CN110643486A

CN110643486A - Ultrasonic sound field device and digital PCR liquid drop array chip manufacturing method

Info

Publication number: CN110643486A
Application number: CN201910914528.2A
Authority: CN
Inventors: 顾忠泽; 田良飞; 杜鑫; 葛芹玉
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2020-01-03
Anticipated expiration: 2039-09-25
Also published as: CN110643486B

Abstract

The invention discloses an ultrasonic sound field device and a method for manufacturing a digital PCR (polymerase chain reaction) liquid drop array chip. The method comprises the steps that a, an ultrasonic sound field device based on a piezoelectric ceramic device is established, and the integral size and the liquid drop density of an obtained liquid drop array are adjusted by controlling the frequency and the size of the ultrasonic device; b. preparing a PCR emulsion; c. and (3) placing the sealed emulsion in an ultrasonic sound field device, starting the device to generate a stable two-dimensional sound field, and gradually forming a uniform two-dimensional array on the node of the standing wave by the water phase in the PCR emulsion. The invention uses a simple piezoelectric ceramic-based sound field device to construct a digital PCR reaction system with a picoliter level, the technology is mature, the cost is low, and the size of the chip can be regulated and controlled in a large range.

Description

Ultrasonic sound field device and digital PCR liquid drop array chip manufacturing method

Technical Field

The invention relates to a method for manufacturing a PCR biochip, in particular to an ultrasonic sound field device and a method for manufacturing a digital PCR liquid drop array chip.

Background

Polymerase Chain Reaction (PCR) is a method for amplifying specific DNA fragments through in vitro enzymatic synthesis, is the most conventional and key technology in the field of molecular biology, plays an irreplaceable role in nucleic acid-related research, and promotes the development of various fields of life science. Quantitative PCR is a technology which is developed on the basis of PCR and can carry out quantitative analysis on nucleic acid molecules more accurately, but the quantitative accuracy is influenced by a plurality of aspects, wherein the influence factors of amplification efficiency are many, and the fact that the amplification efficiency of a sample to be detected is the same as that of a standard sample is difficult to guarantee in actual detection, so that the Ct value (cycle threshold) which is depended on by the quantitative analysis is greatly influenced, therefore, the conventional quantitative PCR is only relatively quantitative, and the accuracy and the reproducibility are difficult to meet the requirements of the current molecular biology quantitative analysis. In addition, because the PCR product has a certain inhibition effect on enzyme catalytic reaction, the existing gene mutation detection method based on quantitative PCR is almost useless for gene variation with low abundance in somatic cells.

With the recent continuous development of biotechnology, absolute quantitative PCR based on a segmentation system has come into play, in which a method of accurately calculating DNA concentration by a limiting dilution method and according to the statistical principle of poisson distribution is called digital PCR (dPCR). The method mainly adopts the current hot microfluidic or micro-droplet method to disperse a large amount of diluted nucleic acid solution into micro-reactors or droplets of a chip, wherein the number of nucleic acid molecules in each reactor is less than or equal to 1. Thus, after PCR amplification, the reactor with the template will give a signal, and the one without the template will not be detected. Based on the relative proportions and the volume of the reactor, the nucleic acid concentration of the original solution can be deduced. Compared with the traditional quantitative PCR technology, the method is not influenced by amplification efficiency, does not need to establish a standard curve, and realizes absolute quantitative analysis without depending on a cycle threshold.

In addition, the extremely high sensitivity and tolerance are also one of the main characteristics of the digital PCR, trace mutant DNA can be identified in a large amount of wild type DNA background, the effective template DNA segmentation system can avoid the amplification inhibition of high-abundance allele nucleic acid on variant nucleic acid, and the detection efficiency of rare mutant nucleic acid is greatly improved. And the limit dilution also greatly reduces the mutual influence of various components among reaction systems, and the interference of background sequences, inhibitors and the like on the reaction is also reduced to the minimum. Droplet digital PCR can detect as low as 0.001% of the mutated fragments, whereas conventional sequencing and fluorescent quantitative PCR cannot detect less than 1% of the mutations at all.

The traditional operation flow of the digital PCR is complex, generally, dilution and sample distribution are carried out manually, then PCR amplification is carried out, corresponding fluorescent signals are read through an instrument after reaction is finished, and finally the concentration or copy number of target molecules to be detected is calculated according to the Poisson distribution principle and the proportion of positive liquid drops. The method has the advantages of complex operation, small flux, low efficiency and limited application range. In recent years, the development of the microfluidic technology and the micro-droplet technology greatly promotes the progress of the digital PCR, the microfluidic technology is automatic, easy to integrate and high in flux, and the detection efficiency of the digital PCR can be greatly improved.

