CN113967487B - Nozzle, liquid drop photo-thermal control system and application thereof - Google Patents
Nozzle, liquid drop photo-thermal control system and application thereof Download PDFInfo
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- CN113967487B CN113967487B CN202111180616.8A CN202111180616A CN113967487B CN 113967487 B CN113967487 B CN 113967487B CN 202111180616 A CN202111180616 A CN 202111180616A CN 113967487 B CN113967487 B CN 113967487B
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- 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
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- 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
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- 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
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- 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
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- 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]
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- 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
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- 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
Abstract
The invention belongs to the technical field related to nucleic acid detection, and discloses a nozzle, a droplet photothermal control system and application thereof, wherein the nozzle comprises: the laser irradiation device comprises a first outlet and a plurality of second outlets, wherein the plurality of second outlets are arranged at the outlet of the first outlet and are communicated with each other, a sieve plate is arranged at the outlet of the first outlet, a plurality of sieve holes are arranged on the sieve plate, the periphery of the sieve plate is detachably connected with the inner wall of the first outlet in a sealing manner, one end of the sieve plate is communicated with the second outlets, and the other end of the sieve plate is communicated with a laser irradiation area; the laser irradiation area is used for receiving laser irradiation to heat the fluid in the first outlet. This application adopts laser heating intensification rapidly and controllable degree is high, can realize DNA heating schizolysis and improved the probability of single nanoparticle of oil pocket through the laser heating district in nucleic acid detects, has removed the process of transferring reagent between different equipment from, has avoided the cross contamination of sample and has revealed the problem.
Description
Technical Field
The invention belongs to the technical field related to nucleic acid detection, and particularly relates to a nozzle, a liquid drop photo-thermal control system and application thereof.
Background
Nucleic acid detection has high sensitivity and high effectiveness, and is the most widely used detection means for pathogen infection.
BEAMing (beads, emulsions, amplification, and magnetics) is a nucleic acid detection technique that combines digital PCR technology with droplet technology, as proposed by Devin Dressman et al in 2003. The technology is to add oil and emulsifier into PCR reaction reagent to form emulsion with the reaction reagent as dispersive phase, and each liquid drop contains at most 1 DNA molecule and 1 magnetic bead. Then, the emulsion is subjected to temperature cycling so that the DNA molecules in each droplet containing both DNA molecules and magnetic beads are individually amplified and adsorbed onto the magnetic beads (emulsion amplification). And finally, removing oil in the system, adding a fluorescent probe, and counting magnetic beads with a fluorescent effect by using a flow cytometer to obtain absolute concentrations and concentration ratios of different types of initial DNA. Compared with nucleic acid detection without emulsification dispersion, the BEAMing technology has higher sensitivity and reliability due to amplification by taking a single nucleic acid molecule as a unit, and can detect a lower content of mutant genes. In addition, the BEAMing technology can detect a lower content of mutant genes due to higher sensitivity and reliability of amplification in units of single nucleic acid molecules compared with detection of nucleic acids without emulsion dispersion. BEAMing also has the ability to screen for the isolation of specific classes of nucleic acid molecules.
However, because it is necessary to generate droplets before amplification and remove the oil phase after the amplification reaction, the BEAMing assay process needs to go through many stirring, washing and resuspending processes, which not only consumes much time, manpower and reagents, but also greatly increases the risk of reagent leakage and cross-contamination.
The advantages of the micro-fluidic technology in biological detection are gradually highlighted due to the development of the micro-nano manufacturing technology. The BEAMing detection process on the microfluidic chip is realized by utilizing the microfluidic technology, and the method mainly has the following advantages:
1) The micro-fluidic chip is easy to realize the automatic control of the detection process, and the labor and time cost is obviously reduced.
2) The micro-fluidic chip avoids the process of transferring reagents among different devices, and avoids the problems of cross contamination and leakage of samples.
