CN115069319A - Method and system for preparing liquid drops by focused ultrasound, detection equipment and storage medium - Google Patents
Method and system for preparing liquid drops by focused ultrasound, detection equipment and storage medium Download PDFInfo
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- CN115069319A CN115069319A CN202210760418.7A CN202210760418A CN115069319A CN 115069319 A CN115069319 A CN 115069319A CN 202210760418 A CN202210760418 A CN 202210760418A CN 115069319 A CN115069319 A CN 115069319A
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- 238000001514 detection method Methods 0.000 title claims abstract description 12
- 238000003860 storage Methods 0.000 title claims abstract description 5
- 239000007788 liquid Substances 0.000 title description 8
- 239000000523 sample Substances 0.000 claims abstract description 52
- 239000006185 dispersion Substances 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
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- 239000012488 sample solution Substances 0.000 claims abstract description 5
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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
- B01L3/502769—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 multiphase flow arrangements
- B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
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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]
Abstract
The invention provides a method, a system, detection equipment and a storage medium for preparing uniform and stable micro-droplets by adopting focused ultrasound, wherein the method comprises the steps of preparing a sample, preparing fluorinated oil phase substances, controlling the temperature and adjusting the ultrasonic environment; carrying out uniform and stable temperature reduction pretreatment on the sample to enable the sample to be in a low-temperature state lower than room temperature; adding a fluorinated oil-containing phase substance into the pretreated low-temperature state sample and performing dispersion treatment; the method comprises the steps of forming a high-frequency short-wave focusing sound field by setting parameters of an ultrasonic forming device, placing a sample in an ultrasonic gathering area under an ultrasonic environment with the duty ratio of 200/500 through a suspended container, enabling a sample solution to be rapidly heated in the ultrasonic focusing environment, and forming uniform stable micro-droplets containing an oil phase and a water phase after a preset time. The invention can avoid sample pollution, reduce the infection risk of experimenters and can help to prepare a unified and standardized sample processing flow.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a droplet generating device for digital PCR detection, a droplet preparation method and a detection device using the droplet generating device.
Background
Polymerase Chain Reaction (PCR) has been proposed for 20 years, during which PCR has developed into a key technology and a conventional technology in the field of molecular biology, and has greatly promoted the development of various fields of life science. Particularly, in the later 90 s, real-time fluorescent quantitative PCR (qPCR) technology and related products proposed by ABI company in America developed PCR from in vitro synthesis and qualitative/semi-quantitative detection technology into a gene analysis technology with high sensitivity, high specificity and accurate quantification.
Although the qPCR technique has been used for diagnosis of all diseases except for trauma and nutritional deficiency through rapid development over a period of ten years, there are many factors affecting the amplification efficiency during PCR amplification, and it cannot be guaranteed that the amplification efficiency remains the same during reaction and that the amplification efficiency is the same between an actual sample and a standard sample and between different samples, thereby causing the basis on which its quantitative analysis depends-the Cycle Threshold (CT) is not constant. Therefore, qPCR is only "relative quantitative", and the accuracy and reproducibility thereof still cannot meet the requirements of molecular biological quantitative analysis.
Vogelstein et al proposed the concept of digital PCR (digital PCR) by dividing a sample into tens to tens of thousands of portions, assigning them to different reaction units, each containing one or more copies of a target molecule (DNA template), performing PCR amplification of the target molecule in each reaction unit, and performing statistical analysis of the fluorescent signals of the reaction units after amplification is complete. Different from qPCR, digital PCR does not depend on CT value, so that the method is not influenced by amplification efficiency, the average concentration (content) of each reaction unit is calculated by direct counting or a Poisson distribution formula after amplification is finished, the error can be controlled within 5%, and absolute quantitative analysis can be realized by digital PCR without reference to a standard sample and a standard curve.
