Microfluidic Chip Fabrication and Method to Detect Influenza

In this paper we present a lab-on-glass system for label free DNA analysis that integrates a polymerase chain reaction (PCR) chamber for DNA amplification, an electrowetting-based droplet-handling system and a hydrogenated amorphous silicon ultraviolet sensor array for DNA detection. The photosensors allow to measure the absorbance of the biomolecules at 254 nm to reveal and quantify the DNA sample. The system is fabricated using thin-film and thick-film technologies on a conventional microscope glass slide and its design has been carried out by taking into account the application requirements as well as the compatibility of the process steps. The PCR chamber incorporates a thin metal film heater optimized for uniform temperature distribution and an amorphous silicon diode for temperature measurement. The diode shows a sensitivity of -3.3 mV/K in the range 30-80degC when biased with a forward current of 10 nA. A successful droplet handling capability was achieved using a hydrophobic Teflon layer deposited over the silicon nitride film used to passivate both the heater and the ultraviolet sensor array. The voltages needed to successfully move a 2 mul water droplet were in the range of 80 Volts. Finally, we demonstrated the sensor ability to distinguish between single- and double-stranded molecules functionalized on a quartz substrate.

A point-of-care, diagnostic system incorporating a portable thermal cycler and a compact fluorescent detector for real-time, polymerase chain reaction (PCR) on disposable, plastic microfluidic reactors with relatively large reaction volume (ranging from 10 µL to 100 µL) is described. To maintain temperature uniformity and a relatively fast temperature ramping rate, the system utilizes double-sided heater that features a master, thermoelectric element and a thermal waveguide connected to a second thermoelectric element. The waveguide has an aperture for optical coupling between a miniature, fluorescent reader and the PCR reaction chamber. The temperature control is accomplished with a modified, feedforward, variable structural proportional-integral-derivative controller. The temperature of the liquid in the reaction chamber tracks the set-point temperature with an accuracy of ± 0.1 °C. The transition times from one temperature to another are minimized with controllable overshoots (< 2 °C) and undershoots (< 5 °C). The disposable, single-use PCR chip can be quickly inserted into a thermal cycler/reader unit for point-of-care diagnostics applications. The large reaction chamber allows convenient pre-storing of dried, paraffin-encapsulated PCR reagents (polymerase, primers, dNTPs, dyes, and buffers) in the PCR chamber. The reagents are reconstituted "just in time" by heating during the PCR process. The system was tested with viral and bacterial nucleic acid targets.

Despite dimethyl siloxane (PDMS) being one of the most used microfluidics chips, its micromachining techniques are infeasible for the mass production of commercial devices. This study proposed Epoxy Resin (ER) as a promising, cost-effective alternative to PDMS in lab-on-chip applications, including Polymerase Chain Reactions (PCR). Moreover, a novel, rapid and non-photolithographic approach has been developed for micromachining epoxy microfluidic chips. A new method for fabricating epoxy resin and polyethylene glycol (PEG) hybrid (ER-PEG) microchannels was developed by casting replication over PMMA molds. A PMMA piece was engraved with a CO2 laser in the raster mode to produce the mold for the ER-PEG casting first. A positive mold was fabricated from a polymethyl methacrylic acid (PMMA) sheet (6mm) by CO2 laser ablation. The microfluidic chips with the negative pattern were replicated by casting thermoset epoxy resin onto the micro-machined PMMA mold. The entire process, from device design conception to working device, can be completed in minutes. Herein, to enhance the surface roughness (Ra) of the ablated mold surface, different Distances to Focus (DF) were tried. The low Ra in the mold results in a low Ra in the replica. Also, the performance of ER-PEG in PCR was evaluated to verify the compatibility of the chip material to the PCR test.

Real-Time PCR: An Essential Guide

This paper presents a new self-compensated thermocycler for polymerase chain reaction (PCR), especially used in DNA amplification process requiring precise thermal control. A new design of micro- heaters to improve the thermal uniformity of the PCR chamber using array-type heaters and a self- compensated mechanism is reported. The feature of this design is that structure change or active thermal compensation is not required. Experimental data show that the spatial variation of temperature distribution is less than ldegC in the PCR chamber at annealing temperature. Besides, the area with thermal variation less than 0.5degC is close to 90 % of the working area.

Rapid Assessment of Minimal Residual Acute Myeloid Leukemia from Peripheral Blood Using a Microfluidic Chip

PCR determination of inactivated RNA coliphage Qß

This paper reports on the design, fabrication, assembly and testing of amicromachined polymerase chain reaction (PCR) system with pigtailed fibreoptics. Two major designs have been fabricated and tested. The first designuses two optical fibres, one for the projection of light into the chamber andthe other for the collection of the light emitted by a fluorescent dye in thereaction chamber. The second design employs a single optical fibre for thedelivery of light into the PCR chamber, with light now being detected using aphotodiode integrated into the base and walls of the reaction chamber withinthe PCR chip. Tests carried out on both systems have produced promisingresults.

