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.     

Compact optical diagnostic device for isothermal nucleic acids amplification

We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of DNA amplification by-product, magnesium pyrophosphate. We have established a correlation curve (R² = 0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. For the applications of rapid pathogens detection, we also have established a standard curve (R² = 0.96) by using LAMP reaction with a standard template in our device. Moreover, we also have successfully used the device on seven clinical serum specimens where HBV DNA levels have been confirmed by real-time PCR. The result indicates that different amounts of HBV DNA can be successfully detected by using this device within 1 h.     

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.     

Rapid glucose concentration detection utilizing disposable integrated microfluidic chip

A novel three-dimensional (3D) disposable glucose concentration detection chip is presented. The chip comprises a four-layer polymethyl methacrylate (PMMA) structure and is fabricated using a commercial CO₂ laser and a hot-press bonding technique. In the proposed device, the glucose solution is injected into a double parallel connection micromixer (DPCM) and is mixed with DNS reagent by means of a self-rotation effect. An experimental platform has been created for multiple reaction process by integrating chip and micro-heater. The fluid streams exiting the two circular mixing chambers of the DPCM are then combined and mixed further at a T-type microchannel outlet before passing to a collection chamber. Numerical simulations are performed to analyze the vortex streamline distribution within the DPCM and to estimate the mixing performance. The numerical results show that a mixing efficiency as high as 92.5% can be obtained at low Reynolds numbers (Re = 12). It is found a good linear relation of R ² = 0.9953 from the chip detection method comparing to the traditional method of R ² = 0.9976 at DNS reagent and glucose solution volume ratio of 1:1. In addition, the experimental results show that the accuracy of the glucose concentration measurements obtained using the proposed microfluidic chip is comparable with that of the measurements obtained using a conventional large-scale detection method. Overall, the results presented in this study indicate that the DPCM chip provides a rapid and low-cost means of detecting the concentration of glucose solutions.     

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.     

Automated genomic and proteomic applications on the Biomek NX laboratory automation workstation

This technical paper describes the utilization of a new automated liquid handler from Beckman Coulter, Inc., the Biomek^® NX Laboratory Automation Workstation, for genomic and proteomic applications. For genomic applications, methodology for plasmid DNA purification using Promega Wizard^® SV 96 reagents was developed for the Biomek NX. A single plate of bacterial pellets can be processed to purified plasmid DNA without user interaction after initial setup. DNA quantity and quality were assessed by spectrophotometric analysis, restriction digestion, PCR (The PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman La Roche, Ltd.), and capillary sequencing. Additionally, the plasmid preparation method was used to purify plasmid DNA from bacterial clones isolated in a bacterial two-hybrid screening procedure. In this case, the system quickly and efficiently prepared clones for rapid identification of target sequences. For proteomic applications, His-tag proteins were purified from bacterial cultures in a 96-well plate format. Following purification, a Bradford assay was used to determine the quantitative yields of the His-tag protein products in each of the aliquots from the purified samples. The AD 340 Automated Labware Positioner (ALP), an integrated absorbance reader, was used for absorbance measurements in the Bradford assay. Given the placement of this ALP on the deck of the Biomek NX, the entire process of protein purification and quantitation was performed in a complete walk-away automated format. Results obtained when purifying proteins, from both uninduced and induced bacterial cultures, on the worksurface of the Biomek NX will be described.     

A magnetic bead-based DNA extraction and purification microfluidic device

This article introduces a novel magnetic bead-based DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 μL in volume, 500 μm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip. Manufacturer’s standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56 °C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.     

