Fast and robust sample self-digitization for digital PCR

Cui, Xu; Wu, Lei; Wu, Yin; Zhang, Jinghong; Zhao, Qiang; Fu, Jing; Yi, Lin; Li, Gang

doi:10.1016/j.aca.2020.02.010

Cited by 27 publications

(22 citation statements)

References 30 publications

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“…The well array was completely primed with sample in less than 5 s. After the completion of priming, the channel layer was removed from the well array layer to immediately isolate all primed wells and achieve a rapid partitioning. Note that in contrast to the previous compartment-based digital platforms which were in monolithic channel-well configurations, 36–39, 41, 42 our DSD chip had a separated channel-well configuration. This configuration allows a direct removal of the excess sample interconnecting the primed wells via detachment without a lengthy flushing step, and thus resulting in an ultra-fast sample self-digitization.…”

Section: Resultsmentioning

confidence: 97%

“…In the reversibly assembled chip, the sample loading channels and partitions holding wells are located on separate layers; we call the device a detachable self-digitization (DSD) chip to distinguish it from the monolithic channel-well configuration reported previously. 36–39, 41, 42 As schematically depicted in Figure 1, the sample loading function layer contains an array of parallel microfluidic channels which are all connected to sample inlet via four main channels, and the partitions holding function layer contains a high-density array of microwells. In the state of assembly, the sample loading channels overlap the microwell array, ensuring the priming of all microwells during sample loading.…”

Section: Figurementioning

confidence: 99%

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A Facile and Rapid Route to Self-Digitization of Samples into a High Density Microwell Array for Digital Bioassays

Cui

Chen

et al. 2021

Preprint

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Digital bioassays are powerful methods to detect rare analytes from complex mixtures and study the temporal processes of individual entities within biological systems. In digital bioassays, a crucial first step is the discretization of samples into a large number of identical independent partitions. Here, we developed a rapid and facile sample partitioning method for versatile digital bioassays. This method is based on a detachable self-digitization (DSD) chip which couples a reversible assembly configuration and a predegassing-based self-pumping mechanism to achieve an easy, fast and large-scale sample partitioning. The DSD chip consists of a channel layer used for loading sample and a microwell layer used for holding the sample partitions. Benefitting from its detachability, the chip avoids a lengthy oil flushing process used to remove the excess sample in loading channels and can compartmentalize a sample into more than 100,000 wells of picoliter volume with densities up to 14,000 wells/cm2 in less than 30 s. We also demonstrated the utility of the proposed method by applying it to digital PCR and digital microbial assays.

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Section: Resultsmentioning

confidence: 97%

Section: Figurementioning

confidence: 99%

A Facile and Rapid Route to Self-Digitization of Samples into a High Density Microwell Array for Digital Bioassays

Cui

Chen

et al. 2021

Preprint

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“…To introduce the sample solution into 3D chamber array, a bidirectional dispensing network was adopted, in which the inlet is located in the middle of the chamber array and the liquid flows in two directions simultaneously. Compared to the unidirectional distribution network, 35,39 the bidirectional one has a half of the path distance of liquid flow in the former. The shortened channels reduce flow resistance and save the time for sample loading.…”

Section: Chip Designmentioning

confidence: 99%

A portable and high-integrated 3D microfluidic chip for bacterial quantification and antibiotic susceptibility testing

Cai

Liu

2021

Preprint

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On-site single-cell antibiotic susceptibility testing (sc-AST) provides unprecedented technical potential to improve the treatment of bacterial infections and study heterogeneous resistance to antibiotics. Herein, we developed a portable and high-integrated 3D polydimethylsiloxane (PDMS) chip to perform fast and on-site bacteria quantification and sc-AST. The 3D arrangement of the chambers significantly improved the integration of reaction units (∼10000/cm2) and widened the dynamic range to 5 orders of magnitude. A capillary valve-based flow distributor was adopted for flow equidistribution in 64 parallel channels and uniform sample loading in as short as 2 s. The degassed PDMS enabled this device to independently dispense the sample into 3D chamber array with almost 100% efficiency. The quantification of Escherichia coli (E. coli) strains with various activity was accomplished in 0.5-2 h, shortened by 20 h in comparison to the traditional plate counting. The functionality of our platform was demonstrated with several effective antibiotics by determining minimum inhibitory concentrations at single-cell level. Furthermore, we utilized the lyophilization of test reagents and needle-mediated reagents rehydration to realize one-step on-site sc-AST. The results indicate that the proposed sc-AST platform is portable, highly sensitive, fast, accurate and user-friendly, thus it has the potential to facilitate precise therapy in time and monitor the treatment. Meanwhile, it could serve as an approach for investigating the mechanisms of heteroresistance at single-cell resolution.

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“…According to previous studies [28], the theoretical formula applicable to LHMC can be obtained as follows:…”

Section: Statistical Processingmentioning

confidence: 99%

“…The chips in this article have been improved over 3 generations. In previous studies [27,28], the speci c structure and performance of the previous generations of chips have been veri ed many times. LHMC has more microcavity structures and more stable performance than before, as well as faster sample loading speed.…”

Section: Introductionmentioning

confidence: 99%

Large Number Hexagonal Cavities Microfluidic Digital Chip for Gene Mutation Ultrasensitive Analysis in Lung Cancer

Liu¹,

Pan²,

Guo³

et al. 2020

Preprint

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Lung cancer is the highest incidence of malignant tumors in the world. Targeted therapy based on gene mutation detection including epidermal growth factor receptor (EGFR) gene mutation is one of the first-line treatments. In this work, took EGFR gene G719S mutation as a representative object, a large number hexagonal cavities microfluidic chip (LHMC) platform based on digital PCR (dPCR) for the absolute quantification of gene mutation in lung cancer was established. In this chip, 113,137 cavities chip was designed to increase the number of absolute quantitative, which was 2~5 times higher than the traditional one. The hexagonal shape of cavities elevates the filling rate and filling speed. A set of primers and probes for G719S with high sensitivity, high specificity and high reliability were designed and screened. Then the PCR parameters were optimized. The results demonstrated that the chip platform shows good performance. The minimum detection concentration of the gene mutant was 3.01 copies/μL, and a good correlation (Y= 0.725X- 0.581, R2= 0.984) was noted between the measured value and the expected value. This chip possesses sensitively detect positive mutations in G719S and completely negative when detecting other substances. The developed LHMC-dPCR chip is a rapid and accurate gene mutation analysis platform, which has faster speed and lower price than classic detection methods, for instance, droplet dPCR, DNA sequencing method. Moreover, LHMC-dPCR is not limited by the number of nucleic acids and droplets and there is no need to estimate the nucleic acid concentration of the sample. This chip platform could also detect other gene mutations, for example, L858R, exon 19 deletions, in other tumors including lung cancer.

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Fast and robust sample self-digitization for digital PCR

Cited by 27 publications

References 30 publications

A Facile and Rapid Route to Self-Digitization of Samples into a High Density Microwell Array for Digital Bioassays

A Facile and Rapid Route to Self-Digitization of Samples into a High Density Microwell Array for Digital Bioassays

A portable and high-integrated 3D microfluidic chip for bacterial quantification and antibiotic susceptibility testing

Large Number Hexagonal Cavities Microfluidic Digital Chip for Gene Mutation Ultrasensitive Analysis in Lung Cancer

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