Halovulum marinum sp. nov., isolated from deep-sea water of the Indian Ocean, and emended description of the genus Halovulum.

A novel Gram-stain-negative, aerobic, motile by peritrichous flagella, oval to rod-shaped bacterium, designated strain 2CG4T, was isolated from a deep-sea water sample collected from the Northwest Indian Ocean. The results of phylogenetic analysis of both 16S rRNA gene and RpoC protein sequences indicated that this strain was affiliated with the genus Halovulum in the Amaricoccus clade of the family Rhodobacteraceae of the class Alphaproteobacteria, sharing 95.3 % similarity at the 16S rRNA gene sequence level with the type strain of Halovulum dunhuangense YYQ-30T, the only species in the genus Halovulum. The predominant fatty acids (>10 %) of 2CG4T were summed feature 8 (C18 : 1ω7c and/ or C18 : 1ω6c; 61.1 %) and cyclo-C19 : 0ω8c (15.6 %). The polar lipids of 2CG4T were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sulfoquinovosyldiacylglycerol. The only isoprenoid quinone of 2CG4T was ubiquinone-10. The DNA G+C content of 2CG4T was determined to be 69.4 %. The central gene pufLM for the photosynthetic reaction was not detected. No growth occurred for 2CG4T in the absence of NaCl. On the basis of these data, it is concluded that the 2CG4T represents a novel species of the genus Halovulum, for which the name Halovulum marinum sp. nov. is proposed. The type strain is 2CG4T (=CGMCC 1.16468T=JCM 32611T).

Hyphobacterium indicum sp. nov., isolated from deep seawater, and emended description of the genus Hyphobacterium.

A novel aerobic, Gram-stain-negative bacterium, designated strain 2ED5T, was isolated from a deep seawater sample in the north-west Indian Ocean. Cells of the strain were oval- to rod-shaped, and motile by a polar flagellum or sessile by a prostheca. The strain formed creamy white colonies on 2216E marine agar plates. It grew at 10-40 °C (optimum 28 °C) and pH 5.0-8.0 (optimum pH 6.0-7.0). The strain required 1-6 % (w/v) NaCl for growth and grew optimally in the presence of 2-3 % NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain 2ED5T was affiliated with the genus Hyphobacterium in the family Hyphomonadaceae of the class Alphaproteobacteria, sharing 95.1 % similarity at the 16S rRNA gene sequence level with the type strain of Hyphobacterium vulgare, the only species in the genus Hyphobacterium. The major fatty acids of the strain were C18 : 1ω7c and iso-C17 : 1ω9c, and the polar lipids included monoglycosyl diglyceride, sulfoquinovosyl diacylglycerol, glucuronopyranosyl diglyceride, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unidentified glycolipid. The strain contained ubiquinone Q-10 as the predominant respiratory quinone. The G+C content of the genomic DNA of the strain was 60.9 mol%. Based on the results of this polyphasic analysis, strain 2ED5T represents a novel species in the genus Hyphobacterium, for which the name Hyphobacterium indicum sp. nov. is proposed. The type strain is 2ED5T (=CGMCC 1.16466T=JCM 32612T).

Multifunctional picoliter droplet manipulation platform and its application in single cell analysis.

We developed an automated and multifunctional microfluidic platform based on DropLab to perform flexible generation and complex manipulations of picoliter-scale droplets. Multiple manipulations including precise droplet generation, sequential reagent merging, and multistep solid-phase extraction for picoliter-scale droplets could be achieved in the present platform. The system precision in generating picoliter-scale droplets was significantly improved by minimizing the thermo-induced fluctuation of flow rate. A novel droplet fusion technique based on the difference of droplet interfacial tensions was developed without the need of special microchannel networks or external devices. It enabled sequential addition of reagents to droplets on demand for multistep reactions. We also developed an effective picoliter-scale droplet splitting technique with magnetic actuation. The difficulty in phase separation of magnetic beads from picoliter-scale droplets due to the high interfacial tension was overcome using ferromagnetic particles to carry the magnetic beads to pass through the phase interface. With this technique, multistep solid-phase extraction was achieved among picoliter-scale droplets. The present platform had the ability to perform complex multistep manipulations to picoliter-scale droplets, which is particularly required for single cell analysis. Its utility and potentials in single cell analysis were preliminarily demonstrated in achieving high-efficiency single-cell encapsulation, enzyme activity assay at the single cell level, and especially, single cell DNA purification based on solid-phase extraction.

Automated microfluidic screening assay platform based on DropLab.

