SIZE-CONTROLLED DROPLET GENERATION IN A MICROFLUIDIC DEVICE FOR RARE DNA AMPLIFICATION BY OPTIMIZING ITS EFFECTIVE PARAMETERS

Versatility and portability of microfluidic devices play a dominant role in their widespread use by researchers. Droplet-based microfluidic devices have been extensively used due to their precise control over sample volume, and ease of manipulating and addressing each droplet on demand. Droplet-based polymerase chain reaction (PCR) devices are particularly desirable in single DNA amplification. If the droplets are small enough to contain only one DNA molecule, single molecule amplification becomes possible, which can be advantageous in several cases such as early cancer detection. In this work, flow-focusing microfluidic droplet generation’s parameters are numerically investigated and optimized for generating the smallest droplet possible, while considering fabrication limits. Taguchi design of experiment method is used to study the effects of key parameters in droplet generation. By exploiting this approach, a droplet with a radius of 111[Formula: see text]nm is generated using a 3[Formula: see text][Formula: see text]m orifice. Since the governing physics of the droplet generation process is not totally understood yet, by means of analysis of variance (ANOVA) analysis, a generalized linear model (GLM) is proposed to predict the droplet radius, given the values of eight major parameters affecting the droplet size. The proposed model shows a correlation of 95.3% and 64.95% for droplets of radius greater than and lower than 5[Formula: see text][Formula: see text]m, respectively. Finally, the source of this variation of behavior in different size scales is identified.

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A 3D tubular structure with droplet generation and temperature control for DNA amplification

Microfluidics and Nanofluidics ◽

10.1007/s10404-021-02454-7 ◽

2021 ◽

Vol 25 (7) ◽

Author(s):

Shaw-Hwa Parng ◽

Ping-Jung Wu ◽

Yu-Yin Tsai ◽

Ruey-Shyan Hong ◽

Su-Jan Lee

Keyword(s):

Temperature Control ◽

Dna Amplification ◽

Tubular Structure ◽

Droplet Generation

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Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

Clinical Chemistry ◽

10.1373/clinchem.2014.228304 ◽

2015 ◽

Vol 61 (1) ◽

pp. 145-153 ◽

Cited By ~ 104

Author(s):

Jared S Farrar ◽

Carl T Wittwer

Keyword(s):

High Resolution ◽

Single Molecule ◽

High Resolution Melting ◽

Dna Amplification ◽

Temperature Cycling ◽

High Yield ◽

Temperature Cycle ◽

Amplification Efficiency ◽

Analytical Sensitivity ◽

Agarose Gels

Abstract BACKGROUND PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62 °C–76 °C and denaturation temperatures of 85 °C–92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

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Self-Aligned Interdigitated Transducers for Acoustofluidics

10.20944/preprints201611.0086.v2 ◽

2016 ◽

Author(s):

Zhichao Ma ◽

Adrian J. T. Teo ◽

Say Hwa Tan ◽

Ye Ai ◽

Nam-Trung Nguyen

Keyword(s):

Acoustic Wave ◽

Surface Acoustic Wave ◽

Particle Separation ◽

Microfluidic Devices ◽

Current Approach ◽

Droplet Generation ◽

Microfluidic Channels ◽

Clean Room ◽

Precise Location ◽

Innovative Method

Surface acoustic wave (SAW) is effective for the manipulation of fluids and particles in microscale. The current approach of integrating interdigitated transducers (IDTs) for SAW generation into microfluidic channels involves complex and laborious microfabrication steps. These steps often require the full access to clean room facilities and hours to align the transducers to the precise location. This work presents an affordable and innovative method for fabricating SAW-based microfluidic devices without the need of clean room facilities and alignment. The IDTs and microfluidic channels are fabricated in the same process and thus precisely self-aligned in accordance with the device design. With the use of the developed fabrication approach, a few types of different SAW-based microfluidic devices have been fabricated and demonstrated for particle separation and active droplet generation.

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Simulation and Validation of Droplet Generation Process for Revealing Three Design Constraints in Electrohydrodynamic Jet Printing

Micromachines ◽

10.3390/mi10020094 ◽

2019 ◽

Vol 10 (2) ◽

pp. 94 ◽

Cited By ~ 8

Author(s):

Yanqiao Pan ◽

Liangcai Zeng

Keyword(s):