The microdroplet technique is the use of two immiscible phases to form relatively stable individual microdroplets of water-in-oil. There are various ways of emulsification, of which droplet generation techniques based on microfluidic chips are most commonly used. The principle is that one continuous liquid flow is broken to form liquid drops under the mutual extrusion action of a certain angle between two liquid flows. The current common liquid drop preparation methods include an orthogonal structure, flow focusing and the like. The methods have the disadvantages of complex system of the whole equipment, high requirement on a fluid control system, troublesome manufacturing of the digital PCR liquid drop chip, high cost and difficulty in preventing liquid drop evaporation.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention aims to provide an ultrasonic field device, and the invention also aims to provide a digital PCR liquid drop array chip manufacturing method based on the ultrasonic field device.

The technical scheme is as follows: according to the ultrasonic field device, the sine wave is applied to the piezoelectric ceramics with the same resonance frequency through the signal generator, and the radiation force generated by the sound wave can capture liquid drops in the emulsion, so that the liquid drops are arranged in a two-dimensional mode at a specific position. The piezoelectric ceramic is PZT lead zirconate titanate.

The digital PCR liquid drop array chip manufacturing method based on the ultrasonic sound field device comprises the following steps:

a. establishing an ultrasonic sound field device based on piezoelectric ceramics, and adjusting the overall size and the droplet density of the obtained droplet array by controlling the frequency and the size of the ultrasonic device;

b. preparing a PCR emulsion which comprises an oil phase and a water phase, wherein the water phase is a mixed solution of a digital PCR reaction kit template DNA, the oil phase is one or more of silicone oil, mineral oil and vegetable oil, and the mass ratio of the water phase to the oil phase is 1: 2-5;

c. the sealed emulsion is placed in an ultrasonic sound field device, the device is started, two groups of piezoelectric ceramics generate two beams of orthogonal standing waves, a stable two-dimensional sound field is generated in the sound field device, and water phase droplets in the PCR emulsion gradually form a uniform two-dimensional array on nodes of the standing waves under the combined action of sound radiation force and gravity. The spacing between the droplets can be achieved by controlling the wavelength of the standing wave, and the size of the droplets can be achieved by controlling the wavelength of the standing wave and the water-oil ratio.

Preferably, the oil phase of the PCR emulsion comprises 90-96 wt% of mineral oil, 4-6 wt% of surfactant Sp80 and the balance of Tween 80. The oil phase of the PCR emulsion comprises AbileEM 903-4 wt%, Triton X-1000.1-0.5 wt% and the balance of water phase. The PCR emulsion comprises 35-60 wt% of silicone oil, 20-30 wt% of a mixture of a silicone compound and trimethylsiloxy silicate and 20-30 wt% of dimethylpolysiloxane.

Has the advantages that: compared with the prior art, the invention has the following remarkable characteristics:

1. the piezoelectric ceramic-based sound field device is used for constructing a digital PCR reaction system with a pico-liter level, the technology is mature, the cost is low, and the size of a chip can be regulated and controlled in a large range;

2. the method can form a droplet array with controllable size, density and position in a PCR emulsion mixture, the process is simple and quick, the whole system can be closed after the emulsion is configured, and various problems caused by droplet evaporation can be greatly reduced;

3. the method of the invention does not directly contact with organisms, and can effectively avoid the pollution of samples.

Drawings

FIG. 1 is a schematic structural diagram of an ultrasonic field apparatus of the present invention;

fig. 2 is a process diagram of the present invention.

Detailed Description

As shown in FIG. 1, four sets of PZT lead zirconate titanate piezoelectric ceramics 4 are disposed on a substrate and are adhered to four sides of an ultrasonic field device 5, and a sealed glass box containing a PCR emulsion 3 is provided in the ultrasonic field device 5. And starting the device, and forming a liquid drop array in situ after the sound field is formed.

Example 1

a. Establishing an ultrasonic sound field device 5 based on a piezoelectric ceramic device, and adjusting the overall size and the droplet density of the obtained droplet array by controlling the frequency and the size of the ultrasonic device;

b. preparing a PCR emulsion 3, wherein an oil phase 1 comprises mineral oil (Sigma-Aldrich M-3516)95.05 wt%, a surfactant Sp80 (Mecline) 4.5 wt%, Tween80 (Mecline) 0.45 wt%, and a water phase 2 is a mixed solution of template DNA of the digital PCR reaction kit, and the mass of the mixed solution is 1/3 of the oil phase 1;

c. the sealed emulsion is placed in an ultrasonic sound field device 5, the device is started, two groups of piezoelectric ceramic devices generate two orthogonal standing waves, and a stable two-dimensional sound field is generated in the sound field device, in the specific two-dimensional sound field, small droplets of the water phase 2 in the PCR emulsion 3 are subjected to the combined action of acoustic radiation force and gravity, and a uniform two-dimensional array is gradually formed on nodes of the standing waves, as shown in figure 2.

And c, replacing the mineral oil in the step b by any content of 90-96 wt%, replacing the surfactant Sp80 by any content of 4-6 wt%, and balancing Tween 80.

