Fabrication and characterization of a polymethyl methacrylate continuous-flow PCR microfluidic chip using CO2 laser ablation

An improved microfabrication method was used to fabricate a continuous-flow PCR (polymerase chain reaction) microfluidic chip on the PMMA substrate using the low-power CO₂ laser ablation technique. The use of the low-power CO₂ laser and the PMMA material could reduce the cost and the time of the fabrication process, especially at the step of laboratory research because of the high flexibility of the laser fabrication technique and the low cost of PMMA. A CO₂ laser output power of 4.5 W and a laser scanning velocity of 76.2 mm/s were chosen to fabricate the chip in this work. The micromachining quality could satisfy the microfluidic requirement of the PCR mixture within the microchannel. Good temperature distribution and gradient were obtained on the PMMA chip with a home-built integrated heating system. An amplification of DNA template with a 990 base pair fragment of Pseudomonas was successfully performed with this chip to characterize its availability and performance with various flow rates.     

Highly sensitive identification of foodborne pathogenic Listeria monocytogenes using single-phase continuous-flow nested PCR microfluidics with on-line fluorescence detection

Highly sensitive detection of foodborne pathogens such as Listeria monocytogenes (L. monocytogenes) is crucial to the prevention and recognition of problems related to public health and legal repercussions, due to “zero tolerance” standard adopted for food safety in many countries. Here we first propose a single-phase continuous-flow nested polymerase chain reaction (SP-CF-NPCR) strategy for identification of the low level of L. monocytogenes on an integrated microfluidic platform. The PCR reactor is constructed by a disposable capillary embedded in the grooved heating column, coupled with a fluorescence microscopy for on-line semi-quantitative end-point fluorescence detection. As a proof-of-concept microfluidic system, the nested PCR is performed in a continuous-flow format without the need of any non-aqueous oil or solvent. On this device, the performance of nested PCR amplification has been evaluated by investigating the effect of reaction parameters, including polymerase concentration, flow rates, and template DNA concentration. In addition, the types of samples the presented system can accept, such as the unpurified DNA samples and artificially contaminated clinical stool samples were also evaluated. With the optimized reaction parameters, 0.2 copies/μL of genomic DNA from L. monocytogenes can be detected on the presented device. To our knowledge, this is the highest detection sensitivity in single-phase continuous-flow PCR microsystems reported so far. The high sensitivity of the analysis method, combined with the flexibility of reaction volumes and convenience of continuous operation, renders it to be further developed for potential analytical and diagnostic applications.     

Bead-based polymerase chain reaction on a microchip

We present a bead-based approach to microfluidic polymerase chain reaction (PCR), enabling fluorescent detection and sample conditioning in a single microchamber. Bead-based PCR, while not extensively investigated in microchip format, has been used in a variety of bioanalytical applications in recent years. We leverage the ability of bead-based PCR to accumulate fluorescent labels following DNA amplification to explore a novel DNA detection scheme on a microchip. The microchip uses an integrated microheater and temperature sensor for rapid control of thermal cycling temperatures, while the sample is held in a microchamber fabricated from (poly)dimethylsiloxane and coated with Parylene. The effects of key bead-based PCR parameters, including annealing temperature and concentration of microbeads in the reaction mixture, are studied to achieve optimized device sensitivity and detection time. The device is capable of detecting a synthetically prepared section of the Bordetella pertussis genome in as few as 10 temperature cycles with times as short as 15?min. We then demonstrate the use of the procedure in an integrated device; capturing, amplifying, detecting, and purifying template DNA in a single microfluidic chamber. These results show that this method is an effective method of DNA detection which is easily integrated in a microfluidic device to perform additional steps such as sample pre-conditioning.     

