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.     

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.     

Simulation and experimental validation of a SU-8 based PCR thermocycler chip with integrated heaters and temperature sensor

We present a SU-8 based polymerase chain reaction (PCR) chip with integrated platinum thin film heaters and temperature sensor. The device is fabricated in SU-8 on a glass substrate. The use of SU-8 provides a simple microfabrication process for the PCR chamber, controllable surface properties and can allow on chip integration to other SU-8 based functional elements. Finite element modeling (FEM) and experiments show that the temperature distribution in the PCR chamber is homogeneous and that the chip is capable of fast thermal cycling. With heating and cooling rates of up to 50 and 30 °C/s, respectively, the performance of the chip is comparable with the best silicon micromachined PCR chips presented in the literature. The SU-8 chamber surface was found to be PCR compatible by amplification of yeast gene ribosomal protein S3 and Campylobacter gene cadF. The PCR compatibility of the chamber surfaces was enhanced by silanization.     

Identification of respiratory pathogen Bordetella Pertussis using integrated microfluidic chip technology

Integrated PCR–CE chip technology has immense potential to be applied in clinical diagnostics. In this work we demonstrate the application of our integrated PCR–CE chip for the detection of the respiratory pathogen Bordetella pertussis. A series of experiments with varying cell concentrations (200,000–2 cfu) were performed to obtain the analytical detection limits of the chip. We find that the chip technology is well suited for sensitive detection of Bordetella pertussis, using genetic material from less than even 2 cfu. We also utilized an off-chip real-time PCR method to compare and validate our on-chip approach.     

Decreasing microfluidic evaporation loss using the HMDL method: open systems for nucleic acid amplification and analysis

Evaporation is of great importance when dealing with microfluidic devices with open air/liquid interfaces due to the large surface-to-volume ratio. For devices utilizing a thermal reaction (TR) reservoir to perform a series of biological and chemical reactions, excessive heat-induced microfluidic evaporation can quickly lead to reaction reservoir dry out and failure of the overall device. In this study, we present a simple, novel method to decrease heat-induced fluid evaporation within microfluidic systems, which is termed as heat-mediated diffusion-limited (HMDL) method. This method does not need complicated thermal isolation to reduce the interfacial temperature, or external pure water to be added continuously to the TR chamber to compensate for evaporation loss. The principle of the HMDL method is to make use of the evaporated reaction content to increase the vapor concentration in the diffusion channel. The experimental results have shown that the relative evaporation loss (V _loss/V _ini) based on the HMDL method is not only dependent on the HMDL and TR region’s temperatures (T _HMDL and T _TR), but also on the HMDL and TR’s channel geometries. Using the U-shaped uniform channel with a diameter of 200 μm, the V _loss/V _ini within 60 min is low to 5% (T _HMDL = 105°C, T _TR = 95°C). The HMDL method can be used to design open microfluidic systems for nucleic acid amplification and analysis such as isothermal amplification and PCR thermocycling amplification, and a PCR process has been demonstrated by amplifying a 135-bp fragment from Listeria monocytogenes genomic DNA.     

A reduced-order model for whole-chip thermal analysis of microfluidic lab-on-a-chip systems

This paper presents a Krylov subspace projection-based reduced-order model (ROM) for whole microfluidic chip thermal analysis, including conjugate heat transfer. Two key steps in the reduced-order modeling procedure are described in detail: (1) the acquisition of a 3D full-scale computational model in the state-space form to capture the dynamic thermal behavior of the entire microfluidic chip; and (2) the model order reduction using the block Arnoldi algorithm to markedly lower the dimension of the full-scale model. Case studies using practically relevant thermal microfluidic chip are undertaken to establish the capability and to evaluate the computational performance of the reduced-order modeling technique. The ROM is compared against the full-scale model and exhibits good agreement in spatiotemporal thermal profiles (<0.5 % relative error in pertinent time scales) and over three-orders-of-magnitude acceleration in computational speed. The salient model reusability and real-time simulation capability render it amenable for operational optimization and in-line thermal control and management of microfluidic systems and devices.     