We present a disposable polymeric microfluidic device capable of reversibly binding and purifying Salmonella DNA through solid phase extraction (SPE). The microfluidic channels are first oxygen plasma treated and simultaneously micro-nanotextured, and then functionalized with amine groups via modification with L-histidine or poly-L-histidine. L-Histidine and poly-L-histidine bind on the plasma treated chip surface, and are not detached when rinsing with DNA purification protocol buffers. A pH-dependent protocol is applied on-chip to purify Salmonella DNA, which is first bound on the protonated amines at a pH (5.0) lower than their pKa of surface amine-groups which is 6.0 and then released at a pH higher than the pKa value (10.5). It was found that modification with poly-L-histidine resulted in higher surface density of amine groups onto microfluidic channel. Using the chip modified with poly-L-histidine, high recovery efficiency of at least 550ng of isolated Salmonella DNA as well as DNA purification from Salmonella cell lysates corresponding to less than 5000 cells or 0.026ng of Salmonella DNA was achieved. The protocol developed does not require ethanol or chaotropic solutions typically used in DNA purification, which are known inhibitors for downstream operations such as polymerase chain reactions (PCR) and which can also attack some polymeric microfluidic materials. Therefore, the microfluidic device and the related protocol hold promise for facile incorporation in microfluidics and Lab-on-a-chip (LOC) platforms for pathogen detection or in general for DNA purification.

Cholangiocyte Endothelin 1 and Transforming Growth Factor β1 Production in Rat Experimental Hepatopulmonary Syndrome

Here, we report the developed cyclo olefin polymer (COP) microfluidic chip on a fabricated rotating heater stage that utilizes centrifugation-assisted thermal cycle in a ring-structured microchannel for polymerase chain reaction (PCR). The PCR solution could be driven by thermal convection and continuously exchanged high/low temperatures in a ring structured microchannel without the use of typical syringe pump. More importantly, the flow rate was controlled by the relative gravitational acceleration only. The platform enables amplification within 10 min at 5G and has a detection limit of 70.5 pg/channel DNA concentration (β-actin, 295 bp). The current rotating system is capable of testing four different samples in parallel. The microfluidic chip can be preloaded with the PCR premix solution for on-site utility, and, with all of the features integrated to the system, the test can be conducted without the need for specialized laboratory and trained laboratory staff. In addition, this innovative chemical reaction technique has the potential to be utilized in other micromixing applications.

In this paper, we present an integrated and automated prototype system which has been developed for real-time polymerase chain reaction (PCR) analysis based on microfluidic PCR array chips. The system integrates the PCR thermal cycling and optical detection capabilities to enable real-time fluorescence imaging and image processing for data analysis. The main advantage of the system is that it provides a solution that can rapidly perform and evaluate PCR experiment simultaneously on microfluidic PCR array chips. The system has demonstrated fast and efficient on-chip real-time PCR analysis using human genomic DNA samples. The implementation of the system integration is a multi-thread Windows software with component structure which is written in Visual C++.

Rapid determination of biogenic amines in ossotide injections by microfluidic chip-mass spectrometry platform: Optimization of microfluidic chip derivatization using response surface methodology

We have fabricated Si-based micromachined DNA polymerase chain reaction (PCR) chips with different groove depths. The platinum thin-film micro heater and the temperature sensor have been integrated on the chip. The volume of the PCR chamber in the chip is about 3.6 µl and the chip size is 17 × 40 mm2. The effects of groove geometry, including width, depth and position, on the thermal characteristics of the PCR chip have been investigated by numerical analysis and experimental measurement. From the results, the power consumption required for the PCR chip is reduced with the increase of groove depth. Compared with results for the case of no groove, the power consumption of the chip with a groove of 280 µm is reduced by 24.0%, 23.3% and 25.6% with annealing, extension and denaturation, respectively. The heating rate is increased rapidly with the increase of the groove depth. In particular, it is revealed that this effect is predominant for depths in the region above 280 µm. For a more precise control of chip temperature, the nonlinear feedback proportional-integral control scheme is used. The obtained heating and cooling rates are about 36 °C s−1 and 22 °C s−1, respectively. The overshoot and the steady state error are less than 0.7 °C and ±0.1 °C, respectively. In the experiment, the effects of the PCR buffer and the bubbles in the chamber on the temperature uniformity have also been studied. From the temperature measurement, it is revealed that the temperature difference between the thin-film sensor (on the lower plate) and the PCR buffer can be neglected if there is no air bubble in the PCR buffer. With such a high performance control scheme, we could implement a remarkable thermal cycling of conducting 30 cycles for 3 min. Finally, the chip PCR of plasmid DNA was successfully performed with no additives using the temperature control system.

Additive manufacturing for microfluidics shows potential to boost research and development in research biology and molecular diagnostics. This paper reports on novel process and material optimisation techniques in the creation of a monolithic microfluidic chip geometry for polymerase chain reaction (PCR) thermocycling using stereolithography (SLA). A two-stage printing protocol with projection SLA is assessed in printing disposable oscillating-flow microfluidic cartridges for PCR. Print performance was characterized in terms of critical channel dimensions and surface quality. Post-treatment with ultraviolet light and solvent washes was shown to reduce PCR inhibiting residuals and facilitate the reaction, indicating material compatibility for fluidic and milli-fluidic PCR architectures. Residuals leaching from the polymer were shown via quantitative PCR that interact with enzyme activity. Passivation of channel surfaces with a polyethylene glycol and a silane static coating reduced the leaching interface improving overall PCR efficiency. The discussed protocols can serve as a low-cost alternative to clean-room and micromachined microfluidic prototypes for various microfluidic concepts.