Since digital PCR is an absolute nucleic acid molecule quantification technique, compared to qPCR, the number of DNA molecules can be directly counted, which is an absolute quantification of the starting sample, and thus is particularly suitable for application fields that cannot be well resolved depending on CT values, such as copy number variation, mutation detection, gene relative expression studies (e.g., allele imbalance expression), second-generation sequencing result verification, miRNA expression analysis, single-cell gene expression analysis, and the like.
The core principle of dPCR is as follows: processing a reaction system containing nucleic acid molecules into nano-upgraded droplets, enabling each droplet to contain or not contain target molecules to be detected, enabling each droplet to serve as an independent PCR reactor, detecting the droplets one by adopting a droplet reader after PCR amplification, and taking the existence or nonexistence of an end signal as a judgment standard (the droplet with a fluorescence signal is interpreted as '1', and the droplet without the fluorescence signal is interpreted as '0'). And finally, calculating the concentration or copy number of the target molecules to be detected by utilizing analysis software according to the Poisson distribution principle and the proportion of the positive microdroplets, thereby obtaining the final result.
The current digital PCR on the market is classified into the following three types according to the sample distribution pattern:
(1) magnetic bead mode: mainly comprises a carrier mode which takes a 96-well plate, a 384-well plate or even a 1536-well plate as a dispersion reaction, and a magnetic bead Emulsion Amplification method (Beads, Emulsion, Amplification, magnetism) which takes Beads, Emulsion, Amplification and magnetism as main operation processes. However, neither of the above two methods can meet the more elaborate requirements in terms of the degree of dispersion and the size of the data population.
(2) Droplet mode: the microdroplet mode is a new mode developed with the development of nano fabrication technology (nanofabrication), microfluidics, and especially the second generation sequencing technology, and thus dPCR is also commonly referred to as "droplet digital PCR (ddPCR)". The quantailife company developed the ddPCR platform at the earliest time. 2011, after the company Bio-Rad purchased QuantaLife, the company was named QX100 TM A micro-drop digital PCR instrument. In 2013, the grade is QX 200. In 2012, RainDance introduced a Raindrop model device that can segment each standard reaction system into reaction emulsions containing from 100 to 1000 thousand picoliter-scale droplets, thereby achieving ultra-high droplet counts.
(3) Chip mode: in 1997, Kalinina et al used a nano-upgrade chip for monoclonal template PCR amplification. In 2013, Life Technologies introduced the latest QuantStaudio 3D digital PCR system. The system can uniformly distribute the samples into 2 ten thousand reaction holes which are completely and independently isolated, can effectively prevent cross contamination while simplifying operation steps as much as possible, and effectively avoids the problem of pipeline blockage possibly faced by a micro-drop system.
However, there are limitations to the drop formation speed or throughput of the current three methods. In addition, the three techniques mentioned above, without exception, rely on multiple large instruments. This not only increases the cost of purchase of the instrument, but also limits the widespread use of digital PCR; but also increases the complexity of the experimental operation.
How to break through the technical barriers and provide a digital PCR droplet forming technology and improve the utilization rate of digital PCR oil becomes an important technical problem to be solved urgently by technical personnel in the field.
Disclosure of Invention
The invention aims to provide a method for preparing uniform and stable micro-droplets by using focused ultrasound, a micro-droplet preparation system and detection equipment so as to improve the problems. In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in one aspect, the present application provides a method for preparing uniform and stable micro-droplets using focused ultrasound, comprising preparing a sample, including a fluorinated oil phase, a temperature control environment, and an adjustable ultrasound environment; carrying out uniform and stable temperature reduction pretreatment on the sample to enable the sample to be in a low-temperature state lower than room temperature; adding a fluorinated oil-containing phase substance into the pretreated low-temperature state sample and performing dispersion treatment; the method comprises the steps of forming a high-frequency short-wave focusing sound field by setting parameters of an ultrasonic forming device, placing a sample in an ultrasonic gathering area under an ultrasonic environment with the duty ratio of 200/500 through a suspended container, enabling a sample solution to be rapidly heated in the ultrasonic focusing environment, and forming uniform stable micro-droplets containing an oil phase and a water phase after a preset time.
