Oil phase composition for preparing droplet digital PCR liquid drops

A composition and micro-droplet technology, which is applied in the field of microfluidics, can solve the problems of increasing the cost of preparing droplets, fluorinated oil is volatile, and the price of surfactants is expensive, and achieves uniform size, good thermal stability, and excellent properties. stable effect

Active Publication Date: 2017-05-31
领航医学科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, fluorinated oils are volatile, and soluble gases will generate a large number of bubbles when heated. On the other hand, some surfactants related to fluorinated oils are relatively expensive, which increases the cost of preparing droplets.

Method used

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  • Oil phase composition for preparing droplet digital PCR liquid drops
  • Oil phase composition for preparing droplet digital PCR liquid drops
  • Oil phase composition for preparing droplet digital PCR liquid drops

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] At present, fluorinated oils are commonly used in the preparation of droplets, such as FC-40 and FC-7500. The surfactants used are: PFPE-PEG-PFPE, 1,1,2,2-tetrahydroperfluorodecyl alcohol, etc. . On the one hand, fluorinated oils are volatile, and soluble gases will generate a large number of bubbles when heated. On the other hand, some surfactants related to fluorinated oils are relatively expensive, which increases the cost of preparing droplets. Mineral oil has the advantages of non-polarity, insoluble in water, non-volatile, stable properties, no inhibitory effect on PCR amplification, and low kinematic viscosity (cps) of light oil, etc., and is often used for liquid sealing of PCR reaction solution, etc. . Therefore, the present invention chooses to use mineral oil as the basis of the main component. However, the kinematic viscosity of mineral oil alone as the main component of the oil phase is still high, and the droplet formation and sedimentation speed are low...

Embodiment 2

[0030] According to the experimental results of Example 1, further add different surfactants to the oil phase composition, according to the solubility of different surfactants and oil phase main components, design the following several oil phase compositions (all in mass):

[0031] (1) 1 / 2 mineral oil + 1 / 2 n-tetradecane + 3% EM90

[0032] (2) 1 / 2 mineral oil + 1 / 2DEC + 3% EM90

[0033] (3) 1 / 2 mineral oil + 1 / 2 n-tetradecane + 3% TritonX-100

[0034] (4) 1 / 2 mineral oil + 1 / 2DEC + 3% TritonX-100

[0035] (5) 1 / 2 mineral oil + 1 / 2 n-tetradecane + 3% EQ503

[0036] (6) 1 / 2 mineral oil + 1 / 2DEC + 3% EQ503

[0037] In each oil phase composition, the ratio of mineral oil and n-tetradecane refers to the mass ratio of the main component, and the ratio of the surfactant is the ratio of the respective mass to the main component.

[0038] Results: The surfactants in the oil phase compositions (5) and (6) were incompatible with the oil phase, and the rest could be better miscible.

Embodiment 3

[0040] The oil phase compositions of (1) to (4) groups screened in Example 2 were used to perform droplet preparation experiments and perform PCR amplification. The specific process is as follows:

[0041] (1) Prepare the oil phase according to the schemes (1)-(4) above.

[0042] (2) Prepare the aqueous phase, that is, the PCR reaction system.

[0043] The template comes from non-small cell lung cancer (NSCLC) cell line H1975, which has two mutations, T790M and L858R.

[0044] The primer sequences are:

[0045] F: 5'-GCCTGCTGGGCATCTG-3';

[0046] R: 5'-TCTTTGTGTTCCCGGACATAGAC-3'

[0047] The probe sequence is:

[0048] 5'-FAM-ATGAGCTGCATGATGAG-MGB-NFQ-3', wherein FAM is a fluorescent reporter group, and NFQ is a fluorescent quencher group.

[0049] Water Phase Formulation:

[0050]

[0051]

[0052] (3) Spread the oil phase in the droplet generating device.

[0053] (4) Use a syringe pump to add the water phase to the oil phase by step emulsification and form dro...

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Abstract

The invention discloses an oil phase composition for preparing droplet digital PCR liquid drops. The oil phase composition comprises main constituents and surfactants. The main constituents include mineral oil and alkane, and the surfactants include EM90 and TritonX-100, wherein the alkane is one of 12-16 alkane or a mixture of 12-16 alkane. Fluorinated oil is replaced by mineral oil, and the oil phase composition has the advantages that the oil phase composition is nonpolar, insoluble in water, not prone to volatilization, stable in property and free of inhibiting effect on PCR amplification, and the kinematic viscosity of light oil is low; meanwhile, kinematic viscosity is further reduced by adding proper alkane, then the proper surfactants EM90 and TritonX-100 are added, and finally, the oil phase composition is used for preparing liquid drops. The obtained liquid drops have the advantages that sizes are uniform, thermal stability is high, volatilization and foam generation do not occur easily, and amplification inhibition is avoided.

Description

technical field [0001] The invention belongs to the field of microfluidic technology, and in particular relates to an oil phase composition used for preparing droplets in microdrop digital PCR. Background technique [0002] Emulsification is the process by which a first liquid is dispersed into a second immiscible liquid. Common emulsified droplets are water-in-oil, oil-in-water, etc. Water-in-oil (W / O) droplets are water dispersed in oil in the form of small droplets. The emulsification system consists of oil phase, water phase and surfactant. The water phase is the internal phase or dispersed phase, and the oil is the external phase or dispersion medium. Surfactants are generally composed of non-polar, lipophilic hydrocarbon chain parts and polar, hydrophilic groups. Oleophilic dual nature. The addition of surfactant is crucial to the formation and stability of droplets, which can greatly reduce the tension of the oil / water interface and form an interfacial film by ads...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2563/159C12Q2527/125
Inventor 赵然卢旖
Owner 领航医学科技(深圳)有限公司
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