Development of a passive micromixer based on repeated fluid twisting and flattening,and its application to DNA purification

We have developed a three-dimensional passive micromixer based on new mixing principles, fluid twisting and flattening. This micromixer is constructed by repeating two microchannel segments, a “main channel” and a “flattened channel”, which are very different in size and are arranged perpendicularly. At the intersection of these segments the fluid inside the micromixer is twisted and then, in the flattened channel, the diffusion length is greatly reduced, achieving high mixing efficiency. Several types of micromixer were fabricated and the effect of microchannel geometry on mixing performance was evaluated. We also integrated this micromixer with a miniaturized DNA purification device, in which the concentration of the buffer solution could be rapidly changed, to perform DNA purification based on solid-phase extraction.     

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 μL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a β-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.     

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification.     

Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization

Chromatography is one of the key operations in the downstream processing of plasmid DNA (pDNA). However, the increased demand for highly purified pDNA experienced in recent years has made clear the need for alternative processes capable of retaining the advantages of conventional chromatography, such as selectivity, while providing increased throughput at a lower cost. The work presented in this article outlines the development and optimization of an alternative hydrophobic interaction membrane chromatography process for the purification of pDNA. The studies included the modification of functionalized membrane supports with a linear alkyl chain ligand and the testing of chromatographic performance of these membranes. Three modification procedures were tested and the membranes were screened for their capacity and selectivity. The modified membranes could separate the model plasmid pVAX1‐LacZ (6050 bp) from impurities in clarified Escherichia coli cell lysates (specifically RNA), with good resolution. Subsequent optimization of elution profiles with the best‐performing modified membrane, resulted in a high purification factor of 4.7, competitive with its bead process counterpart, and a plasmid yield of 73%.     

Plasma modification of polypropylene surfaces and grafting copolymerization of styrene onto polypropylene

The surface ofpolypropylene (iPP) is modified with glow discharge plasma of Ar,so that the modified surfaces of iPP films are obtained.The studies of scanning electron microscopy (SEM) show the surface etching pattern of iPP films.The chemical structures of iPP films are confirmed by X-ray photoelec     

DNA removal on different chromatography supports used for EPO purification by spike studies

Chromatographic techniques are used in the purification step of human recombinant erythropoietin production process to obtain a reliable product with high purity. Anion-exchange chromatography supports have proved high efficient in removing contaminants such as DNA. For that reason, the DNA removal was determined by spike studies, on three anion-exchange chromatographic supports: gel, membrane, and monolithic column, which is used in intermediate purification stage. This study showed that membrane and monolith columns have very good results in the removal of contaminants at this step. Log removal values (LRV) greater than 3.5 were obtained from DNA spike clearance studies. Monolithic column was determined as the best technological proposal, with more than 4 LRV, 7.72?mg DNA per milliliter of adsorbent and 85% protein recovery in nonspike run. The results of this study may be used as a guide in the selection of commercially available chromatography supports for intermediate purification steps in recombinant protein production.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

Trapping DNA with a high throughput microfluidic device

Long strands of DNA can be trapped and concentrated near the inlet of a microfluidic channel by applying a pressure gradient and an opposing electric field. The mechanism for trapping involves a migration of DNA perpendicular to both the fluid flow and the electric field. Migration leads to a highly nonuniform distribution of DNA within a cross section of the channel, with the bulk of the DNA concentrated in a thin (10 μm) layer next to the walls of the channel. This highly concentrated layer generates an electrophoretic flux toward the inlet to the device, despite the much larger fluid flow in the opposite direction. In this paper, the extent to which DNA can be trapped and concentrated by this means has been characterized by fluorescence measurements. At short times (<2 hours) nearly all the incoming DNA remains trapped within the device until the electric field is turned off. The DNA largely accumulates near the inlet, but after 30–60 minutes additional DNA starts to accumulate deeper into the channel. Eventually DNA leaks from the device itself, but ≈80% of the incoming DNA can be retained for up to 5 hours. Optimizing the electric field strength can increase the amount of DNA that can be trapped, but the efficiency is not affected by the channel cross‐section.     