Preferably, the set power of the ultrasonic environment is 40W to 60W.
Preferably, the first temperature is 8-12 ℃.
Preferably, the preset time for performing the ultrasonic treatment is not less than 30 s.
Preferably, the temperature controlled environment is a water bath.
In another aspect, the present invention provides a system for preparing uniform and stable micro-droplets by using focused ultrasound, the system comprising a control system, an ultrasound generation module, a refrigeration module, and a motion module, wherein the motion module is used for transferring a sample to the refrigeration module or the ultrasound generation module, the refrigeration module is used for forming a temperature controlled environment, and the ultrasound generation module is used for forming an ultrasound environment.
In another aspect, the present invention provides a detection apparatus, comprising:
at least one processor; and
a memory communicatively coupled to the at least one processor; wherein, the first and the second end of the pipe are connected with each other,
the memory stores instructions executable by the at least one processor to enable the at least one processor to perform a method as any one of above.
In another aspect, the present invention provides a non-transitory computer readable storage medium having stored thereon computer instructions for causing the computer to perform the method as described above. As an optional implementation manner, the self-attention module performs operation between each frame of input features to obtain weights, and then performs weighted summation on each frame of input features to obtain an output result.
The invention has the following beneficial effects:
the method for preparing uniform and stable micro-droplets by adopting focused ultrasound is quiet, repeatable and controllable. The non-contact liquid drop preparation is realized through temperature control and ultrasonic adjustment, for example, sample pollution can be avoided, the dispersion process is simple to operate, and time and labor are saved; reduce the risk of infection of laboratory personnel and can help prepare a unified and standardized sample processing flow.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a flow chart of a method for preparing uniform and stable micro-droplets using focused ultrasound according to the present invention;
FIG. 2 is a schematic diagram of a system for preparing micro-droplets according to an embodiment of the present invention.
Detailed Description
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
The corresponding meanings of each reference number are as follows: 1. sample, 2 oil phase matter, 3 sample container, 4 sample frame, 5 ultrasonic sound field focusing area
It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. Specific structural and functional details disclosed herein are merely illustrative of example embodiments of the invention. This invention may, however, be embodied in many alternate forms and should not be construed as limited to the embodiments set forth herein.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "comprising," "includes," and/or "including," when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, numbers, steps, operations, elements, components, and/or groups thereof.
In the following description, specific details are provided to provide a thorough understanding of example embodiments. However, it will be understood by those of ordinary skill in the art that the example embodiments may be practiced without these specific details. For example, systems may be shown in block diagrams in order not to obscure the examples in unnecessary detail. In other instances, well-known processes, structures and techniques may be shown without unnecessary detail in order to avoid obscuring example embodiments.
Examples
The present embodiment provides a micro-droplet preparation system and a micro-droplet preparation system using the method for preparing uniform and stable micro-droplets using focused ultrasound.
As shown in fig. 1, which is a flow chart of the method for preparing uniform and stable micro-droplets by using focused ultrasound according to the present invention, the micro-droplet preparation using the system comprises the following steps:
s01 preparing a sample, a fluorinated oil phase, a temperature control environment and an adjustable ultrasonic environment;
s02, setting the temperature control environment to be a first temperature lower than the room temperature, and placing the sample in the temperature control environment for uniform and stable temperature reduction pretreatment to enable the sample to be in a low-temperature state lower than the room temperature;
wherein the first temperature is 8-12 ℃, in the embodiment, 10 ℃ is selected as the target temperature of the cooling treatment, and a water bath mode is adopted to perform uniform, continuous and stable cooling treatment.
S03, adding the oil phase containing fluoride into the pretreated low-temperature state sample and performing dispersion treatment;
s04, suspending the sample solution after dispersion treatment in an ultrasonic environment with the duty ratio of 200/500, controlling the temperature at the ultrasonic focus in the ultrasonic environment, so that the sample solution is rapidly heated in the ultrasonic focused environment, and forming uniform stable micro-droplets containing an oil phase and a water phase after a preset time.
