A Portable Continuous-Flow Polymerase Chain Reaction Chip Device Integrated with Arduino Boards for Detecting Colla corii asini.

Food security is a significant issue in modern society. Because morphological characters are not reliable enough to distinguish authentic traditional Chinese medicines, it is essential to establish an effective and applicable method to identify them to protect people's health. Due to the expensive cost of the manufacturing process and the large volume of the analytical system, the need to build a portable and cheap device is urgent. This work describes the development of a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a novel microfluidic polymerase chain reaction (PCR) chip with symmetric isothermal zones. The total chip volume is small, and only one Arduino board is needed for thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. In the proposed operation, the Nusselt number of the sample flow is less than one, and the heat transfer is conduction only. Then we can ensure temperature uniformity in specific reaction regions. A Colla corii asini DNA segment of 200 bp is amplified to evaluate the PCR performance under the various operational parameters. The initial concentration for accomplishing the PCR process is at least 20 ng/µL at the flow rate of 0.4 µL/min in the portable continuous flow PCR (CFPCR) device. To our knowledge, our group is the first to introduce Arduino boards into the heat control and sample pumping modules for a CFPCR device.

Fabrication of an Oscillating Thermocycler to Analyze the Canine Distemper Virus by Utilizing Reverse Transcription Polymerase Chain Reaction.

The reverse transcription-polymerase chain reaction (RT-PCR) has been utilized as an effective tool to diagnose the infectious diseases of viruses. In the present work, the oscillating thermocycler is fabricated and performed to carry out the one-step RT-PCR process successfully. The ribonucleic acid (RNA) mixture is pipetted into the fixed sample volume inside an aluminum reaction block. The sample oscillates the pathway onto the linear motion control system and through the specific RT-PCR heating zones with individual homemade thermal control modules. The present oscillating thermocycler combines the merits of the chamber type and the CF type systems. Before PCR, the reaction chamber moves to the low-temperature zone to complete the RT stage and synthesize the complementary deoxyribonucleic acid (DNA). Next, the low-temperature zone is regulated to the annealing zone. Furthermore, the reactive sample is moved back and forth among three isothermal zones to complete PCR. No extra heating zone is required for the RT stage. The total length of the moving displacement of the chamber is within 100 mm. The miniaturization of the oscillating thermocycler can be expected. In our oscillatory device, the denaturation zone located between the annealing and extension zones is suggested as the appropriate arrangement of the heating blocks. Heat management without thermal cross-talk is easy. Finally, an improved oscillating device is demonstrated to execute the RT-PCR process directly, utilized to amplify the canine distemper virus templates successfully, which could be well applied to a low-cost DNA analysis system in the future.

Light Energy Conversion Surface with Gold Dendritic Nanoforests/Si Chip for Plasmonic Polymerase Chain Reaction.

Surfaces with gold dendritic nanoforests (Au DNFs) on Si chips demonstrate broadband-light absorption. This study is the first to utilize localized surface plasmons of Au DNFs/Si chips for polymerase chain reaction (PCR) applications. A convenient halogen lamp was used as the heating source to illuminate the Au DNFs/Si chip for PCR. A detection target of Salmonella spp. DNA fragments was reproduced in this plasmonic PCR chip system. By semi-quantitation in gel electrophoresis analysis, the plasmonic PCR with 30 cycles and a largely reduced processing time provided results comparable with those of a commercial PCR thermal cycler with 40 cycles in more than 1 h. In the presence of an Au DNFs/Si chip, the plasmonic PCR provides superior results in a short processing time.

Analysis of PCR Kinetics inside a Microfluidic DNA Amplification System.

In order to analyze the DNA amplification numerically with integration of the DNA kinetics, three-dimensional simulations, including flow and thermal fields, and one-dimensional polymerase chain reaction (PCR) kinetics are presented. The simulated results are compared with experimental data that have been applied to the operation of a continuous-flow PCR device. Microchannels fabricated by Micro Electro-Mechanical Systems (MEMS) technologies are shown. Comprehensive simulations of the flow and thermal fields and experiments measuring temperatures during thermal cycling are presented first. The resultant velocity and temperature profiles from the simulations are introduced to the mathematical models of PCR kinetics. Then kinetic equations are utilized to determine the evolution of the species concentrations inside the DNA mixture along the microchannel. The exponential growth of the double-stranded DNA concentration is investigated numerically with the various operational parameters during PCR. Next a 190-bp segment of Bartonella DNA is amplified to evaluate the PCR performance. The trends of the experimental results and numerical data regarding the DNA amplification are similar. The unique architecture built in this study can be applied to a low-cost portable PCR system in the future.