3) The micro-fluidic structure has larger area-volume ratio, thereby having stronger heat exchange performance and being suitable for the reaction needing quick heat absorption/release or temperature rise/reduction.
However, most of the temperature-variable PCR microfluidic nucleic acid detection chips involved in the existing patents adopt a heating mode based on current thermal effect (resistance wire, peltier element). However, the mode belongs to external contact heating, and has the main defects that:
1) The gap between the chip and the heating element can influence the contact thermal resistance, and the assembly precision requirement on the heating part of the chip is higher;
2) The heat conduction power is limited, and the rapid temperature rise is difficult to realize;
3) The denaturation temperature zone (about 95 ℃) dissipates most of the heat to the environment, and the energy conversion efficiency is low.
Therefore, it is urgently needed to design a new droplet heating and amplification mode.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides a nozzle, a liquid drop photothermal control system and application thereof.
To achieve the above object, according to one aspect of the present invention, there is provided a nozzle comprising: the laser irradiation device comprises a first outlet and a plurality of second outlets, wherein the plurality of second outlets are arranged at the outlet of the first outlet and are communicated with each other, a sieve plate is arranged at the outlet of the first outlet, a plurality of sieve holes are arranged on the sieve plate, the periphery of the sieve plate is detachably connected with the inner wall of the first outlet in a sealing manner, one end of the sieve plate is communicated with the second outlets, and the other end of the sieve plate is communicated with a laser irradiation area; and the laser is used for irradiating the laser irradiation area to heat the fluid in the first outlet.
Preferably, the angle between the first outlet and the plurality of second outlets is 60 to 90 °.
Preferably, the inlets of the first outlet and the plurality of second outlets are provided with one-way valves.
Preferably, the sieve holes are one layer or a plurality of layers, and the cross section of each sieve hole is in a tapered shape; the laser irradiation region includes a lens for focusing the laser light.
According to another aspect of the invention there is provided a droplet photothermal manipulation system for a nozzle, the system comprising: the inlet reversing valve is arranged on an inlet pipeline of the first outlet, and the outlet reversing valve is arranged on an outlet pipeline of the nozzle; the thermostatic tube is used for maintaining the temperature of the fluid, an inlet of the thermostatic tube is connected with an outlet of the outlet reversing valve, and an outlet of the thermostatic tube is connected with an inlet of the inlet reversing valve; the inlet of the inlet reversing valve is also connected with the first reagent inlet; the inlet pipe of the second outlet is connected with the second reagent inlet, and the first reagent and the second reagent are mixed at the outlet of the nozzle.
Preferably, a micro pump is further arranged between the nozzle and the outlet reversing valve, and an outlet channel is further arranged at an outlet of the outlet reversing valve.
According to a further aspect of the present invention, there is provided a use of a droplet photothermal manipulation system for nucleic acid detection, wherein the first reagent is a PCR reaction reagent, the PCR reaction reagent comprises nanoparticles coated with DNA molecules on the surface, and the nanoparticles are a metal, a semiconductor, an alloy, a metal oxide or a composite material thereof with local surface plasmon resonance property; the second reagent is a dispersant; the nano particles are irradiated in a laser irradiation area of the nozzle, the temperature is raised, DNA molecules on the surfaces of the nano particles are uncoiled into single chains, meanwhile, micro bubbles are generated inside a PCR reaction reagent, so that the volume is expanded, pressure is generated to overflow a sieve mesh and be mixed with the dispersing agent to obtain liquid drops coated with the nano particles by oil, and the liquid drops are subjected to DNA single chain amplification in the thermostatic tube.
Preferably, the diameter of the sieve pore is 5 to 10 times of the diameter of the nano particle; more preferably, the nanoparticles have a particle size of 0.5 to 5 μm.
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 as follows:
wherein S is w The flow cross-sectional area of the plurality of second outlets, S o Is the cross-sectional area of the first outlet, n is the number of screen holes, f is the frequency of the laser, v is the volume of the liquid drop, Q p K is the compensation coefficient, and the value of k is 1.6-2.0.