Pre-programmable polymer transformers as on-chip microfluidic vacuum generators

This paper develops novel polymer transformers using thermally actuated shape memory polymer (SMP) materials. This paper applies SMPs with thermally induced shape memory effect to the proposed novel polymer transformers as on-chip microfluidic vacuum generators. In this type of SMPs, the morphology of the materials changes when the temperature of materials reaches its glass transition temperature (T _g). The structure of the polymer transformer can be pre-programmed to define its functions, which the structure is reset to the temporary shape, using shape memory effects. When subjected to heat, the polymer transformer returns to its pre-memory morphology. The morphological change can produce a vacuum generation function in microfluidic channels. Vacuum pressure is generated to suck liquids into the microfluidic chip from fluidic inlets and drive liquids in the microchannel due to the morphological change of the polymer transformer. This study adopts a new smart polymer with high shape memory effects to achieve fluid movement using an on-chip vacuum generation source. Experimental measurements show that the polymer transformer, which uses SMP with a T _g of 40°C, can deform 310 μm (recover to the permanent shape from the temporary shape) within 40 s at 65°C. The polymer transformer with an effective cavity volume of 155 μl achieved negative pressures of −0.98 psi. The maximum negative up to −1.8 psi can be achieved with an effective cavity volume of 268 μl. A maximum flow rate of 24 μl/min was produced in the microfluidic chip with a 180 mm long channel using this technique. The response times of the polymer transformers presented here are within 36 s for driving liquids to the end of the detection chamber. The proposed design has the advantages of compact size, ease of fabrication and integration, ease of actuation, and on-demand negative pressure generation. Thus, this design is suitable for disposable biochips that need two liquid samples control. The polymer transformer presented in this study is applicable to numerous disposable microfluidic biochips.     

New disposable microfluidic chip without evaporation effect for semen analysis in clinics and homes

The number of infertile couples considering using assisted reproductive technologies (ARTs) is growing. Several key indices, such as sperm concentration and motility, are considered when determining an appropriate technique among the existing ARTs. While microscopy is the only way to observe sperms, this method tends to overlook the actual swimming ability of sperms because sperms can be observed only within a very narrow field of view (FOV). In this paper, we propose a microfluidic chip capable of measuring the motility of sperms by inducing the actual swimming ability of sperms in microchannels. To determine whether sperms swim by themselves and reach the target point, 5–10 min is required in an incubator at 37 °C, which inevitably causes the evaporation of the fluid at the microfluidic chip inlet or outlet. A unique structure has been added to the microfluidic chip to prevent unwanted fluid flow due to evaporation, and counting and sorting capabilities of the fabricated device have been experimentally demonstrated. The microfluidic chip is shown to have a good agreement with commercial chips in total sperm counting. Another feature of sorting motile and progressive sperm to 95% on one chip is also verified. This feature differentiates our solution from the existing commercial chips and can help increase the success rate of ARTs. The developed MFC can provide a way to determine the actual swimming motility of sperms using a microscope in small clinics or a portable kit which is publicly available without the expensive sperm analysis equipment.

     

Magnetically controlled valve for flow manipulation in polymer microfluidic devices

A simple, external in-line valve for use in microfluidic devices constructed of polydimethylsiloxane (PDMS) is described. The actuation of the valve is based on the principle that flexible polymer walls of a liquid channel can be pressed together by the aid of a permanent magnet and a small metal bar. In the presence of a small NdFeB magnet lying below the channel of interest, the metal bar is pulled downward simultaneously pushing the thin layer of PDMS down thereby closing the channel stopping any flow of fluid. The operation of the valve is dependent on the thickness of the PDMS layer, the height of the channel, the gap between the chip and the magnet and the strength of the magnet. The microfluidic channels are completely closed to fluid flows ranging from 0.1 to 1.0 μL/min commonly used in microfluidic applications.     