Wherein, the set power of the ultrasonic environment is 40W to 60W, specifically in the present embodiment, 50W is adopted, and after 30 seconds of ultrasonic treatment under the condition of 200/500 duty ratio, uniform and stable micro-droplets can be obtained.
Further, referring to fig. 2, the present invention also provides a micro-droplet preparation system and a detection apparatus using the same. The micro-droplet preparation system comprises a control system, an ultrasonic generation module, a refrigeration module and a motion module.
The control system is used for adjusting parameters of the ultrasonic generation module, so that the frequency, the intensity, the duty ratio, the energy size, the application time and the like of a formed focusing sound field are adjusted, and the sample processing system is guaranteed to be adjusted to a sound field meeting the requirements according to different sample processing requirements.
The ultrasonic generation module is a sound field exciter with adjustable power and forms focused sound energy with high frequency and short wavelength.
The motion module is used to move the sample container into the sound field generated by the refrigeration module or the ultrasound generation module.
The refrigeration module is temperature controllable, and can provide the temperature control device of the stable temperature control environment of homogeneity, specifically in this embodiment, for the water bath, its effect lies in, provides the stable cooling environment of homogeneity for the refrigeration treatment of sample.
In the process of preparing the micro-droplets, a sample 1 and an oil phase object 2 are respectively placed in a closed sample tube 3, the sample tube 3 is placed on a sample rack 4 of a motion module, and the bottom of the sample tube, namely the part for containing the oil phase object 2 and the sample 1, enters the range of an ultrasonic focusing area 5 of the system through the movement of the sample rack 4.
When a sample 1 is placed in an ultrasonic focusing area, focused sound energy can be transmitted to a closed container through a liquid medium and acts on the sample to form an ultrasonic cavitation effect on the sample, namely micro gas core cavitation bubbles in liquid vibrate under the action of sound waves, when sound pressure reaches a certain value, growth and collapse occur, liquid in the bubbles impacts at high speed in all directions to generate extremely high pressure, and the water-in-oil sample after cooling treatment has the effects of dispersion and deagglomeration. Due to the fact that the oil phase substance is in a flowing state, the microorganism units finally form a dispersed microorganism solution in a uniform state under the continuous ultrasonic cavitation action. During the dispersion, the liquid medium temperature is kept constant.
Compared with the prior art, the method for preparing uniform and stable micro-droplets by adopting focused ultrasound is quiet, repeatable and controllable. The non-contact liquid drop preparation is realized through temperature control and ultrasonic adjustment, for example, sample pollution can be avoided, the dispersion process is simple to operate, and time and labor are saved; reduce the risk of infection of laboratory personnel and can help prepare a unified and standardized sample processing flow.
The present invention is not limited to the above-described alternative embodiments, and various other embodiments can be obtained by those skilled in the art from the above-described embodiments in any combination, and any other embodiments can be obtained in various forms while still being within the spirit of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined by the appended claims, which are intended to be interpreted according to the breadth to which the description is entitled. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for preparing uniform and stable micro-droplets by adopting focused ultrasound is characterized by comprising the steps of preparing a sample, preparing a fluorinated oil phase substance, controlling the temperature environment and adjusting the ultrasonic environment; carrying out uniform and stable temperature reduction pretreatment on the sample to enable the sample to be in a low-temperature state lower than room temperature; adding a fluorinated oil-containing phase substance into the pretreated low-temperature state sample and performing dispersion treatment; the method comprises the steps of forming a high-frequency short-wave focusing sound field by setting parameters of an ultrasonic forming device, placing a sample in an ultrasonic gathering area under an ultrasonic environment with the duty ratio of 200/500 through a suspended container, enabling a sample solution to be rapidly heated in the ultrasonic focusing environment, and forming uniform stable micro-droplets containing an oil phase and a water phase after a preset time.
2. The method of claim 1, wherein the ultrasonic environment is set at a power of 40W to 60W.
3. The method for preparing uniform and stable micro-droplets using focused ultrasound according to claim 1, wherein the first temperature is 8-12 ℃.