Preferably, the surface tension of the dispersant is less than the surface tension between the dispersant and the flow-through conduit, and the dispersant comprises a surfactant and an emulsifier to prevent the dispersant from adsorbing at the oil-droplet interface and the flow-through channel walls.
Generally, compared with the prior art, the nozzle, the liquid drop photo-thermal control system and the application thereof provided by the invention have the following beneficial effects:
1. a plurality of second exports are located in the nozzle in this application the exit of first export and communicate each other, the exit of first export is provided with the orifice plate, can realize the exit of first export and second export, can realize the liquid homogeneous mixing that flows out in the first export and the liquid homogeneous mixing that flows out in the second export.
2. Be provided with laser irradiation area in the first export and can realize fixing a point, regularly need not contact heating as required to the fluid in the first export, overcome among the prior art heating slow, there is the problem that the clearance heating efficiency is low in chip and heating element.
3. The liquid drop light and heat control system in this application can realize DNA molecule unwinding and amplification integrated design through devices such as entry switching-over valve and export switching-over valve, nozzle and thermostatic tube, has avoided the cross contamination risk that frequent transfer brought.
4. The overflow of single nanoparticles can be realized by controlling the proportion of the sieve pores and the nanoparticles, so that the probability of single nanoparticles in oil can be improved, and the obtained liquid drops are more uniform and more beneficial to later detection and observation.
5. The nozzle that this application provided only has mechanical structure, and is simple reliable, easily processing. The nozzle is detachably connected with the microfluidic chip, so that the cleaning and the replacement are convenient, and the size of the generated liquid drop can be changed by replacing nozzles with different screen hole sizes.
6. The liquid drop photo-thermal control system provided by the application adopts a periodic optical signal and a sieve pore structure to control the generation and circulation of liquid drops, ensures that the liquid drops are heated by a light source at least once in each circulation, and further ensures the sufficient amplification of DNA in each liquid drop.
Drawings
FIG. 1 is a schematic view showing the structure of a bending nozzle according to the present embodiment;
FIG. 2 is a schematic view showing the structure of a straight nozzle according to the present embodiment;
figure 3A schematically illustrates a mesh panel of the single layer mesh of this embodiment;
FIG. 3B is a schematic view of a perforated deck with multiple layers of screen openings according to this embodiment;
fig. 4 schematically shows a cross-sectional view of a screen aperture of the present embodiment;
fig. 5 schematically shows a structural schematic diagram of the droplet photothermal manipulation system of the present embodiment.
The same reference numbers will be used throughout the drawings to refer to the same or like elements or structures, wherein:
100-nozzle: 110 — a first outlet; 120-a second outlet; 130-a perforated screen; 140-laser irradiation area;
200-inlet selector valve; 300-an outlet selector valve; 400-a thermostatic tube; 500-a first reagent inlet; 600-a second reagent inlet; 700-micropump; 800-an outlet channel; 900-one-way valve.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Referring to fig. 1 and fig. 2, a first aspect of the present invention provides a nozzle 100, where the nozzle 100 may be a straight nozzle or a bent nozzle, the nozzle 100 includes a first outlet 110 and a plurality of second outlets 120, the plurality of second outlets 120 are disposed at an outlet of the first outlet 110 and are communicated with each other, a sieve plate 130 is disposed at the outlet of the first outlet, a plurality of sieve holes are disposed on the sieve plate 130, the periphery of the sieve plate 130 is detachably connected to the inner wall of the first outlet 120 in a sealing manner, one end of the sieve plate 130 is communicated with the second outlet 120, and the other end of the sieve plate 120 is communicated with a laser irradiation region 140; the laser irradiation region 120 is used for receiving laser irradiation to heat the fluid in the first outlet 110.