Non-destructive on-chip imaging flow cell-sorting system for on-chip cellomics

We have developed a non-destructive imaging flow cell-sorting system using an ultra-high-speed camera (shutter speed of 1/10,000 s) with a real-time image analysis unit and a poly(methyl methacrylate) (PMMA)-based disposable microfluidic chip for single-cell-based on-chip cellomics. It has a 3-D micropipetting device that supports fully automated sorting and collection of samples. The entire fluidic system is implemented in a disposable plastic chip, enabling biological samples to be lined up in a laminar flow using hydrodynamic focusing. Its optical system enables direct observation-based cell identification using specific image indexes and phase-contrast/fluorescence microscopy, real-time image processing. It has a non-destructive, wider dynamic range, sorting procedure using mild electrostatic force in a laminar flow; agarose gel electrodes are used to prevent electrode loss and electrolysis bubble formation. The microreservoir used for recultivating collected target cells is contamination-free. An integrated ultra-high-speed droplet polymerase chain reaction measurement module is used for DNA/mRNA analysis of the collected target cells. This system was used to separate cardiomyocyte cells from a mixture of various cells. All the operations were automated using the 3-D micropipetting device. The results demonstrate that this imaging flow cell-sorting system is practically applicable for biological research and clinical diagnosis.     

The design and fabrication of polymerase chain reaction platform

Thermalcycler was extensively used machine for amplify DNA sample. One of the major problems in the working time was that it spent most of time for cooling and heating. In order to improve the efficient, the study presented a novel method for amplify DNA sample. For this concept, the DNA sample in the silicon chamber which was pushed by a tappet through the three temperature regions around a center and then the DNA segments could be amplified rapidly after 30 cycles. The polymerase chain reaction (PCR) platform was composed of the thin-film heaters, Cu plates, DC powers, and temperature controllers. The photolithography and bulk etching technologies were utilized to construct the thin-film heater and DNA reaction chambers. Finally, 1 μl 100 bp DNA segment of Escherichia coli K12 was amplified successfully within 36 min on this PCR platform.     

Automated Lab-on-a-Chip Analysis of DNA Fragments

The design of a fully automated system for the analysis of DNA fragments in 96-well plates is described. Microfluidic technology is used to integrate sample loading, electrophoretic analysis, and fragment detection onto a miniature lab-on-a-chip device. The microfluidic chip operates in an instrument platform that automates sample access, data collection, and data reporting. Each microfluidic chip provides sizing and concentration values for more than 1000 DNA samples.     

Inhibition of on-chip PCR using PDMS–glass hybrid microfluidic chips

In the course of developing a microfluidic analytical platform incorporating the polymerase chain reaction (PCR) and subsequent capillary electrophoresis (CE) analysis for a variety of bio-assays, we examined PCR inhibition through surface interactions with the chip materials. Our devices perform PCR in a three-layer chip, a glass–poly(dimethylsiloxane)–glass sandwich in which the poly(dimethylsiloxane) (PDMS, a silicone rubber) layer is used for pneumatic membrane pumping and valving of the PCR reagents. Initial on-chip PCR–CE tests of BK virus replicated in multiple uncoated chips showed variable results, usually yielding no detectable product at the target sample concentrations used. Subsequent “chip-flush” experiments, where water or reagents were flushed through a chip and subsequently incorporated in off-chip PCR, highlighted bovine serum albumin (BSA) amongst other pre-treatments, chip materials and PCR recipes as being effective in mitigating inhibition. When the BSA channel pre-coating was applied to on-chip PCR–CE experiments, a substantial improvement (10× to 40×) in signal-to-noise (S/N) of the CE product peak was conferred, and was shown with high confidence despite high S/N variability. This is the first study to quantitatively examine BSA’s ability to reduce inhibition of PCR performed on PDMS chips, and one of very few microfluidic PCR inhibition studies of any kind to use a large number of microfluidic chips (~400). The simplicity and effectiveness of our BSA coating suggest that passivating materials applied to microfluidic device channel networks may provide a viable pathway for development of bio-compatible devices with reduced complexity and cost.     

An automatic microfluidic system that continuously performs the systematic evolution of ligands by exponential enrichment

The systematic evolution of ligands by exponential enrichment (SELEX) technique has been extensively used to screen molecule-specific aptamers from combinatorial libraries of synthetic nucleic acids. Aptamers are single-stranded DNA or RNA, which have a high affinity to a large variety of molecules ranging from small drugs or metabolites to cells. Therefore, they have a variety of promising applications such as for diagnostics and targeted therapeutics. In this study, a new microfluidic chip was developed to perform continuous screening of DNA-based aptamers in an automatic format. When compared with the existing manual procedure, the developed microfluidic chip has several advantages including a rapid and efficient screening process, automation, and less consumption of samples/reagents. Experimental data showed that an aptamer specific to alpha-fetoprotein was successfully screened from a random DNA pool. The entire screening process (five continuous, repetitive rounds) can be completed within 6?h, which is much faster than the traditional methods (more than 15?h). An automatic, rapid and efficient SELEX process was performed by this developed microfluidic chip, which may enable a generalized platform for the fast screening of DNA-based biomarkers in the future.     