Numerical Simulation ◽

Simulation Model ◽

High Performance ◽

Droplet Generation ◽

Generation Process ◽

Design Constraints ◽

Whole Process ◽

Velocity Magnitude ◽

Electrohydrodynamic Jet Printing ◽

Jet Printing

Droplet generation process can directly affect process regulation and output performance of electrohydrodynamic jet (E-jet) printing in fabricating micro-to-nano scale functional structures. This paper proposes a numerical simulation model for whole process of droplet generation of E-jet printing based on the Taylor-Melcher leaky-dielectric model. The whole process of droplet generation is successfully simulated in one whole cycle, including Taylor cone generation, jet onset, jet break, and jet retraction. The feasibility and accuracy of the numerical simulation model is validated by a 30G stainless nozzle with inner diameter ~160 μm by E-jet printing experiments. Comparing numerical simulations and experimental results, period, velocity magnitude, four steps in an injection cycle, and shape of jet in each step are in good agreement. Further simulations are performed to reveal three design constraints against applied voltage, flow rate, and nozzle diameter, respectively. The established cone-jet numerical simulation model paves the way to investigate influences of process parameters and guide design of printheads for E-jet printing system with high performance in the future.

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Microfluidic droplet generation based on non-embedded co-flow-focusing using 3D printed nozzle

Scientific Reports ◽

10.1038/s41598-020-77836-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Adrien Dewandre ◽

Javier Rivero-Rodriguez ◽

Youen Vitry ◽

Benjamin Sobac ◽

Benoit Scheid

Keyword(s):

Stokes Equations ◽

Axisymmetric Flow ◽

Droplet Generation ◽

Droplet Radius ◽

Planar Geometry ◽

Flow Focusing ◽

Glass Capillary ◽

New Device ◽

3D Printed ◽

Droplet Generators

AbstractMost commercial microfluidic droplet generators rely on the planar flow-focusing configuration implemented in polymer or glass chips. The planar geometry, however, suffers from many limitations and drawbacks, such as the need of specific coatings or the use of dedicated surfactants, depending on the fluids in play. On the contrary, and thanks to their axisymmetric geometry, glass capillary-based droplet generators are a priori not fluid-dependent. Nevertheless, they have never reached the market because their assembly requires fastidious and not scalable fabrication techniques. Here we present a new device, called Raydrop, based on the alignment of two capillaries immersed in a pressurized chamber containing the continuous phase. The dispersed phase exits one of the capillaries through a 3D-printed nozzle placed in front of the extraction capillary for collecting the droplets. This non-embedded implementation of an axisymmetric flow-focusing is referred to non-embedded co-flow-focusing configuration. Experimental results demonstrate the universality of the device in terms of the variety of fluids that can be emulsified, as well as the range of droplet radii that can be obtained, without neither the need of surfactant nor coating. Additionally, numerical computations of the Navier-Stokes equations based on the quasi-steadiness assumption allow to provide an explanation to the underlying mechanism behind the drop formation and the mechanism of the dripping to jetting transition. Excellent predictions were also obtained for the droplet radius, as well as for the dripping-jetting transition, when varying the geometrical and fluid parameters, showing the ability of this configuration to enventually enhance the dripping regime. The monodispersity ensured by the dripping regime, the robustness of the fabrication technique, the optimization capabilities from the numerical modelling and the universality of the configuration confer to the Raydrop technology a very high potential in the race towards high-throughput droplet generation processes.

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Single-Molecule DNA Amplification and Analysis Using Microfluidics

Chemical Reviews ◽

10.1021/cr900081z ◽

2010 ◽

Vol 110 (8) ◽

pp. 4910-4947 ◽

Cited By ~ 113

Author(s):

Chunsun Zhang ◽

Da Xing

Keyword(s):

Single Molecule ◽

Dna Amplification

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Analytical Currents: STED microscopy illuminates vesicle fate | Imaging of the oxidant peroxynitrite in living cells | Solid immersion lenses made from PDMS | Molecular sorting in microfluidic devices | DNA sequencing on a chip | Single-molecule binding assay in femtoliter arrays | Simple derivatization of glycopeptides for MALDI | Direct electrical detection of DNA synthesis

Analytical Chemistry ◽

10.1021/ac069417z ◽

2006 ◽

Vol 78 (13) ◽

pp. 4239-4242

Keyword(s):

Dna Sequencing ◽

Dna Synthesis ◽

Single Molecule ◽

Binding Assay ◽

Microfluidic Devices ◽

Living Cells ◽

Electrical Detection ◽

Sted Microscopy

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Cyclic Olefin Copolymer Microfluidic Devices for Forensic Applications

Biosensors ◽

10.3390/bios9030085 ◽

2019 ◽

Vol 9 (3) ◽

pp. 85 ◽

Cited By ~ 5

Author(s):

Brigitte Bruijns ◽

Andrea Veciana ◽

Roald Tiggelaar ◽

Han Gardeniers

Keyword(s):

Chemical Resistance ◽

Multiple Displacement Amplification ◽

Visible Region ◽

Dna Amplification ◽

Microfluidic Devices ◽

Cyclic Olefin Copolymer ◽

Color Test ◽

Cyclic Olefin ◽

On Chip ◽

Amplification Reaction

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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