The angle between the first outlet 110 and the plurality of second outlets 120 is preferably 60 to 90 °, and more preferably 90 °, i.e. the first outlet 110 and the second outlet 120 form a T-shape.
The inlets of the first outlet 110 and the plurality of second outlets 120 are each provided with a check valve.
The screen holes are one layer or multiple layers (as shown in fig. 3A and fig. 3B), the cross section of each screen hole is tapered (as shown in fig. 4), and a plurality of screen holes can be uniformly arranged and the screen hole plates can also be arranged according to a certain rule as required. For example, the inner ring may be arranged more sparsely than the outer ring.
The laser irradiation area comprises a lens for focusing the laser, and the output wavelength range of the laser is preferably 740-830 nm.
The first outlet is made of a transparent material resistant to high temperature and high pressure impact, preferably quartz glass, sieve holes are machined by adopting a photoetching technology, and the sieve holes can be detached and are convenient to clean and replace.
Another aspect of the present application provides a droplet photothermal manipulation system comprising the above-described nozzle, as shown in fig. 5, the system comprising a nozzle 100, an inlet directional valve 200, an outlet directional valve 300, and a thermostatic tube 400, wherein:
an inlet direction changing valve 200 and an outlet direction changing valve 300, wherein the inlet direction changing valve 200 is arranged on an inlet pipeline of the first outlet 110, and the outlet direction changing valve 300 is arranged on an outlet pipeline of the nozzle 100;
a thermostatic tube 400 for maintaining the temperature of the fluid, an inlet of the thermostatic tube 400 being connected to an outlet of the outlet selector valve 300, and an outlet of the thermostatic tube 400 being connected to an inlet of the inlet selector valve 200; the inlet of the inlet selector valve 200 is also connected to a first reagent inlet 500;
the inlet duct of the second outlet 120 is connected to a second reagent inlet 600, the first and second reagents being mixed at the outlet of the nozzle 100.
A micro pump 700 is further disposed between the nozzle 100 and the outlet directional valve 300 as a driving system, and an outlet channel 800 is further disposed at an outlet of the outlet directional valve 300. Micropump 700 includes, but is not limited to, use of an S-type pneumatic micropump, piezoelectric micropump actuation, electromagnetic actuation. Preferably, an S-type pneumatic micro-pump is used as the drive system.
During operation, the change of the flow direction of the fluid can be realized by controlling the valve steering of the inlet reversing valve 200 and the outlet reversing valve 300, and further, the circulating motion of the fluid in the thermostatic tube can be realized.
One-way valves 900 are provided between the inlet selector valve 200 and the first reagent inlet 500, between the second reagent inlet 600 and the second outlet 120, and between the nozzle 100 and the inlet selector valve 200.
In yet another aspect, the present application provides for the use of a droplet photothermal manipulation system, which is particularly useful for nucleic acid detection.
The first reagent is a PCR reaction reagent, the PCR reaction reagent comprises nanoparticles with DNA molecules coated on the surfaces, and the nanoparticles are metals, semiconductors, alloys, metal oxides or composite materials of the above materials with local surface plasmon resonance properties; the second reagent is a dispersant; the nano particles are irradiated in a laser irradiation area of the nozzle, the temperature is raised, DNA molecules on the surfaces of the nano particles are uncoiled into single chains, meanwhile, micro bubbles are generated inside a PCR reaction reagent, so that the volume is expanded, pressure is generated to overflow a sieve mesh and be mixed with the dispersing agent to obtain liquid drops coated with the nano particles by oil, and the liquid drops are subjected to DNA single chain amplification in the thermostatic tube.
In this embodiment, the PCR reagent includes, but is not limited to, a DNA template, free nucleotides, DNA polymerase, and PCR buffer.
The nano particles are metal, semiconductor, alloy, metal oxide with local surface plasmon resonance property and composite material composed of the above materials, and the particle diameter of the nano particles is between 0.5 and 5 mu m. Preferably, superparamagnetic ferroferric oxide nanoparticles (hereinafter referred to as magnetic beads) are used, having a particle size of 1.05 μm. + -. 0.1. Mu.m.