Liquid�Cliquid fluorescent waveguides using microfluidic-drifting-induced hydrodynamic focusing

We demonstrate fluorescent liquid-core/liquid-cladding (L ²) waveguides focused in three-dimensional (3-D) space based on a 3-D hydrodynamic focusing technique. In the proposed system, the core and vertical cladding streams are passed through a curved 90° corner in a microfluidic channel, leading to the formation of a pair of counter rotating vortices known as the Dean vortex. As a result, the core fluid is completely confined within the cladding fluid and does not touch the top and bottom poly(dimethylsiloxane) (PDMS) surfaces of the microfluidic channel. Because the core stream was not in contact with the PDMS channel, whose refractive index contrast and optical smoothness with the core fluid are lower than that between the core and the cladding fluids, the 3-D focused L ² waveguide exhibited a higher captured fraction (η) and lower propagation loss when compared to conventional two-dimensional (2-D) focused L ² waveguides. Because the proposed 3-D focused L ² waveguides can be generated in planar PDMS microfluidic devices, such optofluidic waveguides can be integrated with precise alignment together with other in-plane microfluidic and optical components to achieve micro-total analysis systems (μ-TAS).     

On-chip CO<Subscript>2</Subscript> incubation for pocket-sized microfluidic cell culture

We have developed an on-chip CO₂ incubation system based on mass/heat transfer from aqueous solutions of bicarbonate source to cell culture media through a permeable poly(dimethylsiloxane) (PDMS) wall. Heating a carbonate-buffered bicarbonate solution successfully regulated CO₂ generation without any feedback control. Because a microfluidic cell culture chip with the incubation system does not require an external chamber or gas supply, the entire microfluidic cell culture setup becomes pocket sized. Using 5 ml of 0.8 M sodium bicarbonate with 65 mM sodium carbonate as the water jacket, the chip maintained the temperature, osmolality, and pH of 750 μl cell culture medium within physiological levels when the chip was placed on a 37°C surface. The osmolality shift and pCO₂ of the media reservoir stabilized within <5 mmol/kg and 5.0 ± 1.0% over at least 9 days. The incubation capabilities were demonstrated through microfluidic culture of COS-7 epithelial cells under an inverted microscope for 17 days.     

Microfabrication of single-use plastic microfluidic devices for high-throughput screening and DNA analysis

 Modern drug discovery and genomic analysis depend on rapid analysis of large numbers of samples in parallel. The applicability of microfluidic devices in this field needs low cost devices, which can be fabricated in mass production. In close collaboration, Greiner Bio-One and Forschungszentrum Karlsruhe have developed a single-use plastic microfluidic capillary electrophoresis (CE) array in the standardized microplate footprint. Feasibility studies have shown that hot embossing with a mechanical micromachined molding tool is the appropriate technology for low cost mass fabrication. A subsequent sealing of the microchannels allows sub-microliter sample volumes in 96-channel multiplexed microstructures. Received: 16 May 2001 / Accepted: 3 July 2001     

Exploitation of a microfluidic device capable of generating size-tunable droplets for gene delivery

This study presents a new microfluidic chip that generates micro-scale emulsion droplets for gene delivery applications. Compared with conventional methods of droplet formation, the proposed chip can create uniform droplets (size variation <7.1%) and hence enhance the efficiency of the subsequent gene delivery. A new microfluidic chip was developed in this study, which used a new design with a pneumatic membrane chamber integrated into a T-junction microchannel. Traditionally, the size of droplets was controlled by the flow rate ratio of the continuous and disperse phase flows, which can be controlled by syringe pumps. In this study, a pneumatic chamber near the intersection of the T-junction channel was designed to locally change the flow velocity and the shear force. When the upper air chamber was filled with compressed air, the membrane was deflected and then the droplet size could be fine-tuned accordingly. Experimental data showed that using the new design, the higher the air pressure applied to the active tunable membrane, the smaller the droplet size. Finally, droplets were used as carriers for DNA to be transfected into the Cos-7 cells. It was also experimentally found that the size of the emulsion droplets plays an important role on the efficiency of the gene delivery. The preliminary results of this paper have been presented at the 2007 IEEE International Conference of Nano/Molecular Medicine and Engineering (IEEE NANOMED 2007), Macau, China, 6–9 August, 2007.     

