Rapid detection of bacterial cell from whole blood: Integration of DNA sample preparation into single micro-PCR chip

A novel bacterial cell detection method from blood samples has been developed for molecular diagnostics. Functional integration of DNA sample preparation into polymerase chain reaction (PCR) chip enabled detection of pathogenic bacterial cells in a single microchip. Surface-modified micropillars possessing affinity for bacterial cells were fabricated inside a PCR chip, and reaction conditions were optimized to render the microchip with high surface-to-volume ratio PCR-compatible. After bacterial cells were captured on the micropillars from whole blood and PCR inhibitors were washed out, PCR mixture was injected to allow real-time amplification of DNA extracted from the isolated cells. Cell enrichment effect produced by volume reduction from large initial sample to small micro-PCR chip chamber led to increased detection sensitivity. Moreover, the developed method from sample preparation to detection of bacterial cells from whole blood took less than 1 h. These results demonstrated that the surface-modified pillar-packed microchip would be a practical approach for integration into Lab-On-a-Chip (LOC) to enable point-of-care genetic analysis.     

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.     

pH-ChemFET-based analysis devices for the bacterial activity monitoring

Silicon and polymer microtechnologies have been developed in order to integrate pH-metry techniques in the frame of medical diagnosis. Thus, a fluidic analysis device has been designed and realised in order to monitor pH-related bacterial activities. It includes a SiO₂/Si₃N₄ pH-sensitive chemical field effect transistor (pH-ChemFET), its titanium/gold pseudo-reference gate electrode and a poly-dimethylsiloxane (PDMS) integrated flow-cell (total volume: ≤2 mm³). The whole analysis device has been used to detect the biological activities of the Lactobacillus crispatus bacteria and to estimate its sensitivity to antibiotics. Results demonstrate the detection of pH-related bacterial metabolisms in microvolumes, enabling to reduce significantly the analysis response time with regards to standard procedures and to the development of pH-ChemFET-metry for medical analysis.     

A simple and cost-effective method for fabrication of integrated electronic-microfluidic devices using a laser-patterned PDMS layer

We report a simple and cost-effective method for fabricating integrated electronic-microfluidic devices with multilayer configurations. A CO₂ laser plotter was employed to directly write patterns on a transferred polydimethylsiloxane (PDMS) layer, which served as both a bonding and a working layer. The integration of electronics in microfluidic devices was achieved by an alignment bonding of top and bottom electrode-patterned substrates fabricated with conventional lithography, sputtering and lift-off techniques. Processes of the developed fabrication method were illustrated. Major issues associated with this method as PDMS surface treatment and characterization, thickness-control of the transferred PDMS layer, and laser parameters optimization were discussed, along with the examination and testing of bonding with two representative materials (glass and silicon). The capability of this method was further demonstrated by fabricating a microfluidic chip with sputter-coated electrodes on the top and bottom substrates. The device functioning as a microparticle focusing and trapping chip was experimentally verified. It is confirmed that the proposed method has many advantages, including simple and fast fabrication process, low cost, easy integration of electronics, strong bonding strength, chemical and biological compatibility, etc.     

Fabrication and flow test of long-term hydrophilic fluidic chip without using any surface modification treatment

In order to effectively pump liquid in a fluidic chip, the PDMS or SU8 channels were frequently modified by surface treatments to obtain the hydrophilic surface but encountered the problem of the hydrophobic recovery. In this article, long-term highly hydrophilic fluidic chips were demonstrated using rapid fabrication of low-power CO₂ laser ablation and low-temperature glass bonding with an interlayer of liquid crystal polymer (LCP). The intrinsic hydrophilic materials of glass and LCP were beneficial for self-driven flow in the long-term fluidic chip by surface-tension force with no extra fluidic pumps. The higher viscosity fluid could increase the difficulty of self-driven capability. The stability of contact angle and flow test of the chip after 2 months is similar to that at beginning. The high-viscosity human whole blood was successfully driven at an average moving velocity of about 1.89 mm/s for the beginning and at 2.04 mm/s after 2 months. Our fluidic chip simplifies the traditional complex fabrication procedure of glass chips and conquers the problem of traditional hydrophobic recovery.     