The surface of the nanoparticle is modified with at least one PCR primer of initial DNA of the DNA. Preferably, the modification is performed using biotin-streptavidin binding and the PCR primers comprise one of the forward or reverse primers of the original DNA. Further, the distance between the primer and the surface of the nanoparticle is controlled by biotin and a connecting sequence with a certain length, so that in a time-varying temperature field in which a single optical signal excites the nanoparticle, the highest temperature of a region where a target nucleic acid hybridized with the primer is located is near the nucleic acid denaturation temperature (about 94 ℃, which is related to the G-C content in DNA).
The light source may emit a light signal with a certain total energy at a constant frequency and be focused on the reagent nozzle by means of a lens or the like. Preferably, the light source is a laser. Further, the output wavelength of the light source is preferably in a range of 740 to 830nm. Preferably, the focusing lens uses line focusing, and the focusing position is located at the position of the nozzle close to the outlet.
The diameter of the sieve pore is 5 to 10 times of the diameter of the nano particle; more preferably, the nanoparticles have a particle size of 0.5 to 5 μm.
The relationship between the flow cross-sectional areas of the plurality of second outlets and the flow cross-sectional area of the first outlet is as follows:
wherein S is w Cross-sectional area of a plurality of second outlets, S o Is the cross-sectional area of the first outlet, n is the number of screen holes, f is the frequency of the laser, v is the volume of the liquid drop, Q p K is the compensation coefficient, and the value of k is 1.6-2.0.
The dispersant may be a liquid satisfying the following conditions:
1) Is immiscible with water;
2) Does not affect the normal work of DNA polymerase;
3) The surface tension with the dispersant is less than the surface tension between the dispersant and the channel walls.
Preferably, a mixture of sodium bis (2-ethylhexyl) carbonate and mineral oil is used as the dispersant. In addition, a surfactant and an emulsifier are added into the dispersing agent and are used for preventing PCR reaction reagent components from being adsorbed on an oil-liquid drop interface and the wall surface of the micro-channel.
The constant temperature tube is annular and can be formed by sealing an upper sheet and a lower sheet with annular grooves. The material is hydrophobic oleophilic material, so that the contact angle between the dispersing agent and the micro-channel is larger than that between the reagent and the micro-channel, and PDMS (polydimethylsiloxane) is preferably used. The thermostatic tube is sequentially provided with a bent tube (cooling area), a constant-temperature heating area (thermostatic area), a cylindrical cavity (buffer area), a micro reversing valve and a micro one-way valve along the flowing direction of fluid, and finally returns to the reagent nozzle.
The constant temperature heating area is sputtered on the glass substrate through ITO (indium tin oxide), a heater and a temperature sensor are formed by matching with a photoetching circuit, and constant temperature heating is realized through chip control.
The top of the cylindrical cavity is also coated with a PCR sealing plate film which is communicated with the outside air and used for exhausting gas generated by thermal foaming and maintaining the pressure balance in the channel.
The system in the application can achieve the following purposes:
1) Based on the principle of thermal foaming, water-in-oil droplets of a certain size and dimension are generated, containing at most 1 of said nanoparticles.
2) The generation (or passage) rate of the liquid drop is controlled by using a periodic optical signal, and meanwhile, the double-stranded DNA fragments attached to the nanoparticles are rapidly heated to a denaturation temperature, so that the double strands of the double-stranded DNA fragments are uncoiled into two single strands, and the double strands are combined with the nanoparticles after cooling.
3) The droplets are passed through a constant temperature zone, allowing the DNA polymerase to amplify the single-stranded DNA molecules bound to the nanoparticles into double strands at an optimal activation temperature.
4) Repeating 2) and 3) until the number of DNA fragments attached to each nanoparticle is sufficient for fluorescent probe detection.
