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Editorial

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2 pages, 145 KiB

Open AccessEditorial

Application of Microfluidic Methodology for the Analysis of DNA

by Kirsty Shaw, Yi Heng Nai and Stephen Haswell

Micromachines 2018, 9(1), 18; https://doi.org/10.3390/mi9010018 - 2 Jan 2018

Cited by 6 | Viewed by 3564

Abstract

Over the past 20 years, many of the developments and potential applications of microfluidic methodology have incorporated nucleic acid processes which have, in their own right, undergone a number of innovative changes [...]
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(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

Research

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17 pages, 3275 KiB

Open AccessArticle

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

by Darren W. Branch, Erika C. Vreeland, Jamie L. McClain, Jaclyn K. Murton, Conrad D. James and Komandoor E. Achyuthan

Micromachines 2017, 8(7), 228; https://doi.org/10.3390/mi8070228 - 21 Jul 2017

Cited by 12 | Viewed by 7206

Abstract

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use [...] Read more.

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP and the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples. Full article

(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

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11 pages, 1545 KiB

Open AccessFeature PaperArticle

Development of Temperature Control Solutions for Non-Instrumented Nucleic Acid Amplification Tests (NINAAT)

by Tamás Pardy, Toomas Rang and Indrek Tulp

Micromachines 2017, 8(6), 180; https://doi.org/10.3390/mi8060180 - 7 Jun 2017

Cited by 11 | Viewed by 5402

Abstract

Non-instrumented nucleic acid amplification tests (NINAAT) are a novel paradigm in portable molecular diagnostics. They offer the high detection accuracy characteristic of nucleic acid amplification tests (NAAT) in a self-contained device, without the need for any external instrumentation. These Point-of-Care tests typically employ [...] Read more.

Non-instrumented nucleic acid amplification tests (NINAAT) are a novel paradigm in portable molecular diagnostics. They offer the high detection accuracy characteristic of nucleic acid amplification tests (NAAT) in a self-contained device, without the need for any external instrumentation. These Point-of-Care tests typically employ a Lab-on-a-Chip for liquid handling functionality, and perform isothermal nucleic acid amplification protocols that require low power but high accuracy temperature control in a single well-defined temperature range. We propose temperature control solutions based on commercially available heating elements capable of meeting these challenges, as well as demonstrate the process by which such elements can be fitted to a NINAAT system. Self-regulated and thermostat-controlled resistive heating elements were evaluated through experimental characterization as well as thermal analysis using the finite element method (FEM). We demonstrate that the proposed solutions can support various NAAT protocols, as well as demonstrate an optimal solution for the loop-mediated isothermal amplification (LAMP) protocol. Furthermore, we present an Arduino-compatible open-source thermostat developed for NINAAT applications. Full article

(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

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12 pages, 3634 KiB

Open AccessArticle

A Programmable Digital Microfluidic Assay for the Simultaneous Detection of Multiple Anti-Microbial Resistance Genes

by Sumit Kalsi, Samuel L. Sellars, Carrie Turner, J. Mark Sutton and Hywel Morgan

Micromachines 2017, 8(4), 111; https://doi.org/10.3390/mi8040111 - 1 Apr 2017

Cited by 38 | Viewed by 8612

Abstract

The rapid emergence of antimicrobial resistant bacteria requires the development of new diagnostic tests. Nucleic acid-based assays determine antimicrobial susceptibility by detecting genes that encode for the resistance. In this study, we demonstrate rapid and simultaneous detection of three genes that confer resistance [...] Read more.

The rapid emergence of antimicrobial resistant bacteria requires the development of new diagnostic tests. Nucleic acid-based assays determine antimicrobial susceptibility by detecting genes that encode for the resistance. In this study, we demonstrate rapid and simultaneous detection of three genes that confer resistance in bacteria to extended spectrum β-lactam and carbapenem antibiotics; CTX-M-15, KPC and NDM-1. The assay uses isothermal DNA amplification (recombinase polymerase amplification, RPA) implemented on a programmable digital microfluidics (DMF) platform. Automated dispensing protocols are used to simultaneously manipulate 45 droplets of nL volume containing sample DNA, reagents, and controls. The droplets are processed and mixed under electronic control on the DMF devices with positive amplification measured by fluorescence. The assay on these devices is significantly improved with a Time to Positivity (TTP) half that of the benchtop assay. Full article

(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

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19 pages, 3609 KiB

Open AccessArticle

Electrophoretic Concentration and Electrical Lysis of Bacteria in a Microfluidic Device Using a Nanoporous Membrane

by Md. Shehadul Islam, Ali Shahid, Kacper Kuryllo, Yingfu Li, M. Jamal Deen and P. Ravi Selvaganapathy

Micromachines 2017, 8(2), 45; https://doi.org/10.3390/mi8020045 - 3 Feb 2017

Cited by 24 | Viewed by 7342

Abstract

Pathogenic bacteria such as Escherichia coli O157, Salmonella and Campylobacter are the main causes for food and waterborne illnesses. Lysis of these bacteria is an important component of the sample preparation for molecular identification of these pathogens. The pathogenicity of these bacteria [...] Read more.

Pathogenic bacteria such as Escherichia coli O157, Salmonella and Campylobacter are the main causes for food and waterborne illnesses. Lysis of these bacteria is an important component of the sample preparation for molecular identification of these pathogens. The pathogenicity of these bacteria is so high that they cause illness at very low concentrations (1–10 CFU/100 mL). Hence, there is a need to develop methods to collect a small number of such bacterial cells from a large sample volume and process them in an automated reagent-free manner. An electrical method to concentrate the bacteria and lyse them has been chosen here as it is reagent free and hence more conducive for online and automated sample preparation. We use commercially available nanoporous membranes sandwiched between two microfluidic channels to create thousands of parallel nanopore traps for bacteria, electrophoretically accumulate and then lyse them. The nanopores produce a high local electric field for lysis at moderate applied voltages, which could simplify instrumentation and enables lysis of the bacteria as it approaches them under an appropriate range of electric field (>1000 V/cm). Accumulation and lysis of bacteria on the nanoporous membrane is demonstrated by using the LIVE/DEAD BacLight Bacterial Viability Kit and quantified by fluorescence intensity measurements. The efficiency of the device was determined through bacterial culture of the lysate and was found to be 90% when a potential of 300 V was applied for 3 min. Full article

(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

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8 pages, 4028 KiB

Open AccessCommunication

Rapid Fabrication of Electrophoretic Microfluidic Devices from Polyester, Adhesives and Gold Leaf

by Christopher Birch, Jacquelyn A. DuVall, Delphine Le Roux, Brandon L. Thompson, An-Chi Tsuei, Jingyi Li, Daniel A. Nelson, Daniel L. Mills, James P. Landers and Brian E. Root

Micromachines 2017, 8(1), 17; https://doi.org/10.3390/mi8010017 - 9 Jan 2017

Cited by 19 | Viewed by 6154

Abstract

In the last decade, the microfluidic community has witnessed an evolution in fabrication methodologies that deviate from using conventional glass and polymer-based materials. A leading example within this group is the print, cut and laminate (PCL) approach, which entails the laser cutting of [...] Read more.