Novel polymeric ionic liquid microspheres with high exchange capacity for fast extraction of plasmid DNA

A novel polymeric ionic liquid (PIL) microsphere, poly(1-vinyl-3-(2-methoxy-2-oxyl ethyl)imidazolium) hexafluorophosphate, is prepared via W/O emulsion polymerization. Rapid ion-exchange between the anionic moieties of PIL and DNA fragments is demonstrated facilitating the exchange equilibrium to be reached within 1 min. The PIL microspheres exhibit a high capacity of 190.7 μg mg⁻¹ for DNA adsorption. A fast DNA isolation protocol is thus developed with the PIL microspheres as solid phase adsorbent. It is feasible to facilitate DNA adsorption or stripping from the microspheres by simply regulating the concentration of salt. DNA adsorption is facilitated at low salt concentration, while higher concentration of salt entails DNA recovery from the microspheres. In practice, the retained DNA could be readily recovered with 1.0 mol L⁻¹ NaCl as stripping reagent, giving rise to a recovery of ca. 80.7%. The PIL microspheres are used for the adsorption/isolation of plasmid DNA from E. coli cell culture, demonstrating a superior adsorption performance with respect to that achieved by a commercial Plasmid Miniprep Kit.     

DNA fragment‐assisted microfluidic chip for capture and release of circulating tumor cells

Circulating tumor cells are specifically referred as cells that detached from the primary tumor and are present in the bloodstream. They could be isolated from blood and used as representative biomarker for predicting cancer prognoses. Here, we developed a microfluidic chip with multiple curved channels, in which DNA fragments and antibody‐based enrichment are exploited to capture circulating tumor cells in blood sample. By introducing DNA fragments as long tentacles, the active antibody could be extended into the microchannel stereoscopically, which could greatly increase the chances of adhesion in a multidirectional way and improve the capture efficacy. Several pivotal factors for cell capturing were optimized to the best state. Compared to conventional chips for planar capturing, the capture efficiency of MCF‐7 cells was greatly increased from 37.17 to 85.10%. For the detection of MCF‐7‐containing artificial blood sample detection, the capture efficiency of tumor cells was about 74.19 ± 2.13%, which was obviously better than the result of flow cytometry (29.67 ± 4.02%). Captured cells were easily released from the surface of microfluidic chip with high cell viability, which could be investigated for the molecular analysis in the field of tumor diagnosis.     

Cationic polymer coatings for design of electroosmotic flow and control of DNA adsorption

A difficulty with the design and operation of an electrokinetically operated DNA hybridization microfluidic chip is the opposite direction of the electroosmotic flow and electrophoretic mobility of the oligonucleotides. This makes it difficult to simultaneously deliver targets and an appropriate hybridization buffer simultaneously to the probe sites. In this work we investigate the possibility of coating the inner walls of the microfluidic system with hexadimentrine bromide (polybrene, PB) and other cationic polymers in order to reverse the direction of electroosmotic flow so that it acts in the same direction as the electrophoretic transport of the oligonucleotides. The results indicated that the electroosmotic flow (EOF) in channels that were coated with the polymer could be reversed in 1× TBE buffer or 1× SSC buffer. Under these conditions, the DNA and EOF move in the same direction, and the flow can be used to deliver DNA to an area for selective hybridization within the channel. The effects of coating the surface of a nucleic acid microarray with polybrene were also studied to assess non-selective adsorption and stability. The polybrene coating significantly reduced the extent of non-selective adsorption of oligonucleotides in comparison to adsorption onto a glass surface, and the coating did not alter the extent of hybridization. The results suggest that use of the coating makes it possible to achieve semi-quantitative manipulation of nucleic acid oligomers for delivery to an integrated microarray or biosensor.     

Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples

An important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. We compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity, and time requirement. A special focus was on methods that are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment sizeof approx 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the eDNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in-gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18SrDNA groups under standard polymerase chain reaction conditions.     

Recent advances in polymeric facilitated transport membranes for carbon dioxide separation and hydrogen purification

Membrane and membrane process have been widely considered as one of the best candidates for mitigating CO₂ emissions from the combustion or utilization of fossil fuels. Various amine-containing polymers constitute an important class of membranes, where the highly selective CO₂ transport is achieved by the facilitated transport mechanism. In this review, the amine–CO₂ chemistry is discussed in conjunction with the mechanism of the reaction-mediated CO₂ transport. A wide variety of amine-containing polymers are discussed based on two synthesis motifs: (a) polyamines with amino groups covalently bound to the polymer backbone and (b) small molecule amines embedded in a polymer matrix. This review concludes with the remarks on the facilitated transport membranes for post-combustion carbon capture (CO₂/N₂) and hydrogen purification (CO₂/H₂).     

Plasma Characteristics and Antenna Electrical Characteristics of an Internal Linear Inductively Coupled Plasma Source with a Multi-Polar Magnetic Field

The development of a large-area plasma source with high density plasmas is desired for a variety of plasma processes from microelectronics fabrication to flat panel display device fabrication. In this study, a novel internal-type linear inductive antenna referred to as “double comb-type antenna” was used for a large-area plasma source with the substrate area of 880 mm × 660 mm and the effect of plasma confinement by applying multi-polar magnetic field was investigated. High density plasmas on the order of 3.2 × 10¹¹ cm^?3 which is 50% higher than that obtained for the source without the magnetic field could be obtained at the pressure of 15 mTorr Ar and at the inductive power of 5,000 W with good plasma stability. The plasma uniformity <3% could be also obtained within the substrate area. When SiO₂ film was etched using the double comb-type antenna, the average etch rate of about 2,100 Å/min could be obtained with the etch uniformity of 5.4% on the substrate area using 15 mTorr SF₆, 5,000 W of rf power, and ?34 V of dc-bias voltage. The higher plasma density with an excellent uniformity and a lower rf antenna voltage obtained by the application of the magnetic field are related to the electron confinement in a direction normal to the antenna line.     

DNA purification on a lab-on-valve system incorporating a renewable microcolumn with in situ monitoring by laser-induced fluorescence

Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers. The practical applicability of the entire system was demonstrated by separation/purification of λ-DNA in a simulated matrix and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification. When DNAs in a simulated matrix (10.0 ng μl⁻¹ λ-DNA, 50 ng μl⁻¹ bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution flow rate of 0.5 μl s⁻¹, along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples were performed under similar conditions. Figure Lab-on-valve mesofluidic system employed for DNA separation and purification integrating a demountable fluorescence flow cell for in-situ laser induced fluorescence detection     

Platinum black coated microdisk electrodes for the determination of high concentrations of hydrogen peroxide in phosphate buffer solutions