The application method of the system specifically comprises the following steps:
1) The light source periodically emits light signals to focus on the nozzle.
2) And switching the inlet reversing valve to the left position, injecting the PCR reaction reagent into the first outlet through the one-way valve, and injecting the dispersing agent into the second outlet through the one-way valve.
3) Each time when the first outlet receives an optical signal, light energy is converted into heat energy due to a local plasmon resonance effect of the nanoparticle, a temperature field is formed around the nanoparticle, and the temperature of DNA bound to the nanoparticle sharply rises to the vicinity of a boiling point in a short time. At this time, the DNA attached to the nanoparticles in the PCR reaction reagent reaches a denaturation temperature, and the double strand unwinds into single strands (one of which is immobilized on the magnetic beads, and the other is free single strand); meanwhile, micro bubbles are generated in the PCR reaction reagent, so that the volume is expanded, pressure is generated, the reaction reagent overflows the sieve pores, and is mixed with the dispersing agent to form liquid drops with the size slightly larger than the sieve pores.
4) Due to the high surface area-to-volume ratio (S) of the flow channel through which the droplets flow Watch (A) /V Channel ≈4/D Channel ) And the secondary flow phenomenon formed by flowing through the bent pipe, the emulsion is rapidly cooled in the bent pipe area, and the free DNA single chain is hybridized with the primer modified on the nano-particles. Preferably, after passing through the elbow, the emulsion will pass sequentially through:
a) In the isothermal region, DNA polymerase synthesizes a DNA double strand from the 3' -OH end of the primer-hybridized DNA in the 5' -3' direction.
b) A buffer area for discharging the gas existing in the microchannel when the liquid flows into the thermostatic tube for the first time; and bubbles generated by thermal foaming are removed in the circulation process, and the liquid pressure is maintained.
When the emulsion enters the buffer area, the inlet reversing valve is switched to the right position, the inflow of the PCR reaction reagent and the dispersing agent is cut off, and the emulsion enters the internal circulation.
5) The emulsion returns to the nozzle through a one-way valve. Because the size of the liquid drops is slightly larger than the sieve holes, the liquid drops can not directly flow out through the sieve holes and are gathered at the inner side of the sieve holes. And (4) when receiving the next optical signal, the liquid drops close to the sieve holes are heated and expanded, and are deformed to pass through the sieve holes under mutual extrusion, and the step returns to the step 4).
6) And (4) repeating the steps 4) and 5) under the control of the optical signal, so that the liquid drop periodically undergoes the processes of heating, cooling and keeping constant temperature, and the DNA in the liquid drop is amplified.
7) After a plurality of cycles, when the DNA concentration in a single liquid drop reaches the standard of fluorescence detection, the outlet reversing valve is switched to the right position, and all the emulsion in the annular micro-channel is led out for subsequent fluorescence probe detection and counting.
Example 1
The present embodiments provide an application of a droplet photothermal manipulation system. The method specifically comprises the following steps:
1) A droplet photothermal manipulation system is shown in FIG. 5, and has a screen size of about 10 + -0.5 μm using a linear nozzle as shown in FIG. 2. A near-infrared fiber laser of 808nm,3000mW was used, and a stripe-shaped spot of about 10mm in length and about 2mm in width was focused on the nozzle. The laser is modulated so that the spot transmits the optical signal at a frequency of 2 Hz. The constant temperature heating sheet was turned on until the temperature of the constant temperature zone reached 65 ℃. The pneumatic micro pump is turned on.
2) The inlet selector valve was switched to the left, and the PCR reaction reagents and dispersants were mixed in a ratio of about 3: the speed ratio of 1 is injected into the nozzles from the syringe through the one-way valves respectively.