Zone electrophoresis of proteins in poly(dimethylsiloxane) (PDMS) microchip coated with physically adsorbed amphiphilic phospholipid polymer

The zone electrophoresis of protein in poly(dimethylsiloxane) (PDMS) microchip coated with the physically adsorbed amphiphilic phospholipid polymer (PMMSi) was investigated. PMMSi was composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 3-(methacryloyloxy) propyltris (trimethylsiloxy) silane (MPTSSi) units in a random fashion. The membrane of PMMSi can be formed on the PDMS surface by a simple and quick dip-coating method. The membrane showed high hydrophilicity and good stability in water, as determined by contact angle measurement, fourier-transformed infrared absorption by attenuated total reflection (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS) analysis. High suppression of protein adsorption to the PDMS surface and reduction in electroosmotic flow (EOF) were achieved by PMMSi coating due to an increase of hydrophilicity, and a decrease of the ζ-potential on the surface of PDMS. For zone electrophoresis, the PMMSi30 containing 30 % hydrophilic MPC was the most suitable molecular design in terms of the stability of the coated membrane on PDMS surface. The average value of EOF mobility of PDMS microchip coated with PMMSi30 was 1.4 × 10^?4 cm² V^?1 s^?1, and the RSD was 4.1 %. Zone electrophoresis of uranine was further demonstrated with high repeatability and reproducibility. Separation of two FITC-labeled proteins (BSA and insulin) was performed with high efficiency and resolution compared with non-treated PDMS microchip.     

Study and design of resistive switching behaviors in PMMA-based conducting-bridge random-access memory (CBRAM) devices

The fabrication and the performance of PMMA resistive switching device have been studied by using FR-4 (copper), PMMA (poly methyl methacrylate) and aluminum as the active anode, the solid electrolyte and the inert cathode respectively. By etching the copper surface with the acid solution [4HNO₃ + 11H₃PO₄ (98 %) + 5CH₃COOH] at 60 °C for 2 min, a good performance of Cu/PMMA/Al device, which can switch until 2300 cycles, has been realized. The spin rate for forming the PMMA coating plays a decisive role in the performance of Cu/PMMA/Al device. The best performance of the Cu/PMMA/Al device was obtained only when the spin rate of deposition of PMMA reached 4000 rpm (low thickness).

     

Device for oxide dots fabrication with copper wire as cathode probe

A device has been fabricated to perform large area local oxidation with copper wire (thickness 7 μm) as cathode probe. Ability of this device was studied, by silicon oxide (SiO₂) dots fabrication on silicon (Si) surface and patterning of model microorganism, Mycobacterium smegmatis on the predetermined positions (oxide dots) with preferable surface monolayer. Positive patterning of Mycobacterium smegmatis attained on the fabricated substrate due to the charge difference between SiO₂ dot and Si surface, which was confirmed by atomic force microscopy and scanning electron microscopy (SEM) analysis. This device shows possibilities of overcoming the inherent limitations of large area local oxidation by AFM.     

New approach for batch microfabrication of silicon-based micro fuel cells

This paper reports a novel and straightforward approach to the development of a compact micro direct methanol fuel cell. The device consists of a hybrid polymer membrane as a feasible microintegrable electrolyte to be used together with silicon current collectors. These current collectors consist in microfabricated silicon chips that incorporate a fine electrode grid. The membrane combines two polymers with different functionalities, Nafion^® as a proton conducting material and PDMS as a flexible mechanical support. The compatibility of this membrane with MEMS fabrication processes lies in the acknowledged bonding capabilities of the PDMS polymer to materials typically used in microsystems technologies—such as silicon, silicon dioxide and glass—as well as its ability to withstand variations of the Nafion^® volume. The compatibility of all the components with microfabrication processes will permit the application of batch fabrication techniques for the whole device, so contributing to a significant lowering of the fabrication costs.     