Automatic detection of Salmonella enterica in sprout irrigation water using a nucleic acid sensor

A nucleic acid sensor capable of automated sample and reagent loading, real-time PCR, automated detection, and sample line cleaning was tested. Real-time PCR reactions were performed with Salmonella enterica in autoclaved and spent alfalfa sprout irrigation water. S. enterica boiled cells were detected over a range of approximately 10⁴ to 10⁸ CFU/reaction (rxn). It was possible to generate enough PCR product to visualize a band on a gel at the expected size over approximately five orders of magnitude from 3.2 × 10³ to 10⁸ CFU/rxn. Automated detection experiments yielded correct identification of 9/9 positive control reactions over a range of 10⁴ to 10⁸ CFU/rxn, correctly identified a negative control reaction, and a sample of 3.2 × 10³ CFU/rxn was incorrectly identified as negative. Primer dimers were not seen in positive or negative control reactions with sprout irrigation water, suggesting that it may be possible to improve the detection limit simply by increasing the number of thermal cycles or by lowering the annealing temperature. The system required no interpretation of real-time PCR data by the operator. The entire process of loading, running the PCR, automated data interpretation, and sample line cleaning was completed in under 2 h and 20 min, significantly faster than it would take to ship a sample and have it tested by an independent laboratory.     

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.     

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.     

Environmentally friendly disposable sensors with microfabricated on-chip planar bismuth electrode for in situ heavy metal ions measurement

This paper presents an environmentally friendly disposable heavy metal ion sensor for in situ and online monitoring in the nature and physiological systems. The miniaturized sensor chip consists of a non-toxic microfabricated bismuth (Bi) working electrode that replaces the conventional mercury electrodes, an integrated Ag/AgCl reference electrode, a gold counter electrode, and microfluidic channels. In this work, the electrochemical behavior of the Bi working electrode was characterized in several non-deaerated buffer solutions using cyclic voltammetry. The detection and quantification of Pb (II) and Cd (II) were statically performed using anodic stripping voltammetry inside the microchannels, in the Pb (II) concentration range of 25–400 ppb (R² = 0.991) with limit of detection of 8 ppb for 60 s deposition, and in the Cd (II) concentration range of 28–280 ppb (R² = 0.986) with limit of detection of 9.3 ppb for 90 s deposition. Particularly, the applications of this sensor chip have been reported with the examples of in situ measurement of Cd (II) concentration in soil pore and ground water and online direct measurement of Cd (II) concentration in cell culture media in its native environment.     

Review of “Ethnic Variables in Human Factors Engineering. Edited ” by Alphonse ChApanis-(Baltimore: Johns Hopkins University Press, 1975.) [Pp. xviii + 290.] £9-50.

Measurements were made of the motion of a ship, and of the consequent seasickness experienced by passengers. Data are presented for 17 voyages of up to 6?hours duration, involving 4915 passengers. Vertical motion occurred up to 1·0 m s^?2 r.m.s. and vomiting incidence of up to nearly 40% was encountered.

Both vomiting incidence and illness rating correlated well with root mean square vertical z-axis acceleration. The effect of exposure duration was also investigated, producing suggestions for a combined measure of acceleration and time. Multiple regression analysis with all six axes of motion revealed only a small increase in correlation when all directions of motion in addition to the z-axis were taken into account.     

An efficient parallel algorithm for geometrically characterising drawings of a class of 3-D objects

Labelling the lines of a planar line drawing of a 3-D object in a way that reflects the geometric properties of the object is a much studied problem in computer vision, considered to be an important step towards understanding the object from its 2-D drawing. Combinatorially, the labellability problem is a Constraint Satisfaction Problem and has been shown to be NP-complete even for images of polyhedral scenes. In this paper, we examine scenes that consist of a set of objects each obtained by rotating a polygon around an arbitrary axis. The objects are allowed to arbitrarily intersect or overlay. We show that for these scenes, there is a sequential lineartime labelling algorithm. Moreover, we show that the algorithm has a fast parallel version that executes inO(log³ n) time on an Exclusive-Read-Exclusive-Write Parallel Random Access Machine withO(n ³/log³ n) processors. The algorithm not only answers the decision problem of labellability, but also produces a legal labelling, if there is one. This parallel algorithm should be contrasted with the techniques of dealing with special cases of the constraint satisfaction problem. These techniques employ an effective, but inherently sequential, relaxation procedure in order to restrict the domains of the variables.This research was partially supported by the European Community ESPRIT Basic Research Program under contracts 7141 (project ALCOM II) and 6019 (project Insight II).     

