的概率越小，生成含多个磁珠的液滴的概率越大，容易生成杂交的样本。5) The larger the generated droplet, the smaller the probability of generating an empty droplet, the greater the probability of generating a droplet containing multiple magnetic beads, and it is easy to generate a hybridized sample.
因此，通过控制筛孔层数和激光频率，可以有效地控制生成液滴的直径，使得生成的单磁珠液滴比例最大，从而生成更多的有效样本。Therefore, by controlling the number of sieve layers and the laser frequency, the diameter of the generated droplets can be effectively controlled to maximize the proportion of single magnetic bead droplets generated, thereby generating more effective samples.
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.
Claims (10)
Translated fromChinese
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111180616.8ACN113967487B (en) | 2021-10-11 | 2021-10-11 | A nozzle, droplet photothermal control system and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111180616.8ACN113967487B (en) | 2021-10-11 | 2021-10-11 | A nozzle, droplet photothermal control system and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113967487A CN113967487A (en) | 2022-01-25 |
| CN113967487Btrue CN113967487B (en) | 2022-12-02 |
Family
ID=79587307
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111180616.8AActiveCN113967487B (en) | 2021-10-11 | 2021-10-11 | A nozzle, droplet photothermal control system and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113967487B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1935496A1 (en)* | 2006-12-22 | 2008-06-25 | Eppendorf Array Technologies SA | Device and/or method for the detection of amplified nucleotides sequences on micro-arrays |
| CN103258788A (en)* | 2013-04-17 | 2013-08-21 | 华中科技大学 | Through hole interconnection structure manufacturing method based on bidirectional filling and through hole interconnection structure product |
| CN106457252A (en)* | 2014-03-28 | 2017-02-22 | 好奇诊断有限责任公司 | Device for simultaneous and uniform thermal cycling of samples and uses thereof |
| CN109136352A (en)* | 2018-08-10 | 2019-01-04 | 深圳先进技术研究院 | Sample processing device, micro-fluidic chip and application before a kind of unicellular sequencing |
| CN111778155A (en)* | 2020-07-09 | 2020-10-16 | 墨卓生物科技(上海)有限公司 | PCR amplification mechanism for dPCR integrated micro-fluidic chip |
| CN111836890A (en)* | 2018-03-07 | 2020-10-27 | 威斯康星校友研究基金会 | High-throughput nucleic acid profiling of single cells |
| CN112779125A (en)* | 2021-02-09 | 2021-05-11 | 复旦大学 | Light-driven circulating polymerase chain reaction micro-fluidic device and application thereof |
Family Cites Families (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2385324C (en)* | 1999-09-22 | 2008-03-25 | Miodrag Oljaca | Liquid atomization methods and devices |
| US8900811B2 (en)* | 2000-11-16 | 2014-12-02 | Caliper Life Sciences, Inc. | Method and apparatus for generating thermal melting curves in a microfluidic device |
| US9562837B2 (en)* | 2006-05-11 | 2017-02-07 | Raindance Technologies, Inc. | Systems for handling microfludic droplets |
| US9176504B2 (en)* | 2011-02-11 | 2015-11-03 | The Regents Of The University Of California | High-speed on demand droplet generation and single cell encapsulation driven by induced cavitation |
| WO2012158631A2 (en)* | 2011-05-13 | 2012-11-22 | The Regents Of The University Of California | Photothermal substrates for selective transfection of cells |
| CN102363815B (en)* | 2011-11-25 | 2013-09-18 | 杭州优思达生物技术有限公司 | Reagent for detecting salmonellae by using cross primer nucleic acid isothermal amplification technology, amplification method and detection method for salmonellae |
| ES2749700T3 (en)* | 2012-05-24 | 2020-03-23 | Univ Utah Res Found | Extreme PCR |
| KR101441594B1 (en)* | 2012-12-27 | 2014-09-23 | 매쓰파워 주식회사 | Heat sensitive polymer composite based Nucleic Acid Amplification disc apparatus and analysis method using the same |
| WO2017152357A1 (en)* | 2016-03-08 | 2017-09-14 | Coyote Bioscience Co., Ltd. | Methods and systems for analyzing nucleic acids |
| US11142791B2 (en)* | 2016-08-10 | 2021-10-12 | The Regents Of The University Of California | Combined multiple-displacement amplification and PCR in an emulsion microdroplet |
| DE102016124692B4 (en)* | 2016-12-16 | 2019-05-16 | Gna Biosolutions Gmbh | Method and system for amplifying a nucleic acid |
| WO2019126789A1 (en)* | 2017-12-22 | 2019-06-27 | 10X Genomics, Inc. | Systems and methods for processing nucleic acid molecules from one or more cells |