This paper describes DropLab, an automated microfluidic platform for programming droplet-based reactions and screening in the nanoliter range. DropLab can meter liquids with picoliter-scale precision, mix multiple components sequentially to assemble composite droplets, and perform screening reactions and assays in linear or two-dimensional droplet array with extremely low sample and reagent consumptions. A novel droplet generation approach based on the droplet assembling strategy was developed to produce multicomponent droplets in the nanoliter to picoliter range with high controllability on the size and composition of each droplet. The DropLab system was built using a short capillary with a tapered tip, a syringe pump with picoliter precision, and an automated liquid presenting system. The tapered capillary was used for precise liquid metering and mixing, droplet assembling, and droplet array storage. Two different liquid presenting systems were developed based on the slotted-vial array design and multiwell plate design to automatically present various samples, reagents, and oil to the capillary. Using the tapered-tip capillary and the picoliter-scale precision syringe pump, the minimum unit of the droplet volume in the present system reached ~20 pL. Without the need of complex microchannel networks, various droplets with different size (20 pL-25 nL), composition, and sequence were automatically assembled, aiming to multiple screening targets by simply adjusting the types, volumes, and mixing ratios of aspirated liquids on demand. The utility of DropLab was demonstrated in enzyme inhibition assays, protein crystallization screening, and identification of trace reducible carbohydrates.

Microfluidic picoliter-scale translational spontaneous sample introduction for high-speed capillary electrophoresis.

A novel microfluidic picoliter-scale sample introduction approach was developed by combining the spontaneous injection approach with a capillary electrophoresis (CE) system based on a short capillary and slotted-vial array. A droplet splitting phenomenon at the capillary inlet end during the spontaneous sample introduction process was observed for the first time. On the basis of this phenomenon, a translational spontaneous injection approach was established to reduce sample injection volumes to the sub-100 pL range. A versatile high-speed capillary electrophoresis (HSCE) system was built on the basis of this sample injection approach with separation performance comparable to or even better than those reported in microfluidic chip-based CE systems. The HSCE system was composed of a short fused-silica capillary and an automated sample introduction system with slotted sample and buffer reservoirs and a computer-programmed translational stage. The translational spontaneous sample injection was performed by linearly moving the stage, allowing the capillary inlet first to enter the sample solution and then removing it. A droplet was left at the tip end and spontaneously drawn into the capillary by surface tension effect to achieve sample injection. The stage was continuously moved to allow the capillary inlet to be immersed into the buffer solution, and CE separation was performed by applying a high voltage between the buffer and waste reservoirs. With the use of the novel system, high-speed and efficient capillary zone electrophoresis (CZE) separation of a mixture of five fluorescein isothiocyanate (FITC) labeled amino acids was achieved within 5.4 s in a short capillary with a separation length of 15 mm, reaching separation efficiencies up to 0.40 microm plate height. Outstanding peak height precisions ranging from 1.2% to 3.7% RSD were achieved in 51 consecutive separations. By extension of the separation length to 50 mm, both high-speed and high-resolution CZE separation of eight FITC-labeled amino acids could be obtained in less than 21 s with theoretical plates ranging from 163,000 to 251,000 (corresponding to 0.31-0.20 microm plate heights). The present HSCE system also allowed fast chiral separations of FITC-labeled amino acids under micellar electrokinetic chromatography (MEKC) mode within 6.5 s.

Microfluidic chip-based valveless flow injection analysis system with gravity-driven flows.

In this work, a microfluidic chip-based valveless flow injection analysis (FIA) system with gravity-driven flows and liquid-core waveguide (LCW) spectrometric detection was developed. Automated sample injection in the 0.3-6.4 nL range under gated injection mode was achieved by controlling the vertical position of the waste reservoir fixed on a moving platform and the residence time of the reservoir in each position, without the requirement of microvalves or electrokinetic manipulation. An integrated LCW spectrometric detection system was built on the chip by coupling a 20 mm-long Teflon AF 2400 capillary with the microchannel to function as a LCW flow cell, using a green LED as light source and a photodiode as detector. The performance of the system was demonstrated in the determination of [NO(2)](2-) based on the Saltzman reaction. Linear absorbance response was obtained in the range of 0.1-20 mg L(-1) (R(2) = 0.9910), and a good reproducibility of 0.34% RSD (n = 17) was achieved.

Establishment of a finite element model for extracting chemical reaction kinetics in a micro-flow injection system with high throughput sampling.