The performance of a series of platinum black coated microdisk electrodes (Pt-Bs) was investigated in H₂O₂ solutions over the concentration range 0.1–500 mM, in phosphate buffer media pH 7. The Pt-Bs were prepared by electrodeposition of Pt onto the surface of microdisk electrodes 12.5 μm of nominal radius, from aqueous solutions containing hexachloroplatinic acid. The resulting roughness factors (RF, i.e., the ratio of the effective surface area to the geometric electrode area) varied between about 10 and 100. The voltammograms recorded with these electrodes, at relatively low H₂O₂ concentrations (up to 50–100 mM), displayed rather steep mixed anodic–cathodic waves, which attained well-defined and stable current plateaus. At the higher hydrogen peroxide concentrations, additional waves both in the anodic and cathodic region or dramatic current drop phenomena were observed. The wave split phenomenon was attributed to the insufficient buffering capacity of the base electrolyte solution within the pores of the platinum black, induced by the large amounts of hydrogen ions produced in the oxidation process. The current drop was attributed to either the formation of oxygen bubbles, which limit diffusion of H₂O₂ down the pores, or saturation of the active sites responsible for the decomposition of H₂O₂ to O₂ and H₂O. The H₂O₂ concentration at which the above phenomena occurred depended either on the phosphate buffer concentration in the bulk solution or the RF factor of the electrodes. The latter conditions also affected the dynamic range of detection, the sensitivity and low detection limits. Advantageous analytical characteristics were obtained with a Pt-B of RF of about 24, which provided a dynamic range between 0.5 and 230 mM, a sensitivity of 1.93(±0.06) A M⁻¹ cm⁻² and a low detection limit of 0.05 mM. The reproducibility was also very good, it being within 2–3%. The usefulness of the Pt-Bs for real samples analysis was tested in an antiseptic solution containing large amounts of H₂O₂.     

Influence of polymer structure on electroosmotic flow and separation efficiency in successive multiple ionic layer coatings for microchip electrophoresis

The effect of successive multiple ionic layer (SMIL) coatings on the velocity and direction of EOF and the separation efficiency for PDMS electrophoresis microchips was studied using different polymer structures and deposition conditions. To date, the majority of SMIL studies have used traditional CE and fused-silica capillaries. EOF was measured as a function of polymer structure and number of layers, in one case using the same anionic polymer and varying the cationic polymer and in the second case using the same cationic polymer and varying the anionic polymer. In both situations, the EOF direction reversed with each additional deposited polymer layer. The absolute EOF magnitude, however, did not vary significantly with layer number or polymer structure. Next, different coatings were used to compare separation efficiencies on native and SMIL-coated PDMS microchips. For native PDMS microchips, the average separation efficiency was 4105 +/- 1540 theoretical plates. The addition of two layers of polymer increased the separation efficiency anywhere from two- to five-fold, depending on the polymer structure. A maximum separation efficiency of 12 880 +/- 1050 theoretical plates was achieved for SMIL coatings of polybrene (cationic) and dextran sulfate (anionic) polymers after deposition of six total layers. It was also noted that coating improved run-to-run consistency of the peaks as noted by a reduction of the RSD of the EOF and separation efficiency. This study shows that the use of polyelectrolyte coatings, irrespective of the polymer structure, generates a consistent EOF in the current experiments and dramatically improves the separation efficiency when compared to unmodified PDMS microchips.     

Recent advances in high surface area electrodes for bioelectrochemical applications

The search for high surface area electrodes for bioelectrochemical applications is becoming more intense. In the last few years, new strategies have emerged to develop three-dimensional electrode materials with very well controlled architecture providing at the same time high specific surface, bendability and flexibility. This review will highlight some of the recent work published in the last 2 years and will discuss the issue of mathematical modeling of porous electrodes and what could be the future of high surface area electrodes materials.     

Evaluation of a new polymeric stationary phase with reversed-phase properties for high temperature liquid chromatography

The performance of a polymeric stationary phase with reversed-phase properties (ET-RP1) was evaluated for LC separations at elevated temperature. The most significant observation was that the reduced plate height (h) decreased from 3.4 at 25 °C (optimal flow 0.5 mL/min) to 2.4 at 150 °C (optimal flow 2.5 mL/min) which is comparable to the efficiency obtained with silica-based reversed-phase columns of 4.6 mm ID operated at 0.8 mL/min. The phase showed no deterioration after long use at 150 °C within the pH range 1–9. Catalytic activity originating from the stationary phase material, e.g. as experienced on zirconium columns operated at elevated temperature, was absent. The performance of ET-RP1 is illustrated with the analysis of some pharmaceutical samples by LC and LC–MS. Operation at elevated temperature also allows to reduce the amount of organic modifier or to replace acetonitrile and methanol by the biodegradable ethanol.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.