| CN107999155A (en)* | 2017-12-25 | 2018-05-08 | 四川蓝光英诺生物科技股份有限公司 | Micro-fluidic chip and its control method, drop formation device and microballoon preparation facilities |
| CN108004135B (en)* | 2018-01-16 | 2021-03-05 | 清华大学 | Full-automatic on-line liquid drop digital PCR device based on Inkjet |
| CN111004708B (en)* | 2019-12-24 | 2023-05-30 | 中国科学院长春光学精密机械与物理研究所 | PCR temperature cycle control method and rotary-driven PCR temperature cycle control device |
| CN112791754B (en)* | 2020-12-31 | 2021-09-28 | 苏阳 | Digital microfluidic polymerase chain reaction multi-connection detection system |
- 2021
- 2021-10-11CNCN202111180616.8Apatent/CN113967487B/enactiveActive
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1935496A1 (en)* | 2006-12-22 | 2008-06-25 | Eppendorf Array Technologies SA | Device and/or method for the detection of amplified nucleotides sequences on micro-arrays |
| CN103258788A (en)* | 2013-04-17 | 2013-08-21 | 华中科技大学 | Through hole interconnection structure manufacturing method based on bidirectional filling and through hole interconnection structure product |
| CN106457252A (en)* | 2014-03-28 | 2017-02-22 | 好奇诊断有限责任公司 | Device for simultaneous and uniform thermal cycling of samples and uses thereof |
| CN111836890A (en)* | 2018-03-07 | 2020-10-27 | 威斯康星校友研究基金会 | High-throughput nucleic acid profiling of single cells |
| CN109136352A (en)* | 2018-08-10 | 2019-01-04 | 深圳先进技术研究院 | Sample processing device, micro-fluidic chip and application before a kind of unicellular sequencing |
| CN111778155A (en)* | 2020-07-09 | 2020-10-16 | 墨卓生物科技(上海)有限公司 | PCR amplification mechanism for dPCR integrated micro-fluidic chip |
| CN112779125A (en)* | 2021-02-09 | 2021-05-11 | 复旦大学 | Light-driven circulating polymerase chain reaction micro-fluidic device and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Experimental results on two-phase heat transfer enhancement in microstructured corrugated plate heat exchangers;Polzin,AE等;《EXPERIMENTAL HEAT TRANSFER》;20200930;全文* |
| 芯片层叠塑料封装的MEMS惯性器件的开封方法;林晓玲等;《电子产品可靠性与环境试验》;20180820;第49-53页* |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113967487A (en) | 2022-01-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20240175091A1 (en) | Methods and Systems for Detecting Biological Components | |
| US20220154248A1 (en) | Combined multiple-displacement amplification and pcr in an emulsion microdroplet | |
| CN101903104B (en) | Integrated microfluidic device and methods | |
| EP3253910B1 (en) | Multiple-emulsion nucleic acid amplification | |
| US20140005066A1 (en) | Multiplexed PCR and Fluorescence Detection on a Droplet Actuator | |
| KR101605701B1 (en) | Apparatus and Method for Microdroplet-based Assay of Biomaterials | |
| Jalili et al. | A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR | |
| JP7747522B2 (en) | A platform for deterministic assembly of microfluidic droplets | |
| Fu et al. | Rapid vortex microfluidic mixer utilizing double-heart chamber | |
| CN108004135A (en) | Based on the full-automatic online drop numeral PCR devices of Inkjet | |
| CN105013544B (en) | A kind of microlayer model fusion method based on the induction of hydrophilic fibers silk | |
| Mao et al. | A digitalized isothermal nucleic acid testing platform based on a pump-free open droplet array microfluidic chip | |
| CN113967487B (en) | A nozzle, droplet photothermal control system and its application | |
| Cheng | Photo-responsive Fluorosurfactant based on Plasmonic Nanoparticles for Droplet Microfluidics | |
| AU2016203092B2 (en) | Nano-pcr: methods and devices for nucleic acid amplification and detection | |
| Stumpf | Automated microfluidic nucleic acid analysis for single-cell and sample-to-answer applications | |
| Ozdemir et al. | DNA Analysis in Droplet‐Based Microfluidic Devices | |
| HK1248284B (en) | Multiple-emulsion nucleic acid amplification |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right | Effective date of registration:20250602 Address after:430074 Hubei Province, Wuhan city Hongshan District Luoyu Road No. 1037 Patentee after:HUAZHONG University OF SCIENCE AND TECHNOLOGY Country or region after:China Patentee after:Wuhan Mingzhi Diagnostic Technology Co.,Ltd. Address before:430074 Hubei Province, Wuhan city Hongshan District Luoyu Road No. 1037 Patentee before:HUAZHONG University OF SCIENCE AND TECHNOLOGY Country or region before:China Patentee before:Wuhan nanuode Medical Technology Co.,Ltd. |