Numerical simulation can provide valuable insights for complex microfluidic phenomena coupling mixing and diffusion processes. Herein, a novel finite element model (FEM) has been established to extract chemical reaction kinetics in a microfluidic flow injection analysis (micro-FIA) system using high throughput sample introduction. To reduce the computation burden, the finite element mesh generation is performed with different scales based on the different geometric sizes of micro-FIA. In order to study the contribution of chemical reaction kinetics under non-equilibrium condition, a pseudo-first-order chemical kinetics equation is adopted in the numerical simulations. The effect of reactants diffusion on reaction products is evaluated, and the results demonstrate that the Taylor dispersion plays a determining role in the micro-FIA system. In addition, the effects of flow velocity and injection volume on the reaction product are also simulated. The simulated results agree well with the ones from experiments. Although gravity driven flow is used to the numerical model in the present study, the FEM model also can be applied into the systems with other driving forces such as pressure. Therefore, the established FEM model will facilitate the understanding of reaction mechanism in micro-FIA systems and help us to optimize the manifold of micro-FIA systems.

High-throughput analysis of DNA fragments using a miniaturized CE system combined with a slotted-vial array sample introduction system.

An automated nanoliter sample introduction system was combined to a liquid-core waveguide (LCW)-based microfluidic CE system for high-throughput analysis of DNA fragments. The main component of the sample introduction system was a motor-driven plate, on which a circular array of bottom-slotted vials containing sample/buffer solutions was placed. A 7 cm-long LCW capillary served as both the sample probe and separation channel. The inlet terminal of the capillary could pass through the slots of the vials for electrokinetic sample introduction, and the capillary outlet was immersed in the solution of a reservoir, behind which a PMT facing directly to the outlet was positioned. A diode laser was used as excitation source for LCW LIF detection. Performance of the system was demonstrated through the separation of DNA fragments. Baseline separation was achieved for all 11 fragments of PhiX174-HaeIII digest DNA with a throughput of 33/h. Theoretical plate number for 603 bp fragment was 7.3x10(6)/m, corresponding to a plate height 0.14 microm. The detection limitation for 603 bp fragment was 0.4 ng/microL with a precision of 2.2% RSD for the peak height. Automated sample changing and introduction were achieved with only 0.3 nL gross sample consumption for each cycle.

Fabrication of a monolithic sampling probe system for automated and continuous sample introduction in microchip-based CE.

A fabrication process for producing monolithic sampling probes on glass chips, with tip diameters of a few hundred micrometers was developed, using simple tools including a glass cutter and a bench drill. Microfluidic chips with probes fabricated by this approach were coupled to a linearly moving slotted-vial array sample presentation system for performing continuous sample introduction in the chip-based CE system. On-chip horizontal tubular reservoirs containing working electrolyte and waste were used to maintain a stable hydrostatic pressure in the chip channels during prolonged working periods. The performance of the system was demonstrated in the separation of FITC-labeled amino acids with LIF detection, by continuously introducing a train of different samples without interruption. Throughputs of 30-60/h were achieved with <1.0% carry-over and reproducibilities in peak height of 3.6, 3.3, and 3.5% RSD for arginine, FITC, and phenylalanine, respectively (n = 11). Continuous analysis of a mixture of FITC-labeled amino acids for 2 h, involving 60 analytical cycles, yielded an RSD of 7.5 and 6.8% for arginine and FITC (n = 60), respectively. An extremely low sample consumption of 30 nL for each analysis was obtained. Separation efficiencies in plate numbers were in the range of 0.8-2x10(5)/m. In addition to the application in sample introduction, the sample/reagent introduction system was also used to produce working electrolyte gradients during a CE separation to improve the separation efficiency. Comparing with isocratic electrophoresis separation, gradient CE demonstrated better separation efficiencies for a mixture of FITC-labeled amino acids.

Microfluidic liquid-liquid extraction system based on stopped-flow technique and liquid core waveguide capillary.

In this work, a miniaturized liquid-liquid extraction system under stopped-flow manipulation mode with spectrometric detection was developed. A Teflon AF liquid-core waveguide (LCW) capillary was used to serve as both extraction channel for organic solvent flow and adsorption detection flow cell. Gravity induced hydrostatic pressure was used to drive the organic and aqueous phases through the extraction channels. During extraction process, a stable organic and aqueous phase interface was formed at the outlet of the capillary, through which the analyte in the flowing aqueous stream was extracted into the stationary organic solvent in capillary. The absorbance of the analyte extracted into the organic solvent was measured in situ by a spectrometric detection system with light emitting diode (LED) as light source and photodiode as absorbance detector. The performance of the system was demonstrated in the determination of sodium dodecyl sulfate (SDS) extracted as an ion pair with methylene blue into chloroform. The precision of the measured absorbance for a 5mgL(-1) SDS standard was 6.1% R.S.D. (n=5). A linear response range of 1-10mgL(-1) SDS was obtained with 5min extraction period. The limit of detection (LOD) for SDS based on three times standard deviation of the blank response was 0.25mgL(-1).