Failure of Au RF-MEMS switches subjected to dynamic loading

The dynamic failure of Au RF-MEMS was investigated over a wide range of loading rates by three different experimental setups: a drop weight tower, which induced a maximum peak acceleration of 600g (g: acceleration of gravity), a Hopkinson pressure bar with a maximum peak acceleration of 300,000g, and a pulsed laser loading technique with a maximum peak acceleration of 1.8 × 10⁸g. In the drop weight tower the total load pulse duration was in the milliseconds range – much longer than the 28 μs resonant period of the devices – and no failure of any kind occurred in the RF-MEMS devices or their substrate. At 90,000g (generated in the Hopkinson bar) no damage in either the substrate or the devices was observed. However, at 200,000g, which corresponds to a loading duration of a few microseconds, i.e., comparable to the device resonant period, 10% of the switches failed although postmortem imaging showed no damage to the substrate. Damage increased after this acceleration and at 300,000g 20% of the switches failed, but, in addition, significant failure in the quartz substrate was recorded. Lastly, the pulsed laser loading technique, which has a loading pulse duration of a few tens of nanoseconds, was applied to accelerate the Au switches to 1.8 × 10⁸g, and the probability of failure at this loading ranged from 50% to 80%. At even larger accelerations, 10⁹g, the probability of failure was 100%. The results of this study establish the severity of dynamic failure in MEMS, despite their small mass, and its dependence on the level of acceleration which spanned about 7 orders of magnitude.     

基于纳米磁珠技术的新型微全分析DNA芯片的研究

在微全分析系统的研究中,样品提取及DNA分析技术是非常重要的一个环节.也是目前国内外研究的热点之一.文中介绍了一种新型的基于单芯片的样品制备和扩增方法.采用多层微加工技术制作SU-8模具,通过注模成型,制作出有立体微柱结构的PDMS(聚二甲基硅氧烷)芯片,在芯片微池内填充超顺磁性磁珠,利用固相提取(solid phase extraction,SPE)法,将细胞裂解、DNA提取、PCR反应等功能集成在一个PDMS芯片上.整个流程快速有效,操作简便且易于芯片系统集成,提取产物可以不必洗脱,直接作为下一步PCR反应的模板,在同一芯片上进行扩增反应,实现了样品预处理、DNA提取和PCR扩增的集成.     

Label-free electrochemical impedance spectroscopy using a micro interdigitated electrode inside a PCR chip for real-time monitoring

Conventional real-time PCR using fluorescence detection requires expensive optical detection systems with fluorescence labeling. To simplify this PCR system, we proposed an electrochemical impedance spectroscopy (EIS) using an interdigitated electrode integrated inside the PCR chip. The electrode makes a direct contact with the PCR sample and does not require any labeling or immobilization pretreatment. The input AC voltage for EIS showed the lowest noise at 100 mV. Electrical impedances in a frequency domain were measured during 30 cycles in the PCR of Escherichia coli genomic DNA region (of length 180 bp, 10 ng/μl). From the analysis of EIS data, the magnitude of imaginary value steadily increased with an increase in the PCR cycles and showed the greatest change rate at 186 Hz. For comparing the quantitative performance with previous researches, the figure of merit (FM) was defined as the ratio of normalized sensitivity (NS) to the normalized root mean square error (NRMSE). The performance of the proposed EIS method is similar to that reported in other studies, and the damage of the sample monitored through electrophoresis by EIS measurement was confirmed to be negligible.

     

A miniaturized bulk micromachined triaxial accelerometer fabricated using deep reactive etching through a multilevel thickness membrane

We present the design, fabrication and characterization of an application specific triaxial accelerometer for post-surgery heart monitoring. The accelerometer chip is designed as a 2?×?4?×?1.2?mm³ chip with nominal acceleration range of?±4?g and frequencies below 50?Hz. It has been fabricated using a multiproject wafer service with an additional deep reactive ion etching process to obtain controlled etch-through of membranes of 3, 23 and 400?μm thicknesses simultaneously. The novelty of the work presented here is the bulk micromachining technique using both deep reactive dry etching and alkali-based anisotropic wet etching of single crystal (100) silicon wafers used to obtain a space efficient design. Proof of concept is demonstrated with preliminary testing, with an acceleration sensitivity of?~0.04 mv/V/g for out of plane (z axis) acceleration.