The PCR reaction reagent is prepared according to the following method: mu.L of each of 2.5. Mu.M forward primer and 400. Mu.M reverse primer per 150. Mu.L of PCR reaction reagent, 10. Mu.L of template DNA (synthesized by reverse transcription from rbm39a and rbm39b, the ratio of alleles a and b is about 49.
The dispersant is prepared according to the following method: 7% of polyglycerol-4 isostearate ABIL WE09 emulsifier, 20% of mineral oil and 73% of diethylhexyl carbonate. Tegosoft DEC softener.
3) When the emulsion enters the buffer area, the inlet reversing valve is switched to the right position, the inflow of the reagent and the dispersing agent is cut off, and the emulsion enters the internal circulation.
4) And after 30min, switching an outlet reversing valve to the right position, completely guiding out the emulsion in the annular microchannel, and carrying out average droplet diameter test and magnetic bead inclusion rate test.
5) Adding a droplet breaker into the emulsion, centrifuging, sucking off the upper oil layer, standing by a magnetic frame, removing all water, adding 100 mu L TK buffer solution for resuspension, and storing the sample at 4 ℃.
The drop breaker was formulated as follows: 1% Trition-X surfactant, 1% SDS (sodium dodecyl sulfate), 1mM EDTA (ethylenediaminetetraacetic acid), 10mM Tris-HCl (Tris-HCl), 100mM NaCl (sodium chloride).
The TK buffer was prepared as follows: 20mM Tris-HCl,50mM KCL (potassium chloride)
Example 2
This example is different from example 1 in that the curved nozzle shown in fig. 1 is used and the mesh size is the same.
Example 3
This example is different from example 1 in that the double-layer mesh structure shown in fig. 3B is used at the outlet of the curved nozzle, and the mesh size is the same.
Example 4
This example differs from example 1 in that the mesh size is about 15. + -. 0.5. Mu.m.
Example 5
The present embodiment is different from embodiment 1 in that the frequency of the laser signal is 4Hz.
The examples were tested. Examples 1-5 were tested.
1) Average droplet diameter test, the average diameter of 10 droplets containing magnetic beads in the same emulsion sample was randomly measured using an optical microscope and micrometer cells (Davg 10).
2) The fraction of droplets containing no magnetic beads (n (a + b)) and at least two magnetic beads (n (. Gtoreq.2)) in 50 droplets of the same emulsion sample to the total number of droplets (n) was randomly measured using an optical microscope.
3) Amplification hybridization Rate test, after hybridizing the DNA bound to the magnetic beads with fluorescent probes, the ratio of magnetic beads that bind rbm39a and rbm39b (n (a + b)) simultaneously to magnetic beads that bind rbm39a (n (a)) and rbm39b (n (b)) alone was measured using a flow cytometer.
4) Amplification accuracy test, the ratio of magnetic beads that bind rbm39a (n (a)) only and rbm39b (n (b)) only was measured using a flow cytometer.
The test structures are shown in table 1.
TABLE 1
According to the test results, the following results are obtained:
1) Under normal conditions, the average diameter of the generated liquid drops is determined by the diameter of the sieve pores and is slightly larger than the diameter of the sieve pores;
2) Compared with the single-layer sieve pores, the double-layer sieve pores have the advantages that the total flow area is increased, the pulse pressure is reduced, and the generated liquid drops are reduced;
3) Too high a laser frequency causes flooding and the resulting droplets become smaller.
4) The smaller the generated liquid drop is, the higher the probability of generating an empty liquid drop is, and the fewer effective samples are;
5) The larger the droplet is generated, the lower the probability of generating an empty droplet, and the higher the probability of generating a droplet containing a plurality of magnetic beads, and a hybridized sample is easily generated.
Therefore, by controlling the number of the mesh layers and the laser frequency, the diameter of the generated liquid drop can be effectively controlled, so that the proportion of the generated single magnetic bead liquid drop is maximum, and more effective samples are generated.
It will be understood by those skilled in the art that the foregoing is only an exemplary embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, since various modifications, substitutions and improvements within the spirit and scope of the invention are possible and within the scope of the appended claims.