Coupled flow and reaction during natural convection PCR

Polymerase chain reaction (PCR) in a microfluidic Rayleigh–Benard convection cell represents a promising route towards portable PCR for point-of-care uses. In the present contribution, the coupled fluid mechanics and heat transport processes are solved numerically for a 2-D flow cell. The resultant velocity and temperature fields serve as the inputs to a convection-diffusion-reaction model for the DNA amplification, wherein the reaction kinetics are modeled by Gaussian distributions around the conventional bulk PCR reaction temperatures. These evolution equations are integrated to determine the exponential growth rate of the double-stranded DNA concentration. The predicted doubling time is approximately 10–25 s, increasing with the Péclet number. This effect is attributed to low velocity, slow kinetics “dead zones” located at the center of the reactor. The latter observation provides an alternative rationalization for the use of loop-based natural convection PCR systems.     

Chitosan microfiber fabrication using a microfluidic chip and its application to cell cultures

In this study, a poly-methyl-methacrylate (PMMA) microfluidic chip with a 45° cross-junction microchannel is fabricated using a CO₂ laser machine to generate chitosan microfibers. Chitosan solution and sodium tripolyphosphate (STPP) solution were injected into the cross-junction microchannel of the microfluidic chip. The laminar flow of the chitosan solution was generated by hydrodynamic focusing. The diameter of laminar flow, which ranged from 30 to 50 μm, was controlled by changing the ratio between chitosan solution and STPP solution flow rates in the PMMA microfluidic chip. The laminar flow of the chitosan solution was converted into chitosan microfibers with STPP solution via the cross-linking reaction; the diameter of chitosan microfibers was in the range of 50–200 μm. The chitosan microfibers were then coated with collagen for cell cultivation. The results show that the chitosan microfibers provide good growth conditions for cells. They could be used as a scaffold for cell cultures in tissue engineering applications. This novel method has advantages of ease of fabrication, simple and low-cost process.     

A microvalve for hybrid microfluidic systems

A hybrid valve for lab on chip applications is presented. The valve is assembled by bonding poly (methyl methacrylate), PMMA, and silicon-based elastomers. The process used to promote the hybrid bonding includes the deposition of an organosilane (TMSPM) on the thermoplastic polymer, PMMA to interface PMMA and elastomers. For this study, a membrane in ELASTOSIL^? is bonded in correspondence of the end of two microfluidic channels of a fabricated PMMA microfluidic chip. Prior the bonding, a plasma etching process has been used to remove the TMSPM in a confined circular area. This process made possible to bond selectively the edge of a membrane leaving free to move its central part. Actuating the membrane with an external positive pressure or vacuum is possible, respectively, to obstruct or to connect the microfluidic channels. The microvalve may be simply integrated in microfluidic devices and permits the control of microvolumes of fluid in processes such as transport, separation, and mixing. The deposition of the TMSPM, the bonding of the valve and its actuation has been characterized and tested. The flow rate control of liquids through the valve has been characterized. The results have been discussed and commented. The valve can stand up to 14 psi without showing leakages.     

Abstraction layers for scalable microfluidic biocomputing

Microfluidic devices are emerging as an attractive technology for automatically orchestrating the reactions needed in a biological computer. Thousands of microfluidic primitives have already been integrated on a single chip, and recent trends indicate that the hardware complexity is increasing at rates comparable to Moore’s Law. As in the case of silicon, it will be critical to develop abstraction layers—such as programming languages and Instruction Set Architectures (ISAs)—that decouple software development from changes in the underlying device technology. Towards this end, this paper presents BioStream, a portable language for describing biology protocols, and the Fluidic ISA, a stable interface for microfluidic chip designers. A novel algorithm translates microfluidic mixing operations from the BioStream layer to the Fluidic ISA. To demonstrate the benefits of these abstraction layers, we build two microfluidic chips that can both execute BioStream code despite significant differences at the device level. We consider this to be an important step towards building scalable biological computers.