Evaluation of micromilled metal mold masters for the replication of microchip electrophoresis devices

High-precision micromilling was assessed as a tool for the rapid fabrication of mold masters for replicating microchip devices in thermoplastics. As an example, microchip electrophoresis devices were hot embossed in poly(methylmethacrylate) (PMMA) from brass masters fabricated via micromilling. Specifically, sidewall roughness and milling topology limitations were investigated. Numerical simulations were performed to determine the effects of additional volumes present on injection plugs (i.e., shape, size, concentration profiles) due to curvature of the corners produced by micromilling. Elongation of the plug was not dramatic (< 20%) for injection crosses with radii of curvatures to channel width ratios less than 0.5. Use of stronger pinching potentials, as compared to sharp-corner injectors, were necessary in order to obtain short sample plugs. The sidewalls of the polymer microstructures were characterized by a maximum average roughness of 115 nm and mean peak height of 290 nm. Sidewall roughness had insignificant effects on the bulk EOF as it was statistically the same for PMMA microchannels with different aspect ratios compared to LiGA-prepared devices with a value of ca. 3.7 × 10⁻⁴ cm²/(V s). PMMA microchip electrophoresis devices were used for the separation of pUC19 Sau3AI double-stranded DNA. The plate numbers achieved in the micromilled-based chips exceeded 1 million/m and were comparable to the plate numbers obtained for the LiGA-prepared devices of similar geometry.     

Micromachined SiO<Subscript>2</Subscript> microcantilever for high sensitive moisture sensor

Ultra-sensitive and selective moisture sensors are needed in various industries for processing control or environmental monitoring. As an outstanding sensor platform, surface-stress sensing microcantilevers have potential application in moisture detection. To enlarge the deflection of the microcantilever under surface stress induced by specific reactions, a new SiO₂ microcantilever is developed which features a much lower Young’s modulus than conventional Si or SiN_x microcantilevers. For comparing SiO₂ cantilever with Si cantilevers, a model of the cantilever sensor is given by using both analysis and simulation, resulting in good agreement with the experimental data. The results demonstrate the SiO₂ cantilever can achieve a much higher sensitivity than the Si cantilever. In order to fabricate this device, a new fabrication process using isotropic combined with anisotropic dry etching to release the SiO₂ microcantilever beam by ICP (Inductively Coupled Plasma) was developed and investigated. This new process not only obtains a high etch rate at 9.1 μm/min, but also provides good profile controllability, and a flexibility of device design. Attributed to the high sensitivity, a significant deflection amplitude of the surface modified SiO₂ microcantilever was observed upon exposure to 1% relative humidity. The SiO₂ cantilevers are promising for inexpensive and highly sensitive moisture detection.     

高聚物微流控芯片上集成化气动微阀的研制

报道了一种新型的聚甲基丙烯酸甲酯(PMMA)/聚二甲基硅氧烷(PDMS)复合芯片。该芯片采用PMMA-PDMS…PDMS-PMMA的四层构型,以在芯片上集成气动微阀。具有液路和控制通道网路的PMMA基片与PDMS弹性膜间采用不可逆封接,分别形成液路半芯片和控制半芯片,而2个半芯片则依靠PDMS膜间的粘性实现可逆封接,组成带有微阀的全芯片。这种制备方法解决了制备PMMA-PDMS-PMMA三层结构芯片的封接难题,封接过程简单可靠。其控制部分和液路部分可以单独更换,可进一步降低使用成本,尤其适合一次性应用场合。初步实验表明:该微阀具有良好的开关性能和耐用性。     

Design and fabrication of poly(dimethylsiloxane) electrophoresis microchip with integrated electrodes