Claims (10)
1. A droplet photothermal manipulation system, the system comprising:
the inlet reversing valve (200) is arranged on an inlet pipeline of the first outlet (110), and the outlet reversing valve (300) is arranged on an outlet pipeline of the nozzle (100); wherein the nozzle comprises: the laser irradiation device comprises a first outlet (110) and a plurality of second outlets (120), wherein the plurality of second outlets (120) are arranged at the outlet of the first outlet (110) and are communicated with each other, a sieve plate (130) is arranged at the outlet of the first outlet (110), a plurality of sieve holes are arranged on the sieve plate (130), the periphery of the sieve plate (130) is hermetically and detachably connected with the inner wall of the first outlet (110), one end of the sieve plate (130) is communicated with the second outlet (120), and the other end of the sieve plate (130) is communicated with a laser irradiation area (140); the laser irradiation zone (140) is used for receiving laser irradiation to heat the fluid in the first outlet (110);
a thermostatic tube (400), the thermostatic tube (400) being used to maintain the temperature of the fluid, an inlet of the thermostatic tube (400) being connected to an outlet of the outlet selector valve (300), an outlet of the thermostatic tube (400) being connected to an inlet of the inlet selector valve (200);
the inlet of the inlet reversing valve (200) is also connected with a first reagent inlet (500);
the inlet duct of the second outlet (120) is connected to a second reagent inlet (600), the first and second reagents being mixed at the outlet of the nozzle.
2. The system according to claim 1, characterized in that the angle of the first outlet (110) to the second outlets (120) is 60 to 90 °.
3. A system according to claim 1 or 2, wherein a one-way valve (900) is provided at the inlet of each of the first outlet (110) and the plurality of second outlets (120).
4. The system of claim 1, wherein the screen openings are one or more layers, and each screen opening is tapered in cross-sectional shape; the laser irradiation region (140) includes a lens for focusing the laser light.
5. The system according to claim 1, characterized in that a micro-pump (700) is further provided between the nozzle (100) and the outlet selector valve (300), and an outlet channel (800) is further provided at the outlet of the outlet selector valve (300).
6. The use of the liquid droplet photothermal manipulation system according to claim 5, wherein said system is used for nucleic acid detection, said first reagent is a PCR reagent, said PCR reagent comprises nanoparticles coated with DNA molecules, said 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 are irradiated in a laser irradiation area of the nozzle, the temperature is raised, DNA molecules on the surfaces of the nanoparticles are uncoiled into single chains, meanwhile, micro bubbles are generated in a PCR reaction reagent, so that the volume is expanded, pressure is generated to overflow a sieve pore and is mixed with the dispersing agent to obtain liquid drops with the nanoparticles coated by the oil, and the liquid drops are subjected to DNA single chain amplification in the thermostatic tube.
7. The use according to claim 6, wherein the diameter of the mesh is 5 to 10 times the diameter of the nanoparticle.
8. The use according to claim 7, wherein the nanoparticles have a particle size of 0.5 to 5 μm.
9. Use according to claim 7 or 8, wherein the flow cross-sectional area of the plurality of second outlets (120) is related to the flow cross-sectional area of the first outlet (110) by:
wherein the content of the first and second substances,
the cross-sectional area of the second outlets (120),
is the cross-sectional area of the first outlet (110),nthe number of the screen holes is the same as the number of the screen holes,fis the frequency of the laser light and is,vis the volume of the liquid droplet and,
is the volume flow rate of the micro pump,kthe value of the compensation coefficient is 1.6 to 2.0.
10. Use according to claim 6, wherein the surface tension of the dispersant is less than the surface tension between the dispersant and the flow-through conduit wall, and the dispersant comprises a surfactant and an emulsifier to prevent adsorption of the dispersant at the oil-droplet interface and flow-through conduit wall.
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