Microfluidic sequential injection analysis in a short capillary.

An automated microfluidic sequential injection analysis system that efficiently manipulates sample and reagent solutions in the nanoliter range in approximately 10 s per analytical cycle is described. The system consisted of a 6-cm-long, typically 75-microm i.d., fused-silica capillary (which functioned as a sampling probe and reactor as well as a flow-through detection cell), a horizontally oriented waste reservoir that provided liquid level differences for inducing gravity-driven flows, an autosampling device holding samples and reagents with horizontally fixed slotted microvials, and a laser-induced fluorescence detection system. Sample and reagent zones were sequentially introduced via gravity-driven flow by scanning the capillary tip (functioning as the sampling probe) through the vial slots, while vials containing sample, reagent, and carrier were sequentially rotated to the probe by programmed movement of the vial holders. Sequentially injected nanoliter zones were rapidly mixed by convection and diffusion within the carrier flow, demonstrating a behavior that conformed well to the Taylor dispersion model, and zone penetration effects were characterized and optimized under Taylor's dispersion theory guidelines. For the determination of fluorescein, a high throughput of 400 h(-1) was achieved, rapidly producing calibration curves (five points) within 45 s. Owing to its adaptability to the Taylor's dispersion model, the system was used also for measuring diffusion coefficients of fluorescent species. Potentials for using the system in enzyme inhibition assays were demonstrated by a reaction involving the conversion of fluorescein digalactoside to fluorescent hydrolysates via beta-galactosidase and the inhibition of beta-galactosidase by diethylenetriaminepentaacetic acid.

An automated electrokinetic continuous sample introduction system for microfluidic chip-based capillary electrophoresis.

An automated and continuous sample introduction system for microfluidic chip-based capillary electrophoresis (CE) was developed in this work. An efficient world-to-chip interface for chip-based CE separation was produced by horizontally connecting a Z-shaped fused silica capillary sampling probe to the sample loading channel of a crossed-channel chip. The sample presentation system was composed of an array of bottom-slotted sample vials filled alternately with samples and working electrolyte, horizontally positioned on a programmable linearly moving platform. On moving the array from one vial to the next, and scanning the probe, which was fixed with a platinum electrode on its tip, through the slots of the vials, a series of samples, each followed by a flow of working electrolyte was continuously introduced electrokinetically from the off-chip vials into the sample loading channel of the chip. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine and phenylalanine with LIF detection, by continuously introducing a train of different samples. Employing 4.5 kV sampling voltage (1000 V cm(-1) field strength) for 30 s and 1.8 kV separation voltage (400 V cm(-1) field strength) for 70 s, throughputs of 36 h(-1) were achieved with <1.0% carryover and 4.6, 3.2 and 4.0% RSD for arginine, FITC and phenylalanine, respectively (n = 11). Net sample consumption was only 240 nL for each sample.

High-throughput nanoliter sample introduction microfluidic chip-based flow injection analysis system with gravity-driven flows.

In this work, a simple, robust, and automated microfluidic chip-based FIA system with gravity-driven flows and liquid-core waveguide (LCW) spectrometric detection was developed. The high-throughput sample introduction system was composed of a capillary sampling probe and an array of horizontally positioned microsample vials with a slot fabricated on the bottom of each vial. FI sample loading and injection were performed by linearly moving the array of vials filled alternately with 50-microL samples and carrier, allowing the probe inlet to enter the solutions in the vials through the slots sequentially and the sample and carrier solution to be introduced into the chip driven by gravity. The performance of the system was demonstrated using the complexation of o-phenanthroline with Fe(II) as a model reaction. A 20-mm-long Teflon AF 2400 capillary (50-microm i.d., 375-microm o.d.) was connected to the chip to function as a LCW detection flow cell with a cell volume of 40 nL and effective path length of 1.7 cm. Linear absorbance response was obtained in the range of 1.0-100 microM Fe(II) (r2=0.9967), and a good reproducibility of 0.6% RSD (n=18) was achieved. The sensitivity was comparable with that obtained using conventional FIA systems, which typically consume 10,000-fold more sample. The highest sampling throughput of 1000 h-1 was obtained by using injection times of 0.08 and 3.4 s for sample and carrier solution, respectively, with a sample consumption of only 0.6 nL for each cycle.