Example 2

b. preparing a PCR emulsion 3, wherein an oil phase 1 is mineral oil (Sigma-Aldrich M-3516) and contains AbilEM 903% and Triton X-100 (polyethylene glycol octyl phenyl ether) 0.1% as a surfactant, and a water phase 2 is a PCR reaction system and is 1/2 of the oil phase 1 in mass;

c. the sealed emulsion is placed in an ultrasonic sound field device 5, the device is started, two groups of piezoelectric ceramic devices generate two beams of orthogonal standing waves, a stable two-dimensional sound field is generated in the sound field device, and in the specific two-dimensional sound field, droplets of the water phase 2 in the PCR emulsion 3 are subjected to the combined action of acoustic radiation force and gravity to gradually form a uniform two-dimensional array on nodes of the standing waves.

Wherein, in the step b, AbileEM 90 of the oil phase 1 can be replaced by any content of 3-4 wt%, Triton X-100 can be replaced by any content of 0.1-0.5 wt%, and the balance is mineral oil.

Example 3

b. preparing a mixed solution of 3 g of PCR emulsion, 10g of silicone oil, 7.5g of 749FLUID (silicone compound, trimethylsiloxy silicate) and 7.5g of 5225C Formulation Aid (cyclopenasiloxane and PEG/PPG-18/18dimethicone dimethyl polysiloxane), and 10g of template DNA of a digital PCR reaction kit;

Wherein the silicone oil in the step b can be replaced by any content of 10-12 g, the 749FLUID can be replaced by any content of 7.5-8.5 g, and the dimethylpolysiloxane can be replaced by any content of 7.5-8.0 g.

Example 4

b. preparing a PCR emulsion 3, wherein an oil phase 1 is 95.5 wt% of mineral oil (Sigma-Aldrich M-3516), a surfactant Sp 804 wt% and Tween 800.5 wt%, a water phase 2 is a mixed solution of template DNA of the digital PCR reaction kit, and the mass of the mixed solution is 1/5 of the oil phase 1;

Claims

1. An ultrasonic acoustic field apparatus, characterized by: the signal generator applies sine waves to the piezoelectric ceramics (4) with the same resonance frequency, and the radiation force generated by the sound waves can capture the liquid drops in the emulsion, so that the liquid drops are arranged in a two-dimensional mode at a specific position.

2. An ultrasonic acoustic field apparatus according to claim 1, wherein: the piezoelectric ceramic (4) is PZT lead zirconate titanate.

3. A digital PCR liquid drop array chip manufacturing method based on an ultrasonic sound field device is characterized by comprising the following steps:

(a) establishing an ultrasonic sound field device (5) based on piezoelectric ceramics (4), and adjusting the overall size and the droplet density of the obtained droplet array by controlling the frequency and the size of the ultrasonic device;

(b) preparing a PCR emulsion (3) comprising an oil phase (1) and a water phase (2);

(c) the sealed emulsion is placed in an ultrasonic sound field device (5), the device is started, two groups of piezoelectric ceramics (4) generate two beams of orthogonal standing waves, a stable two-dimensional sound field is generated in the sound field device, and droplets of the water phase (2) in the PCR emulsion (3) are subjected to the combined action of acoustic radiation force and gravity to gradually form a uniform two-dimensional array on nodes of the standing waves.

4. The method for manufacturing the digital PCR liquid drop array chip based on the ultrasonic sound field device according to claim 3, wherein the method comprises the following steps: the water phase (2) is a mixed solution of the template DNA of the digital PCR reaction kit.

5. The method for manufacturing the digital PCR liquid drop array chip based on the ultrasonic sound field device according to claim 3, wherein the method comprises the following steps: the mass ratio of the water phase (2) to the oil phase (1) is 1: 2-5.

6. The method for manufacturing the digital PCR liquid drop array chip based on the ultrasonic sound field device according to claim 3, wherein the method comprises the following steps: the oil phase (1) comprises one or more of silicone oil, mineral oil and vegetable oil.

7. The method for manufacturing the digital PCR liquid drop array chip based on the ultrasonic sound field device according to claim 3, wherein the method comprises the following steps: the oil phase (1) comprises 90-96 wt% of mineral oil, 4-6 wt% of surfactant Sp80 and the balance Tween 80.

8. The method for manufacturing the digital PCR liquid drop array chip based on the ultrasonic sound field device according to claim 3, wherein the method comprises the following steps: the oil phase (1) comprises surfactant Degussa AbileEM 903-4 wt% and Triton X-1000.1-0.5 wt%.

9. The method for manufacturing the digital PCR liquid drop array chip based on the ultrasonic sound field device according to claim 3, wherein the method comprises the following steps: the oil phase (1) comprises 35-60 wt% of silicone oil, 20-30 wt% of a mixture of a silicone compound and trimethylsiloxy silicate, and 20-30 wt% of dimethylpolysiloxane.