On-chip real-time monitoring of multiple displacement amplification of DNA

Bruijns, Brigitte; Costantini, Francesca; Lovecchio, Nicola; Tiggelaar, Roald M.; Timoteo, Gaia Di; Nascetti, A.; Cesare, G. de; Gardeniers, Han; Caputo, D.

doi:10.1016/j.snb.2019.04.144

Cited by 16 publications

(25 citation statements)

References 29 publications

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“…The results of the DNA quantification are shown in Table 2. After a 2 h run, the average DNA concentration for uncoated chips was well above 200 ng/µL, which was in accordance with the run in the thermocycler (Figure 7) and the literature [37,38,59]. For purified DNA, the recommended amplification time by the manufacturer is 1.5 h, since the exonuclease activity of the polymerase enzyme might otherwise start to degrade the amplification product, which explains why after 2 h less DNA was measured than after 1.5 h (Figure 7).…”

Section: Resultssupporting

confidence: 89%

Cyclic Olefin Copolymer Microfluidic Devices for Forensic Applications

et al. 2019

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Microfluidic devices offer important benefits for forensic applications, in particular for fast tests at a crime scene. A large portion of forensic applications require microfluidic chip material to show compatibility with biochemical reactions (such as amplification reactions), and to have high transparency in the visible region and high chemical resistance. Also, preferably, manufacturing should be simple. The characteristic properties of cyclic olefin copolymer (COC) fulfills these requirements and offers new opportunities for the development of new forensic tests. In this work, the versatility of COC as material for lab-on-a-chip (LOC) systems in forensic applications has been explored by realizing two proof-of-principle devices. Chemical resistance and optical transparency were investigated for the development of an on-chip presumptive color test to indicate the presence of an illicit substance through applying absorption spectroscopy. Furthermore, the compatibility of COC with a DNA amplification reaction was verified by performing an on-chip multiple displacement amplification (MDA) reaction.

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Section: Resultssupporting

confidence: 89%

Cyclic Olefin Copolymer Microfluidic Devices for Forensic Applications

et al. 2019

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“…With enough enzymes and primers, the efficiency of the reaction can be guaranteed. It has been shown that performing MDA on a microarray increases the amplification yield of DNA [17]. The reason is that the chip plays the role of physical limitation, which allows a higher efficiency.…”

Section: Discussionmentioning

confidence: 99%

Low bias multiple displacement amplification with confinement effect based on agarose gel

et al. 2021

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Multiple displacement amplification (MDA) is a popular single-cell whole-genome amplification (WGA) technique that can greatly improve the amplification efficiency of single-cell genomes. However, there is an inherent problem that cannot be completely solved, that is, the amplification bias. We here propose an improved MDA method based on low melting agarose gel, named gelMDA. Firstly, the agarose gel and solution were characterized with SEM and fluorescent reagent. Then, we used gelMDA for cDNA amplification in library preparation of RNA-seq, and conventional MDA was used as a comparison. The sensitivity, efficiency of gelMDA, and amplification bias were evaluated with fluorescence curve, product yield, and the sequencing results. Finally, gelMDA was used for single-cell transcriptome sequencing. The results showed that the sensitivity and product yield of gelMDA were significantly higher than those of conventional MDA. A lower coefficient of variation (CV) and a higher reproducibility were obtained from gelMDA sequencing results. A region of 30 μm in diameter was amplified from the tissue sections and successfully sequenced. In conclusion, gelMDA obtained higher amplification efficiency and lower amplification bias in the present study. It suggested the great potential in single-cell RNA amplification and sequencing.

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“…Miniaturized heaters can be integrated as integrated resistive heaters, e.g., as deposited thin-film metal [26][27][28][29] or as laminated Cu foil [30], or as micro-Peltier elements [31,32]. These miniaturized heaters can be implemented directly onto the microfluidic chip [28] or on a different substrate and leter incorporated onto the microfluidic chip [33][34][35][36]. The geometry of such a heater contributes significantly to the uniformity of the heat distribution within the chip [26,37].…”

Section: State-of-the-artmentioning

confidence: 99%

“…Fabrication methods suitable for mass production are thermoforming/embossing or injection molding [41]. One of the materials which is biocompatible and suitable for both industrial scale fabrication technologies is cyclic olefin copolymer (COC) [42], which is one of the materials used in the past as well [28,36,[43][44][45]. Guckenberger et al estimates the costs of injection molding of only 50 simple microfluidic devices on $47, but this becomes cheaper when the mass production stage is reached [41].…”

Section: State-of-the-artmentioning

confidence: 99%

“…For clarity reasons, the two microfluidic structures are colored with food coloring dye. The reaction chamber is based on the work of Bruijns et al [36] and its dimensions are chosen in such way that the internal volume of the reaction chamber is the same as the reaction volume of the used Illustra GenomiPhi V2 DNA amplification kit (GE Healthcare Life Sciences, Eindhoven, The Netherlands) together with the EvaGreen dye solution (Biotium, Fremont, CA, USA), while maintaining an as low as possible surface-area-to-volume ratio [44]. Using SolidWorks 2018 computer-aided design (CAD) software (Dassault Systemes, Vélizy-Villacoublay, France), the 3D image of the chip is drawn and with the use of the AutoDesk HSMWorks computer-aided manufacturing (CAM) plug-in (Autodesk Inc., San Rafael, CA, USA), this image is transferred into a computer numerical control (CNC) milling code.…”

Section: Microfluidic Structure Designmentioning

confidence: 99%

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Disposable DNA Amplification Chips with Integrated Low-Cost Heaters †

et al. 2020

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Fast point-of-use detection of, for example, early-stage zoonoses, e.g., Q-fever, bovine tuberculosis, or the Covid-19 coronavirus, is beneficial for both humans and animal husbandry as it can save lives and livestock. The latter prevents farmers from going bankrupt after a zoonoses outbreak. This paper describes the development of a fabrication process and the proof-of-principle of a disposable DNA amplification chip with an integrated heater. Based on the analysis of the milling process, metal adhesion studies, and COMSOL MultiPhysics heat transfer simulations, the first batch of chips has been fabricated and successful multiple displacement amplification reactions are performed inside these chips. This research is the first step towards the development of an early-stage zoonoses detection device. Tests with real zoonoses and DNA specific amplification reactions still need to be done.

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On-chip real-time monitoring of multiple displacement amplification of DNA

Cited by 16 publications

References 29 publications

Cyclic Olefin Copolymer Microfluidic Devices for Forensic Applications

Cyclic Olefin Copolymer Microfluidic Devices for Forensic Applications

Low bias multiple displacement amplification with confinement effect based on agarose gel

Disposable DNA Amplification Chips with Integrated Low-Cost Heaters †

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