Polymerase chain reaction compatibility of adhesive transfer tape based microfluidic platforms

Laser patterned adhesive transfer tapes are a rapid, versatile, and low cost option to fabricate microfluidic platforms. In this work, we examined the compatibility with polymerase chain reaction (PCR) of different types of adhesive tape materials patterned with a CO₂ laser cutter. Acrylic, polyimide, and silicone-based tapes were considered. We performed a systematic study on off-the-shelf adhesive tapes with respect to fluid handling, PCR inhibition, reagent loss, and on-chip PCR reaction. A novel microfluidic PCR approach was implemented that combines the advantages of previously reported systems. It uses a thermal gradient from a single heating element and the thermocycling was carried out by passing the reaction mixture back and forth in a microfluidic channel strategically placed along the thermal gradient. Only the silicone-based tapes were compatible with on-chip PCR. The overall fabrication process takes less than 30 min, uses only off-the-shelf finished or semi-finished materials, and is amenable to large-scale reel-to-reel processing.     

Fabrication of DNA purification microchip integrated with mesoporous matrix based on MEMS technology

This paper presents a novel microfabricated DNA purification microfluidic chip with enhanced performance based on the principle of micro solid phase extraction. The microchip comprises a layer of mesoporous material as solid phase matrix which is fabricated on the internal wall of the channel of microfluidic chip by electrochemical etching Si in an electrolyte. The conditions of electrochemical etching and porosity of the mesoporous matrix have been investigated. The properties of mesoporous matrix have been characterized by scanning electron microscopy and by BET (Brunauer, Emmet, and Teller) nitrogen adsorption analysis. The pore size of the mesoporous matrix is in the range of 10–30 nm, and the surface area is about 300 m²/g. Compared with the microfluidic chips with micropillar array matrix or non-porous matrix, the microchip with mesoporous matrix is able to extract enough polymerase chain reaction-amplifiable DNA from cultures of rat mesenchymal stem cells in 20 min. This highly efficient, effortless, and flexible technology can be used as a lab-on-a-chip component for initial biologic sample preparation.     

Experimental and numerical investigation into the joule heating effect for electrokinetically driven microfluidic chips utilizing total internal reflection fluorescence microscopy

This paper presents a detection scheme for analyzing the temperature distribution nearby the channel wall in a microfluidic chip utilizing a temperature-dependent fluorescence dye. An advanced optical microscope system—total internal reflection fluorescence microscope (TIRFM) is used for measuring the temperature distribution on the channel wall at the point of electroosmotic flow in an electrokinetically driven microfluidic chip. In order to meet the short working distance of the objective type TIRFM scheme, microscope cover glass slits are used to fabricate the microfluidic chips. The short fluorescence excitation depth from a TIRFM system makes the intensity information obtained using TIRFM is not sensitive to the channel depth variation which ususally biases the measured results while using a conventional Epi-fluorescence microscope (EPI-FM). Therefore, a TIRFM can precisely describe the temperature profile of the distance within 100 nm of the channel wall where consists of the Stern layer and the diffusion layer for an electrokinetic microfluidic system. Results indicate the proposed TIRFM provides higher measurement sensitivity over the EPI-FM. Significant temperature gradient along the channel depth is experimentally observed. In addition, the measured wall temperature distributions can be the boundary conditions for numerical investigation into the joule heating effect. The proposed method gives a precise temperature profile of microfluidic channels and shows the substantial impact on developing a numerical simulation model for precisely predicting the joule heating effect in microfluidic chips.     

