Emerging Loop-Mediated Isothermal Amplification-Based Microchip and Microdevice Technologies for Nucleic Acid Detection

Abstract

Rapid, sensitive, and selective pathogen detection is of paramount importance in infectious disease diagnosis and treatment monitoring. Currently available diagnostic assays based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are time-consuming, complex, and relatively expensive, thus limiting their utility in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been used extensively in the development of rapid and sensitive diagnostic assays for pathogen detection and nucleic acid analysis and hold great promise for revolutionizing point-of-care molecular diagnostics. Here, we review novel LAMP-based lab-on-a-chip (LOC) diagnostic assays developed for pathogen detection over the past several years. We review various LOC platforms based on their design strategies for pathogen detection and discuss LAMP-based platforms still in development and already in the commercial pipeline. This review is intended as a guide to the use of LAMP techniques in LOC platforms for molecular diagnostics and genomic amplifications.

Keywords: LAMP; lab on a chip; microfluidics; molecular diagnostics; nucleic acid tests; pathogen detection.

Figure 1

Search results for number of publications and citations related to LAMP-based technologies. (A) Number of publications with keywords of “loop-mediated isothermal amplification”. (B) Number of publications with keywords of “loop-mediated isothermal amplification” and “lab on a chip”. (C) Number of citations with keywords of “loop-mediated isothermal amplification” and “lab on a chip”. These statistics indicate the exponential growth in the interest in developing LAMP-based techniques. (D) Major contributing countries that published in science and commercial advancement based on LAMP LOCs. All statistical data were collected from SCOPUS.

Figure 2

LAMP-based LOC devices with colorimetric detection modalities. (A) Schematic of color change during amplification using (a) Calcein and (b) HNB dye. (B) Paper microchip for LAMP amplification and detection. (a) Exploded and (b) assembled schematic of paper microchip, which consists of three different layers to wash the lysate, purify the DNA sample, amplify the DNA template, and detect the amplification product using SYBR Green I. (c) image of the prototype. (C) Paper-plastic hybrid assay for detecting H1N1 virus. (a) 3D schematic of the chip, which consists of PDMS chip, glass, and paper chromatography paper. (b) The actual image of a fabricated microchip. Chromatography paper is placed at the amplification zones to preload primers. (D) LAMP-based cassette device (a) Cassette device with an array of chambers on a flexible ribbon for high-throughput bacterial detection. (b) S. aureus pathogen was detected using Calcein with the LOD of 200 CFU/mL. (c) E. coli bacteria were detected using HNB dye with LOD of 30 CFU/mL in 60 min amplification time. Reproduced with the permission from refs and –. Copyright 2008 Nature, 2014 American Chemical Society, 2015 American Chemical Society, and 2014 Royal Society of Chemistry.

Figure 3

LAMP-based microchip devices with electrochemical sensing modalities (end-point detection). (A) 3D schematic of electrochemical sensing of amplified NAs (a) Schematic of binding redox and LAMP amplicon at the end of LAMP reaction, which results in the LSV peak reduction. (b) LSV of Hoechst redox. (B) Microchip for electrochemical detection of maize CBH 351 GMO DNA. (a) Image of DNA stick, which consists of amplification and detection sections that are separated from each other by a valve. (b) The amplification reaction was first performed on a hot plate. (c) by damaging the interface valve with gentle mixing, redox and amplification products were mixed and further electrochemical detection was possible. (C) Microfluidic electrochemical assay for detecting E. coli.. (a) Schematic of microfluidic chip for negative control and sample detection. Each microfluidic chip consists of PDMS chip, glass substrate, and aluminum heater. The detection chamber has a disposable carbon screen-printed electrode. (b) Image of disposable screen-printed electrode. Reproduced with the permission from refs and . Copyright 2009 Royal Society of Chemistry and 2013 American Chemical Society. respectively.

Figure 4

Multiplex RT-LAMP integrated with LFT to control the flow (A) 2D schematic of the CD microfluidic device. (B) (i) An illustration of how the microchannels were connected to the LFT, (ii) cross-sectional schematic for the connection between microchannel, RT-LAMP chamber, and the LFT, and (iii) schematic for the connection between the running buffer reservoir and the LFT. (C) Image of the RT-LAMP-LFT CD microfluidic. (D) Exploded 3D schematic of the RT-LAMP-LFT microchip. Different layers cover the LFT, hold the sample, and control the flow. (E) Actual image of the assay results for detecting (i) H1 gene, (ii) M gene, and (iii) negative control. Reproduced with permission from ref . Copyright 2015 Royal Society of Chemistry.