One-heater flow-through polymerase chain reaction device by heat pipes cooling.

This study describes a novel microfluidic reactor capable of flow-through polymerase chain reactions (PCR). For one-heater PCR devices in previous studies, comprehensive simulations and experiments for the chip geometry and the heater arrangement were usually needed before the fabrication of the device. In order to improve the flexibility of the one-heater PCR device, two heat pipes with one fan are used to create the requisite temperature regions in our device. With the integration of one heater onto the chip, the high temperature required for the denaturation stage can be generated at the chip center. By arranging the heat pipes on the opposite sides of the chip, the low temperature needed for the annealing stage is easy to regulate. Numerical calculations and thermal measurements have shown that the temperature distribution in the five-temperature-region PCR chip would be suitable for DNA amplification. In order to ensure temperature uniformity at specific reaction regions, the Re of the sample flow is less than 1. When the microchannel width increases and then decreases gradually between the denaturation and annealing regions, the extension region located in the enlarged part of the channel can be observed numerically and experimentally. From the simulations, the residence time at the extension region with the enlarged channel is 4.25 times longer than that without an enlarged channel at a flow rate of 2 µl/min. The treated surfaces of the flow-through microchannel are characterized using the water contact angle, while the effects of the hydrophilicity of the treated polydimethylsiloxane (PDMS) microchannels on PCR efficiency are determined using gel electrophoresis. By increasing the hydrophilicity of the channel surface after immersing the PDMS substrates into Tween 20 (20%) or BSA (1 mg/ml) solutions, efficient amplifications of DNA segments were proved to occur in our chip device. To our knowledge, our group is the first to introduce heat pipes into the cooling module that has been designed for a PCR device. The unique architecture utilized in this flow-through PCR device is well applied to a low-cost PCR system.

Analytical study of a microfludic DNA amplification chip using water cooling effect.

A novel continuous-flow polymerase chain reaction (PCR) chip has been analyzed in our work. Two temperature zones are controlled by two external controllers and the other temperature zone at the chip center is controlled by the flow rate of the fluid inside a channel under the glass chip. By employing a water cooling channel at the chip center, the sequence of denaturation, annealing, and extension can be created due to the forced convection effect. The required annealing temperature of PCR less than 313 K can also be demonstrated in this chip. The Poly(methyl methacrylate) (PMMA) cooling channel with the thin aluminum cover is utilized to enhance the temperature uniformity. The size of this chip is 76 mm × 26 mm × 3 mm. This device represents the first demonstration of water cooling thermocycling within continuous-flow PCR microfluidics. The commercial software CFD-ACE+(TM) is utilized to determine the distances between the heating assemblies within the chip. We investigate the influences of various chip materials, operational parameters of the cooling channel and geometric parameters of the chip on the temperature uniformity on the chip surface. Concerning the temperature uniformity of the working zones and the lowest temperature at the annealing zone, the air gap spacing of 1 mm and the cooling channel thicknesses of 1 mm of the PMMA channel with an aluminum cover are recommended in our design. The hydrophobic surface of the PDMS channel was modified by filling it with 20 % Tween 20 solution and then adding bovine serum albumin (BSA) solution to the PCR mixture. DNA fragments with different lengths (372 bp and 478 bp) are successfully amplified with the device.

Optimal designs of staggered dean vortex micromixers.

A novel parallel laminar micromixer with a two-dimensional staggered Dean Vortex micromixer is optimized and fabricated in our study. Dean vortices induced by centrifugal forces in curved rectangular channels cause fluids to produce secondary flows. The split-and-recombination (SAR) structures of the flow channels and the impinging effects result in the reduction of the diffusion distance of two fluids. Three different designs of a curved channel micromixer are introduced to evaluate the mixing performance of the designed micromixer. Mixing performances are demonstrated by means of a pH indicator using an optical microscope and fluorescent particles via a confocal microscope at different flow rates corresponding to Reynolds numbers (Re) ranging from 0.5 to 50. The comparison between the experimental data and numerical results shows a very reasonable agreement. At a Re of 50, the mixing length at the sixth segment, corresponding to the downstream distance of 21.0 mm, can be achieved in a distance 4 times shorter than when the Re equals 1. An optimization of this micromixer is performed with two geometric parameters. These are the angle between the lines from the center to two intersections of two consecutive curved channels, θ, and the angle between two lines of the centers of three consecutive curved channels, ϕ. It can be found that the maximal mixing index is related to the maximal value of the sum of θ and ϕ, which is equal to 139.82°.