A novel poly(dimethylsiloxane) (PDMS) microchip integrated with platinum electrodes has been designed and fabricated using the Micro-electro-mechanical-systems (MEMS) technology. Since high voltage electrodes are integrated on the glass wafer using lift-off process, the microchip is a friendly-to-use system that does not need any extra mechanical apparatus for electrode insertion. To improve the sealing of microchip and ensure the uniformity of microchannel material, one PDMS membrane is formed on glass wafer with electrodes by pressing method. In this study, integrated microchip has been demonstrated as a capillary electrophoresis device for amino acids and satisfactory separation was achieved under separation electrical field strengths of 200 V/cm. The overall performance suggests that this novel microchip is advantageous and practical for the fabrication of lab-on-a-chip.     

Integrated structure of PMMA microchannels for DNA separation by microchip capillary electrophoresis

We proposed and fabricated an integrated structure of microchannels consists of three different functional PMMA layers for post-genome analysis, gene diagnosis, and screenings of useful materials for pharmaceutical. This integrated structure with 96 microchip capillary electrophoresis units in one chip is characterized as the simple structure with low cost and new aspects of the serial unit bio-chemical operation from DNA amplification to their analysis using microchip capillary electrophoresis. The design of the structure was performed using computational fluid dynamics, heat transmission, and electrophoresis simulation. To improve DNA separation resolution, microchannel with narrow width at the corner was adapted. The deep X-ray lithography process using synchrotron radiation “New SUBARU”, nano-imprint, and fusion bonding without bonding adhesive was applied for the fabrication of the integrated structure of microchannels. It was demonstrated that the proposed integrated structure of microchannels results in a good performance of the on-chip DNA amplification and separation in a small MCE unit area of 9 mm × 9 mm.     

Fabrication of flexible light guide plate using CO2 laser LIGA-like technology

The liquid crystal display (LCD) needs the back light module (BLM) for the light source. The light guide plate (LGP) is the main component of BLM to spread light source to the whole LCD surface and requires for the generation trend of lightweight, easy to carry, and bendable for LCD. In this article, we have demonstrated the fabrication of flexible LGP using CO₂ laser LIGA-like technology which includes the laser ablation of micro-groove polymethylmethacrylate (PMMA) master mold, pouring polydimethylsiloxane (PDMS) to the mold and casting the micro-groove microstructure for flexible LGP application. Different laser powers and micro-groove pitches were used to ablate the PMMA mold with varied groove depths and taper angles. Optical microscope was used to examine the morphology and profile of the final bendable LGP microstructure. Under the varied laser power of 1–12 W, the mean taper angles of PMMA micro-grooves ranged from 28° to 70° and the etching depths were from 44.5 to 281.8 μm. The flexible PDMS LGP had good microstructure duplication after casting. The optical uniformity and luminance of flexible LGP was concerned with structure of micro-grooves and measured using BM9 luminance meter. The maximal light uniformity and average luminance of LGP at some microstructure reaches 75 % and 119 cd/m², respectively.     

Optimized design and fabrication of a microfluidic platform to study single cells and multicellular aggregates in 3D

A microfluidic platform for cell motility analysis in a three-dimensional environment is presented. The microfluidic device is designed to study migration of both single cells and cell spheroids, in particular under spatially and temporally controlled chemical stimuli. A layout based on a central microchannel confined by micropillars and two lateral reservoirs was selected as the most effective. The microfluidics have an internal height of 350 μm to accommodate cell spheroids of a considerable size. The chip is fabricated using well-established micromachining techniques, by obtaining the polydimethylsiloxane replica from a Si/SU-8 master. The chip is then bonded on a 170-μm-thick microscope glass slide to allow high spatial resolution live microscopy. In order to allow the cost-effective and highly repeatable production of chips with high aspect ratio (5:1) micropillars, specific design and fabrication processes were optimized. This design permits spatial confinement of the gel where cells are grown, the creation of a stable gel–liquid interface and the formation of a diffusive gradient of a chemoattractant (>48 h). The chip accomplishes both the tasks of a microfluidic bioreactor system and a cell analysis platform avoiding critical handling of the sample. The experimental fluidic tests confirm the easy handling of the chip and in particular the effectiveness of the micropillars to separate the Matrigel? from the culture media. Experimental tests of (i) the stability of the gradient, (ii) the biocompatibility and (iii) the suitability for microscopy are presented.     