A new thermal cycling mechanism for effective polymerase chain reaction in microliter volumes

This study presents a new thermal cycler using infrared (IR) heating and water impingement cooling for polymerase chain reaction (PCR) amplification of 10 l samples in thin glass capillary tubes. The thermal cycling system can achieve a temperature ramping-up rate of 65 °C/s and a ramping-down rate of 80 °C/s. Two other cooling mechanisms, natural convection and forced air convection, can also be used in the present system to obtain a ramping-down rate of 2 °C/s and 6 °C/s, respectively. The amplification of the 439 fragment of hepatitis B virus (HBV) DNA was successful. The PCR amplified products were analyzed by agarose gel electrophoresis with ethidium bromide staining for visualization. A comparison of results of the amplification products at three different ramping-down rates was made and the rapid thermal cycling of the present system can run DNA required amplification in 29 min for 20 thermal cycles that is only 1/3 the time spent in the conventional PCR machine used in comparison.This work was supported by the National Taiwan University College of Medicine in preparing the HBV DNA samples. We thank our research assistance W. L. Chen and Dr. Chen for their technical advice. We gratefully acknowledge the financial support by the Nation Science Council, No. NSC-88–2212-E-002–025.     

A method of water pretreatment to improve the thermal bonding rate of PMMA microfluidic chip

A new method of water pretreatment for thermal bonding polymethylmethacrylate microfluidic chip was proposed in this paper. The bonding rate (effective bonding area) of microfluidic chip under different pretreatment time was studied and the mechanism of this method was discussed. The main thermal bonding parameters were as follows: bonding pressure 1.4 ~ 1.9 Mpa, temperature 91 ~ 93°C, time 360 s. The experimental result shows that this method can increase the effective bonding area, improve the bonding quality of the microfluidic chip compared to the conventional thermal bonding method. The optimal water pretreatment time is 1 h with the bonding rate increased by 34% compared with the conventional thermal bonding method. The pollution to the micro-channels is avoided and the performance of the microfluidic system will be reserved with this water pretreatment method. This method is available for the biochemical analysis of the chip, and holds the benefits of easy-operation, high-efficiency and low-cost properties.     

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.     

A novel integrated microfluidic platform to perform fluorescence in situ hybridization for chromosomal analysis

The fluorescence in situ hybridization (FISH) technique has been commonly employed to detect the chromosomal abnormalities. However, applications of this technique are limited due to its lengthy process and labor-intensive sample preparation. In this study, a novel integrated microfluidic chip capable of performing the entire FISH protocol automatically was reported. This novel technique can achieve several advantages, including reduce the consumption of bio-samples and reagents, automation and rapid analysis compared to the conventional method. In this study, several functional microfluidic devices were integrated on a single chip to perform automatic FISH on the microfluidic platform. Experimental data demonstrated that the developed microfluidic system successfully provided superior performance for probing the chromosomal abnormality of cells. Furthermore, the novel microfluidic system performed the entire process automatically within 3 h, where the conventional method required 10 h to perform the entire protocol manually. This data indicated superior performance of the novel method. Our findings conclude that the novel integrated FISH protocol is more convenient to perform large quantities of samples, which can be used in clinical trials.     

Low-cost polymer microfluidic device for on-chip extraction of bacterial DNA

A polymer microfluidic device for on-chip extraction of bacterial DNA has been developed for molecular diagnostics. In order to manufacture a low-cost, disposable microchip, micropillar arrays of high surface-to-volume ratio (0.152 μm⁻¹) were constructed on polymethyl methacrylate (PMMA) by hot embossing with an electroformed Ni mold, and their surface was modified with SiO₂ and an organosilane compound in subsequent steps. To seal open microchannels, the organosilane layer on top plane of the micropillars was selectively removed through photocatalytic oxidation via TiO₂/UV treatment at room temperature. As a result, the underlying SiO₂ surface was exposed without deteriorating the organosilane layer coated on lateral surface of the micropillars that could serve as bacterial cell adhesion moiety. Afterwards, a plasma-treated PDMS substrate was bonded to the exposed SiO₂ surface, completing the device fabrication. To optimize manufacturing throughput and process integration, the whole fabrication process was performed at 6 inch wafer-level including polymer imprinting, organosilane coating, and bonding. Preparation of bacterial DNA was carried out with the fabricated PDMS/PMMA chip according to the following procedure: bacterial cell capture, washing, in situ lysis, and DNA elution. The polymer-based microchip presented here demonstrated similar performance to Glass/Si chip in terms of bacterial cell capture efficiency and polymerase chain reaction (PCR) compatibility.