Figure 5

LAMP-based microfluidics integrated with optical sensing apparatus. (A) Microchip integrated with fiber optics. (a) Image of Gene-Z device integrated with an iPod dock, a rechargeable port, and a disposable microchip. (b) An actual image of the microfluidic device with four parallel arrays of microchannels and close-up of the reaction wells. (c) 3D schematic of the microchannels demonstrating the working principle behind the Gene-Z technology. After sample loading through the sample inlet the reaction wells are filled within several seconds. Air inside the reaction wells is purged from the vents located downstream of each channel to distribute samples in all chambers equally. (B) A LAMP-based microchip for bacteria detection integrated with a spectrophotometer. (a) Actual image of the integrated microfluidic device connected to an air vent, a heat block, and a magnet. (b) Schematic of the principle behind the technology for MRSA detection. MRSA bacteria are injected into the chip with a specific probe conjugated with magnetic beads and lysed in lysis chamber through heating at 95 °C. After releasing MRSA DNA, the molecular probe is hybridized with DNA at 63 °C. The target DNA is separated from background using magnetic separation. LAMP reagents are added to the solution and amplified targets are optically detected with a spectrophotometer. (c) 2D schematic of the microchip and its components. Reproduced with the permission from refs and . Copyright 2011 and 2012 Royal Society of Chemistry, respectively.

Figure 6

LAMP-based microchips integrated with CCD imaging. (A) The nuclemeter. (a) The nuclemeter has a reaction chamber and a microconduit where reaction-diffusion takes place. (b) NA template is amplified and diffused through the channel. (c) Actual image of a fabricated nuclemeter microchip with four separate microchannels for detecting HIV RNA. (d) Actual image of the portable nuclemeter with its components, includinga flexible thin heater and USB-size microscope. (e) Quantification of HIV-1 in samples based on the diffusion length of amplified amplicons in each channels. (B) Microfluidic device platform for detecting various bacterial DNA using a CCD camera, emission filter, LED with excitation filter, and a microchip. Reproduced with permission from refs and . Copyright 2011 Springer and 2014 Nature, respectively.

Figure 7

LAMP-based microfluidic device with real-time electrochemical sensing modalities. (A) Schematic of the principle behind real-time electrochemical detection using MB active redox compound. Prior to amplification, MB is free in the solution and generates high redox current due to rapid diffusion on the surface of gold working electrodes. During LAMP reaction, MB intercalates with newly formed double stranded product, which leads to a decrease in the current peak. (B) Schematic of the microfluidic electrochemical quantification (MEQ)-LAMP chip and its cross-section (C) Microchip is placed on a heat block to provide the required temperature (65 °C) for amplification. The amplified target is detected through SWV using a potentiostat. Adapted with permission from ref . Copyright 2012 Wiley.

Figure 8

LAMP-based microchip with pH sensing modality. (A) The principle behind the ion sensing platform is illustrated. Schematic of how C–V curve changes throughout the LAMP amplification process due to the production of H⁺. The elongation process yields accumulation of protons. Thus, the pH shift in during the LAMP process is proportional to the number of nucleotides. The hydrogen accumulation can be measured by impedance spectroscopy. (B) Schematic of pH sensing with its ISFET platform. Cross section of array of an ISFET-based system with its gate, source (S), drain (D), temperature sensors (yellow), and controlled heater (blue), all are embedded at the underneath of a silicon nitride-sensing surface. Reproduced with the permission from refs and . Copyright 2013 Nature and 2014 Elsevier, respectively.

Figure 9

Multiplexing and high-throughput LAMP LOC devices. (A) CapitalBio RTisochip. (a) Microfluidic disc platform. Each disk consists of 24 reaction wells, which are attached to the buffer well. The buffer wells are connected with crooked primary channels with a slim capillary channel. The capillary channel is then cut off by thermal shock during the amplification process, which isolates the reaction well to protect the sample from potential contamination. Each reaction well has a volume of 1.414 μL and all the primers are first added into the wells and dehydrated. (b) Schematic of top and bottom coverslips. (c) Actual image of a fabricated CD-based microchip. (d) Image of the CapitalBio RTisochip platform. (B) A LAMP-based microchip for multiplexing biotargets using real-time electrochemical sensing modality. (a) Actual image of a fabricated microchip. (b) 2D schematic of the device and its three sections for detecting three different DNA pathogens. (C) A droplet-LAMP-based microfluidic for high-throughput biotarget detection. Amplified droplets are detected using Calcein fluorescent signal (CFS) analysis with confocal microscopy. Reproduced with the permission from refs , , and . Copyright 2014 Elsevier, 2014 Elsevier, and 2015 Royal Society of Chemistry, respectively.

Figure 10

Electricity-free cartridges for on-chip LAMP amplification. (A) (a) Exploded 3D schematic of the cartridge is demonstrated. (b) Actual image of a fabricated cartridge. (B) NINA electricity free cartridge that uses CaO for heat generation. (C) (a) NINA-PATH cartridge. (b) The cartridge was integrated with lateral flow test for detecting HIV amplicons. Reproduced with the permission from refs , , and . Copyright 2012 Public Library of Science, 2011 Royal Society of Chemistry, and 2014 Public Library of Science, respectively.