Rapid prototyping of PMMA microfluidic chips utilizing a CO<Subscript>2</Subscript> laser

A commercially available CO₂ laser scriber is used to perform the direct-writing ablation of polymethyl-methacrylate (PMMA) substrates for microfluidic applications. The microfluidic designs are created using commercial layout software and are converted into the command signals required to drive the laser scriber in such a way as to reproduce the desired microchannel configuration on the surface of a PMMA substrate. The aspect ratio and surface quality of the ablated microchannels are examined using scanning electron microscopy and atomic force microscopy surface measurement techniques. The results show that a smooth channel wall can be obtained without the need for a post-machining annealing operation by performing the scribing process with the CO₂ laser beam in an unfocused condition. The practicality of the proposed approach is demonstrated by fabricating two microfluidic chips, namely a cytometer, and an integrating microfluidic chip for methanol detection, respectively. The results confirm that the proposed unfocused ablation technique represents a viable solution for the rapid and economic fabrication of a wide variety of PMMA-based microfluidic chips.     

Cleaning of structured templates from nanoparticle accumulation using silicone

Polydimethylsiloxan (PDMS) turned out to be a simple and cost efficient material for the removal of nanoparticles from patterned surfaces. After molding the particle-laden surface using liquid silicone, surface cleaning is realized by curing the PDMS comprising the encapsulated particles and subsequent removal. The method is proven for silicon, SiO₂ and gold surfaces occupied by carbon and Polytetrafluorethylen (PTFE or Teflon) particles. Samples up to 2?inch wafers were successfully cleaned. The effect of PDMS on the surface energy is verified by contact angle measurements showing a clear change in wetting for H₂O. This effect is abolished by oxygen plasma and HF-Dip.     

Locked nucleic acids biosensor for detection of BCR/ABL fusion gene using benzoate binuclear copper (II) complex as hybridization indicator

Benzoate binuclear copper (II) complex, [Cu₂(C₇H₅O₂)₄(C₂H₆O)₂] (abbreviated as CuR₂) was prepared and its interaction with double-stranded salmon sperm DNA (dsDNA) in pH 7.4 phosphate buffer solution was studied by electrochemical experiments at the Au electrode (AuE). It was revealed that CuR₂ presented an excellent electrochemical activity on AuE and could bind with dsDNA by intercalation mode. The CuR₂ was further utilized as a new indicator in the fabrication of an electrochemical DNA biosensor for detection of BCR/ABL fusion gene. The biosensor based on nanogold (NG) modified AuE was developed by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe for hybridization with BCR/ABL fusion gene. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization. The constructed electrochemical DNA biosensor achieved a detection limit of 1.0 × 10⁻¹⁰ M for complementary target DNA with a good stability.     

A new technology for microfluidic structures preparation based on a photoimageable ceramic

A new method for fabrication of hybrid ceramic-polymer structures with diversified geometry of microchannel was elaborated. This method is universal, non-complicated, and utilises commercially available materials and basic equipment for thick film technology and photolithography. A microchip for capillary electrophoresis was prepared as an example of microfluidic structure fabrication. The chip was prepared by using a photosensitive paste (dielectric FODEL 6050) which was screen printed onto a ceramic substrate, exposed through an appropriate mask, developed, fired and then glazed. In this way, we obtained the structure which can be bonded with poly(dimethylsiloxane) PDMS after oxygen plasma treatment. The application of transparent PDMS as a seal of the microchannel enabled the optical detection.