In the last decade, the microfluidic community has witnessed an evolution in fabrication methodologies that deviate from using conventional glass and polymer-based materials. A leading example within this group is the print, cut and laminate (PCL) approach, which entails the laser cutting of microfluidic architecture into ink toner-laden polyester sheets, followed by the lamination of these layers for device assembly. Recent success when applying this method to human genetic fingerprinting has highlighted that it is now ripe for the refinements necessary to render it amenable to mass-manufacture. In this communication, we detail those modifications by identifying and implementing a suitable heat-sensitive adhesive (HSA) material to equip the devices with the durability and resilience required for commercialization and fieldwork. Importantly, this augmentation is achieved without sacrificing any of the characteristics which make the PCL approach attractive for prototyping. Exemplary HSA-devices performed DNA extraction, amplification and separation which, when combined, constitute the complete sequence necessary for human profiling and other DNA-based analyses. Full article

(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

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9 pages, 2175 KiB

Open AccessArticle

Combining Electro-Osmotic Flow and FTA^® Paper for DNA Analysis on Microfluidic Devices

by Ryan Wimbles, Louise M. Melling and Kirsty J. Shaw

Micromachines 2016, 7(7), 119; https://doi.org/10.3390/mi7070119 - 14 Jul 2016

Cited by 7 | Viewed by 5586

Abstract

FTA^® paper can be used to protect a variety of biological samples prior to analysis, facilitating ease-of-transport to laboratories or long-term archive storage. The use of FTA^® paper as a solid phase eradicates the need to elute the nucleic acids from [...] Read more.

FTA^® paper can be used to protect a variety of biological samples prior to analysis, facilitating ease-of-transport to laboratories or long-term archive storage. The use of FTA^® paper as a solid phase eradicates the need to elute the nucleic acids from the matrix prior to DNA amplification, enabling both DNA purification and polymerase chain reaction (PCR)-based DNA amplification to be performed in a single chamber on the microfluidic device. A disc of FTA^® paper, containing a biological sample, was placed within the microfluidic device on top of wax-encapsulated DNA amplification reagents. The disc containing the biological sample was then cleaned up using Tris-EDTA (TE) buffer, which was passed over the disc, via electro-osmotic flow, in order to remove any potential inhibitors of downstream processes. DNA amplification was successfully performed (from buccal cells, whole blood and semen) using a Peltier thermal cycling system, whereupon the stored PCR reagents were released during the initial denaturing step due to the wax barrier melting between the FTA^® disc and PCR reagents. Such a system offers advantages in terms of a simple sample introduction interface and the ability to process archived samples in an integrated microfluidic environment with minimal risk of contamination. Full article

(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

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Review

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14 pages, 688 KiB

Open AccessReview

Electrode Materials in Microfluidic Systems for the Processing and Separation of DNA: A Mini Review

by Christopher Birch and James P. Landers

Micromachines 2017, 8(3), 76; https://doi.org/10.3390/mi8030076 - 3 Mar 2017

Cited by 13 | Viewed by 6928

Abstract

Since the advent of genetic analysis, electrode materials have played an irreplaceable role due to the easily-exploitable negatively-charged backbone of the DNA structure. Initially, the employment of electrophoretic movement lay only in the separation of DNA fragments of differing length; however, the widening [...] Read more.

Since the advent of genetic analysis, electrode materials have played an irreplaceable role due to the easily-exploitable negatively-charged backbone of the DNA structure. Initially, the employment of electrophoretic movement lay only in the separation of DNA fragments of differing length; however, the widening utility of electrokinetic phenomena at the microscale in areas such as fluid transportation and multistep integration led researchers to capitalize further when translating processes to microfluidic or “lab-on-chip” devices. Over the following three decades, the field witnessed a plethora of ways in which the necessary voltages could be transmitted to the sample and reagents with many successes; however, additional demands were then placed on those hoping to bring their microdevices to the market place. A greater emphasis on the cost of all constituent parts along with the increased importance that fluidics be contained hermetically at all times meant groups would become more imaginative when incorporating electrode materials. This review will aim to exactly describe the evolution of how those materials have been employed in DNA-based microfluidic devices. It will focus on how developers began to explore other emerging uses and also discuss how their tactics reflected the progressive demands of their chosen industry. Full article

(This article belongs to the Special Issue Application of Microfluidic Methodology for the Analysis of DNA)

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