4. The method of claim 1, wherein the sample is placed in the focused acoustic field for a period of not less than 30 seconds.
5. The method of claim 1, wherein the temperature controlled environment is a water bath.
6. A micro-droplet preparation system using the method for preparing uniform and stable micro-droplets using focused ultrasound according to any one of claims 1 to 5, the system comprising a control system, an ultrasound generation module, a refrigeration module, and a motion module, the motion module being used for transporting a sample to the refrigeration module or the ultrasound generation module, the refrigeration module being used for forming a temperature controlled environment, and the ultrasound generation module being used for forming an ultrasound environment.
7. A detection apparatus, comprising:
at least one processor; and
a memory communicatively coupled to the at least one processor; wherein the content of the first and second substances,
the memory stores instructions executable by the at least one processor to enable the at least one processor to perform the method of any one of claims 1-5.
8. A non-transitory computer readable storage medium having stored thereon computer instructions for causing the computer to perform the method of any one of claims 1-5.
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| CN202210760418.7A CN115069319A (en) | 2022-06-29 | 2022-06-29 | Method and system for preparing liquid drops by focused ultrasound, detection equipment and storage medium |
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| CN202210760418.7A CN115069319A (en) | 2022-06-29 | 2022-06-29 | Method and system for preparing liquid drops by focused ultrasound, detection equipment and storage medium |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE1001659A6 (en) * | 1989-11-22 | 1990-01-23 | Phyber Holdings Ltd | A device for forming a liquid drop |
| US20020155231A1 (en) * | 2000-09-25 | 2002-10-24 | Ellson Richard N. | Use of immiscible fluids in droplet ejection through application of focused acoustic energy |
| WO2016133783A1 (en) * | 2015-02-17 | 2016-08-25 | Zalous, Inc. | Microdroplet digital pcr system |
| CN205665079U (en) * | 2016-06-03 | 2016-10-26 | 苏州工业园区海纳科技有限公司 | In vitro carries out non -contact ultrasonic wave focusing arrangement for sample preparation |
| CN106047687A (en) * | 2016-04-08 | 2016-10-26 | 周辉 | Ribonucleic acid chain type polymerization amplification reaction detecting device and detecting method thereof |
| CN112426943A (en) * | 2020-11-30 | 2021-03-02 | 深圳达远辰光科技有限公司 | Method for dispersing microorganisms by adopting focused ultrasound and sample processing system |
| CN213091308U (en) * | 2020-09-29 | 2021-04-30 | 深圳达远辰光科技有限公司 | Novel sample tube, sample bearing system and focused ultrasound sample processing system |
-
2022
- 2022-06-29 CN CN202210760418.7A patent/CN115069319A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE1001659A6 (en) * | 1989-11-22 | 1990-01-23 | Phyber Holdings Ltd | A device for forming a liquid drop |
| US20020155231A1 (en) * | 2000-09-25 | 2002-10-24 | Ellson Richard N. | Use of immiscible fluids in droplet ejection through application of focused acoustic energy |
| WO2016133783A1 (en) * | 2015-02-17 | 2016-08-25 | Zalous, Inc. | Microdroplet digital pcr system |
| CN106047687A (en) * | 2016-04-08 | 2016-10-26 | 周辉 | Ribonucleic acid chain type polymerization amplification reaction detecting device and detecting method thereof |
| CN205665079U (en) * | 2016-06-03 | 2016-10-26 | 苏州工业园区海纳科技有限公司 | In vitro carries out non -contact ultrasonic wave focusing arrangement for sample preparation |
| CN213091308U (en) * | 2020-09-29 | 2021-04-30 | 深圳达远辰光科技有限公司 | Novel sample tube, sample bearing system and focused ultrasound sample processing system |
| CN112426943A (en) * | 2020-11-30 | 2021-03-02 | 深圳达远辰光科技有限公司 | Method for dispersing microorganisms by adopting focused ultrasound and sample processing system |
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