基于自-digitization芯片的低成本和robust数字PCR设备

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"数字PCR芯片技术" 本文讨论了一种自-digitization芯片，集成了水化层，用于低成本和robust数字PCR（digital PCR）。该芯片具有自-digitization功能，可以实现低成本和高效的数字PCR检测。 **数字PCR技术** 数字PCR（digital PCR）是一种高灵敏度和高精度的PCR检测技术，能够对少量的核酸样本进行检测。数字PCR技术通过将PCR反应分割成多个微小的反应室，实现对每个反应室中的核酸样本的检测。这种技术可以提供高精度和高灵敏度的检测结果。 **自-digitization芯片** 自-digitization芯片是一种特殊的微流体芯片，可以实现对PCR反应的自动检测和分析。该芯片集成了水化层，可以实现对PCR反应的稳定和可靠的检测。自-digitization芯片可以实现低成本和高效的数字PCR检测。 **水化层** 水化层是一种特殊的材料层，能够稳定PCR反应的温度和湿度环境。水化层可以防止PCR反应中的水分损失，确保PCR反应的稳定和可靠性。 **微流体技术** 微流体技术是一种微尺度流体处理技术，能够实现对PCR反应的微小化和自动化。微流体技术可以实现对PCR反应的高效、低成本和高灵敏度的检测。 **量子点检测** 量子点检测是一种高灵敏度的检测技术，能够对PCR反应中的核酸样本进行检测。量子点检测可以提供高精度和高灵敏度的检测结果。 **低成本和robust数字PCR** 该芯片可以实现低成本和robust数字PCR检测，能够提供高精度和高灵敏度的检测结果。该芯片可以应用于各种领域，例如疾病诊断、基因检测和生命科学研究等。 **结论** 自-digitization芯片集成了水化层，能够实现低成本和robust数字PCR检测。该芯片可以提供高精度和高灵敏度的检测结果，具有广泛的应用前景。

2.3. Chip Operation

The basic operating procedure for the self-partition in the proposed chip is outlined in Fig. 3. Firstly, adopted

from our previously reported “place n play” (PnP) PDMS pump strategy [27], a piece of PDMS pump slab was

attached to the outlets of the chip, which aimed to accelerate the self-partition process of the microfluidic chip.

Meanwhile, a piece of adhesive tape covered the bottom glass slide and sealed all openings connected to the void

layer under the digital PCR array. Afterwards, the chip was placed in a vacuum desiccator and degassed at 10 kPa for

a certain period, and then sealed in air-tight packaging for ready use. After the chip was taken out from vacuum, a 20

μL of PCR premix was immediately dispensed into the inlet port of the chip using a conventional micropipette. After

all the channels and chambers were completely filled with the sample, the sealing tape covering the bottom glass slide

was peeled off and then a few drops of deionized (DI) water was loaded to fill the void layer of the chip. Meanwhile a

10:1:20 weight mixture of PDMS pre-polymer, cross-linker and silicone oil was added in the inlet port of the chip to

remove the excess sample in the channel and isolate each chamber. Once the oil phase arrived at the outlet ports, the

PnP PDMS pump slab was peeled off from the chip. Finally, all inlet/oulet ports of the chip were sealed with tape, and

the chip was transferred to a flat PCR apparatus (Mastercycler nexus flat, Eppendorf, German) for amplification

reaction.

2.4. PCR amplification

In this work, EGFR genomic DNA (gDNA) purified from lung cancer cell line H1975 was selected as DNA

template. The EGFR gDNA was extracted using a QIAprep Blood & Cell Culture DNA Medi Kit according to the

manufacturer’s protocol. To assess the performance of the proposed dPCR chip, the EGFR gDNA solution was

serially diluted to yield dilutions ranging from 10

to 10

copies/L. All DNA samples were stored at −20°C prior to

use.

The primer sequences for EGFR gene in exon 20 used in this study were as follows: forward primer,

5’-ATCTGCCTCACCTCCACC-3’, and reverse primer, 5’-CACCAGTTGAGCAGGTACTG-3’. A HEX-labeled

hydrolysis probe (5’-HEX-CCGGGAACACAAAGACAA-MGB-3’) was used to trace all the PCR products while a

FAM-labeled hydrolysis probe (5’-FAM-CTGCATGATGAGCTGC-MGB-3’) was used to trace the PCR products

with T790M mutation. The reaction mix (20 μL) consisted of 5 × BioDigital dPCR Master Mix 5 μL, forward/reverse

primers 1 μL (0.4 mM), HEX/FAM probes 1 μL (0.4 mM), RNase-free water 10 μL and a serially diluted template 1

μL. Each reaction mix was premixed off-chip before sampling.

After sampling, the dPCR chip was placed on a flat PCR apparatus for PCR reaction with a two-step PCR

thermocycling. Firstly, an initialization step of 10 min at 50°C was used to digest any potential DNA contamination.

Then, a 10 min “hot start” was performed at 95°C, to activate Taq DNA polymerase. After that, forty-five cycles of

95°C for 20 s and 60°C for 40 s were performed to amplify the target DNA. The entire PCR reaction took about 77

min (including the heating and cooling time in each thermal cycling). No template control (NTC) reactions were

performed using sterile, double-distilled water with no template in each run of dPCR, to detect the presence of

contamination. Each experiment was done at least in triplicate.

2.5 Image Acquisition and Analysis

All bright field images were acquired with an inverted microscope (NIB900, Nexcope, USA), and all

fluorescence images were acquired using a monochrome CMOS camera (Nikon DS-Qi2, Japan) mounted to a Nikon

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