Paper-Based Loop Mediated Isothermal Amplification (LAMP) Platforms: Integrating the Versatility of Paper Microfluidics with Accuracy of Nucleic Acid Amplification Tests (original) (raw)

Roll-to-roll fabrication of integrated PDMS-paper microfluidics for nucleic acid amplification

Lab on a chip, 2018

Microfluidic-based integrated molecular diagnostic systems, which are automated, sensitive, specific, user-friendly, robust, rapid, easy-to-use, and portable, can revolutionize future medicine. Current research and development largely relies on polydimethylsiloxane (PDMS) to fabricate microfluidic devices. Since the transition from the proof-of-principle phase to clinical studies requires a vast number of integrated microfluidic devices, there is a need for a high-volume manufacturing method of silicone-based microfluidics. Here we present the first roll-to-roll (R2R) thermal imprinting method to fabricate integrated PDMS-paper microfluidics for molecular diagnostics, which allows production of tens of thousands of replicates in an hour. In order to validate the replicated molecular diagnostic platforms, on-chip amplification of viral ribonucleic acid (RNA) with loop-mediated isothermal amplification (LAMP) was demonstrated. These low-cost, rapid and accurate molecular diagnostic pl...

Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA

Lab on A Chip, 2010

For the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into #30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.

Microfluidic-Based Nucleic Acid Amplification Systems in Microbiology

Micromachines

Rapid, sensitive, and selective bacterial detection is a hot topic, because the progress in this research area has had a broad range of applications. Novel and innovative strategies for detection and identification of bacterial nucleic acids are important for practical applications. Microfluidics is an emerging technology that only requires small amounts of liquid samples. Microfluidic devices allow for rapid advances in microbiology, enabling access to methods of amplifying nucleic acid molecules and overcoming difficulties faced by conventional. In this review, we summarize the recent progress in microfluidics-based polymerase chain reaction devices for the detection of nucleic acid biomarkers. The paper also discusses the recent development of isothermal nucleic acid amplification and droplet-based microfluidics devices. We discuss recent microfluidic techniques for sample preparation prior to the amplification process.

Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests

Biosensors, 2018

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges ('chips') that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed.

LAMP-on-a-chip: Revising microfluidic platforms for loop-mediated DNA amplification

Trends in Analytical Chemistry, 2019

Nucleic acid amplification for the detection of infectious diseases, food pathogens, or assessment of genetic disorders require a laboratory setting with specialized equipment and technical expertise. Isothermal deoxyribonucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), exhibit characteristics ideal for point-of-care (POC) applications, since their instrumentation is simpler in comparison with the standard method of polymerase chain reaction. Other key advantages of LAMP are robustness and the production of pyrophosphate in the presence of the target gene, enabling to detect the reaction products using the naked eye. Polymerase inhibitors, presented in clinical samples, do not affect the amplification process, making LAMP suitable for a simple sample-to-answer diagnostic systems with simplified sample preparation. In this review, we discuss the trends in miniaturized LAMP techniques, such as microfluidic, paper-based, and digital with their advantages and disadvantages, especially for POC applications alongside our opinion of the future development of miniaturized LAMP.

Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection

Molecular (nucleic acid)-based diagnostics tests have many advantages over immunoassays, particularly with regard to sensitivity and specifi city. Most on-site diagnostic tests, however, are immunoassay-based because conventional nucleic acid-based tests (NATs) require extensive sample processing, trained operators, and specialized equipment. To make NATs more convenient, especially for point-of-care diagnostics and on-site testing, a simple plastic microfl uidic cassette ("chip") has been developed for nucleic acidbased testing of blood, other clinical specimens, food, water, and environmental samples. The chip combines nucleic acid isolation by solid-phase extraction; isothermal enzymatic amplifi cation such as LAMP (Loop-mediated AMPlifi cation), NASBA (Nucleic Acid Sequence Based Amplifi cation), and RPA (Recombinase Polymerase Amplifi cation); and real-time optical detection of DNA or RNA analytes. The microfl uidic cassette incorporates an embedded nucleic acid binding membrane in the amplifi cation reaction chamber. Target nucleic acids extracted from a lysate are captured on the membrane and amplifi ed at a constant incubation temperature. The amplifi cation product, labeled with a fl uorophore reporter, is excited with a LED light source and monitored in situ in real time with a photodiode or a CCD detector (such as available in a smartphone). For blood analysis, a companion fi ltration device that separates plasma from whole blood to provide cell-free samples for virus and bacterial lysis and nucleic acid testing in the microfl uidic chip has also been developed. For HIV virus detection in blood, the microfl uidic NAT chip achieves a sensitivity and specifi city that are nearly comparable to conventional benchtop protocols using spin columns and thermal cyclers.

Low-cost bioanalysis on paper-based and its hybrid microfluidic platforms

Talanta, 2015

Low-cost assays have broad applications ranging from human health diagnostics and food safety inspection to environmental analysis. Hence, low-cost assays are especially attractive for rural areas and developing countries, where financial resources are limited. Recently, paper-based microfluidic devices have emerged as a low-cost platform which greatly accelerates the point of care (POC) analysis in low-resource settings. This paper reviews recent advances of low-cost bioanalysis on paper-based microfluidic platforms, including fully paper-based and paper hybrid microfluidic platforms. In this review paper, we first summarized the fabrication techniques of fully paper-based microfluidic platforms, followed with their applications in human health diagnostics and food safety analysis. Then we highlighted paper hybrid microfluidic platforms and their applications, because hybrid platforms could draw benefits from multiple device substrates. Finally, we discussed the current limitations and perspective trends of paper-based microfluidic platforms for low-cost assays.

Miniaturized devices for isothermal DNA amplification addressing DNA diagnostics

Microsystem Technologies, 2015

Microfluidics is an emerging technology enabling the development of Lab-on-a-chip (LOC) systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. Currently, available nucleic acid diagnostic tests take advantage of Polymerase Chain Reaction (PCR) that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. At the same time, isothermal methods for DNA amplification are being developed and are preferred for their simplified protocols and the elimination of thermocycling. Here, we present a low-cost and fast DNA amplification device for isothermal Helicase Dependent Amplification (HDA) implemented in the detection of mutations related to breast cancer as well as the detection of Salmonella pathogens. The device is fabricated by mass production amenable technologies on printed circuit board (PCB) substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zone necessary for isothermal amplification methods.

Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: A review

Analytica Chimica Acta, 2012

Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10-20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics.