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Zhao-Lun Fang

Publications and source records attributed to Zhao-Lun Fang.

At least 19 recordsLinked to original sources
PubMed2007

Direct extraction of double-stranded DNA into ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and its quantification.

Ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6), as a green solvent, was successfully used for the direct extraction of dsDNA. The extraction efficiency and the distribution coefficient values indicated that trace amounts of DNA at the levels of <5 ng microL-1 facilitate quantitative fast extraction, while proteins and metal species do not interfere. A total of 30% of the DNA in ionic liquid at approximately 20 ng microL-1 was back extracted into aqueous phase in phosphate-citrate buffer with a single-stage extraction. The extraction is demonstrated to be endothermic with an enthalpy of 34.3 kJ moL-1. The extraction mechanisms were proposed and verified by 31P NMR and FT-IR spectra. Interactions between cationic 1-butyl-3-methylimidazolium (Bmim+) and P-O bonds of phosphate groups in the DNA strands take place both in the dissolved BmimPF6 in aqueous phase and at the interface of the two phases. This interaction consequently led to the transformation of DNA conformations, along with a reduction of ethidium resonance light scattering at 510 nm, and a procedure for DNA quantification in ionic liquid was developed based on this observation.

Cations↗
PubMed2006

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.

Journal Article↗
PubMed2006

A microfluidic chip based sequential injection system with trapped droplet liquid-liquid extraction and chemiluminescence detection.

A microfluidic chip-based sequential injection system with trapped droplet liquid-liquid extraction preconcentration and chemiluminescence detection was developed for achieving high sensitivity with low reagent and sample consumption. The microfabricated glass lab-chip had a 35 mm long extraction channel, with 134 shrunken opening rectangular recesses (L 100 microm x W 50 microm x D 25 microm) arrayed within a 1 mm length on both sides of the middle section of the channel. Ketonic peroxyoxalate ester solution was filled in the recesses forming organic droplets, and keeping the aqueous sample solution flowing continuously in the extraction channel; analytes were transferred from the aqueous phase into the droplets through molecular diffusion. After liquid-liquid extraction preconcentration, catalyst and hydrogen peroxide solutions were introduced into the channel, and mixed with analytes and peroxyoxalate ester to emit chemiluminescence light. The performance of the system was tested using butyl rhodamine B, yielding a precision of 4% RSD (n = 5) and a detection limit of 10(-9) M. Within a 17 min analytical cycle, the consumptions of sample and peroxyoxalate solutions were 2.7 microL and 160 nL, respectively.

Journal Article↗
PubMed2006

Laser-induced fluorescence detection system for microfluidic chips based on an orthogonal optical arrangement.

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45 degrees in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90 degrees. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N = 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building.

Journal Article↗
PubMed2005

Octadecyl immobilized surface for precipitate collection with a renewable microcolumn in a lab-on-valve coupled to an electrothermal atomic absorption spectrometer for ultratrace cadmium determination.

Octadecyl immobilized surface was, for the first time, proved to be a superb precipitate-collecting medium. Surface charge effect was assumed to dominate the adsorption of cadmium hydroxide precipitate, facilitated by electrostatic interaction between the negatively charged C18 bead surface and positively charged cadmium hydroxide clusters. Residual silanol groups on the C18-immobilized silica surface did not contribute to precipitate adsorption. A novel procedure for ultratrace cadmium preconcentration was proposed by incorporating a renewable microcolumn in a lab-on-valve system. Cd(OH)(2) precipitate was adsorbed onto the C18 surface, which was afterward eluted with 20 microL of nitric acid (1%) and quantified with detection by electrothermal atomic absorption spectrometry. An enrichment factor of 28 and a limit of detection of 1.7 ng L(-1), along with a sampling frequency of 13 h(-1) were obtained with a sample consumption of 600 microL within the concentration range of 0.01-0.2 microg L(-1), achieving a precision of 2.1% RSD at the 0.05 microg L(-1) level. The enrichment factor was further enhanced to 44 by increasing the sample volume to 1200 microL. The procedure was validated by analyzing cadmium in three certified reference materials, that is, river sediment (CRM 320), sea lettuce (CRM 279), and frozen cattle blood (GBW 09140). Good agreement between the obtained results and the certified values was achieved.

Absorption↗
PubMed2005

Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis.

Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H2O2 for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.

Calibration↗
PubMed2005

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.

Electrophoresis, Capillary↗
PubMed2005

Microfluidic chip-based liquid-liquid extraction and preconcentration using a subnanoliter-droplet trapping technique.

A robust and simple approach for microfluidic liquid-liquid (L-L) extraction at the subnanoliter-scale was developed for on-chip sample pretreatment. Organic solvent droplets of a few hundred pL were trapped within micro recesses fabricated in the channel walls of a microfabricated glass chip. L-L extraction was performed by delivering aqueous samples through the channel, with the sample stream continuously flowing adjacent to the droplets. The analytes in aqueous streams were enriched within the droplet with high preconcentration factors owing to both phase transfer and dissolution of organic solvent into the bypassing aqueous sample. An aqueous solution of butyl rhodamine B (BRB) and 1-hexanol were used, respectively, as sample and extractant to demonstrate the performance of the system. The fluorescence intensity of the dye extracted into the droplet was monitored in situ by LIF. The system proved to be an efficient means for achieving high enrichment factors of over 1000, with sample consumption of a few microL. Quantitative measurement of the extracted analyte was achieved with a linear response in the range 1 x 10(-9)-8 x 10(-7) M BRB. The precision of the measured fluorescence values for a 10(-7) M BRB standard with a 12.5 min preconcentration period was 6.6% RSD (n = 5).

Journal Article↗
PubMed2005

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.

Journal Article↗
PubMed2005

Feature characterization of microfabricated microfluidic chips by PDMS replication and CCD imaging.

This paper presents a new approach for the metrological characterization of microfabricated features on microfluidic chips, based on a combination of poly(dimethylsiloxane) (PDMS) replication and charge-coupled device (CCD) imaging. A PDMS replicate was cast from the original chip sample, and a 2-mm thick sample slice was cut from the replica at the cross-section to be studied. The digital image of the revealed structural profile was captured by a CCD camera under a microscope, and the image was processed using specially-developed algorithms for CCD image calibration and edge detection. Depth and width measurements obtained using the method agreed well with those gained using a stylus profiler and universal measuring microscope, with a deviation of below 0.9 mum, while profile distortions of deeper structures using stylus profilers were avoided. The method is reliable, non-destructive, and cheap and simple to implement in any analytical laboratory.

Journal Article↗
PubMed2005

DNA separation with low-viscosity sieving matrix on microfabricated polycarbonate microfluidic chips.

Microfluidic devices have been fabricated on polycarbonate (PC) substrates by use of a hot embossing method using a silicon master template. By adding auxiliary lines around the functional channel on the silicon master, its lifetime was significantly prolonged and the bonding strength of the PC cover plate to the microfluidic chip was greatly improved. More than 300 polycarbonate microfluidic chips have been replicated with the same silicon mold. CE separation of Phi X-174/HaeIII DNA restriction fragments, with high resolution efficiency and good reproducibility, was achieved on these devices using the low-viscosity sieving matrix HPMC-50. Temperature was found to have a significant effect on separation efficiency.

Bacteriophage phi X 174↗
PubMed2005

A simple microfluidic system for efficient capillary electrophoretic separation and sensitive fluorimetric detection of DNA fragments using light-emitting diode and liquid-core waveguide techniques.

A miniaturized CE system has been developed for fast DNA separations with sensitive fluorimetric detection using a rectangle type light-emitting diode (LED). High sensitivity was achieved by combining liquid-core waveguide (LCW) and lock-in amplification techniques. A Teflon AF-coated silica capillary on a compact 6x3 cm baseplate served as both the separation channel for CE separation and as an LCW for light transmission of fluorescence emission to the detector. An electronically modulated LED illuminated transversely through a 0.2 mm aperture, the detection point on the LCW capillary without focusing, and fluorescence light was transmitted to the capillary outlet. To simplify the optics and enhance collection of light from the capillary outlet, an outlet reservoir was designed, with a light transmission window, positioned directly in front of a photomultiplier tube (PMT), separated only by a high pass filter. Automated sample introduction was achieved using a sequential injection system through a split-flow interface that allowed effective release of gas bubbles. In the separation of a phiX174 HaeIII DNA digest sample, using ethidium bromide as labeling dye, all 11 fragments of the sample were effectively resolved in 400 s, with an S/N ratio comparable to that of a CE system with more sophisticated LIF.

DNA↗
PubMed2005

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis.

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.

Adult↗
PubMed2005

Static adsorptive coating of poly(methyl methacrylate) microfluidic chips for extended usage in DNA separations.

A simple and robust static adsorptive (dynamic) coating process using 2% hydroxyethylcellulose was developed for surface modification of poly(methyl methacrylate) (PMMA) microfluidic chips for DNA separations, suitable for usage over extended periods, involving hundreds of runs. The coating medium was also used as a sieving matrix for the DNA separations following the coating process. Four consecutive static treatments, by simply filling the PMMA chip channels with sieving matrix once every day, were required for obtaining a stable coating and optimum performance. The performance of the coated chips at different phases of the coating process was studied by consecutive gel electrophoretic separations with LIF detection using a PhiX-174/HaeIII DNA digest sample. The coated chip, with daily renewal of the sieving matrix, showed high stability in performance during a 25-day period of systematic study, involving more than 100 individual runs. The performance of the coated chip also remained almost the same after 3 months of continuous usage, during which over 200 separations were performed. The average precision of migration time for the 603-bp fragment was 1.31% RSD (n = 6) during the 25-day study, with a separation efficiency of 6.5 x 10(4) plates (effective separation length 5.4 cm).

Adsorption↗
PubMed2004

Bonding of glass microfluidic chips at room temperatures.

A simple, room-temperature bonding process was developed for the fabrication of glass microfluidic chips. High-quality bonding with high yields (>95%) was achieved without the requirement of clean room facilities, programmed high-temperature furnaces, pressurized water sources, adhesives, or pressurizing weights. The plates to be bonded were sequentially prewashed with acetone, detergent, high-flow-rate (10-20 m/s) tap water, and absolute ethyl alcohol and were soaked in concentrated sulfuric acid for 8-12 h. The plates were again washed in high-flow-rate tap water for 5 min and, finally, with demineralized water. The plates were bonded by bringing the cleaned surfaces into close contact under a continuous flow of demineralized water and air-dried at room temperature for more than 3 h. This bonding process features simple operation, good smoothness of the plate surface, and high bonding yield. The procedures can be readily applied in any routine laboratory. The bonding strength of glass chips thus produced, measured using a shear force testing procedure, was higher than 6 kg/cm(2). The mechanism for the strong bonding strength is presumably related to the formation of a hydrolyzed layer on the plate surfaces after soaking the substrates in acid or water for extended periods. Microfluidic chips bonded by the above procedure were tested in the CE separation of fluorescein isothiocyanate-labeled amino acids.

Journal Article↗
PubMed2003

Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip.

A microfluidic system was developed for the analysis of single biological cells, with functional integration of cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection in microfabricated channels of a single glass chip. Channels were 12 microm deep and 48 microm wide, with a simple crossed-channel design. The effective separation channel length was 35 mm. During sampling with a cell suspension (cell population 1.2 x 10(5) cells per mL in physiological salt solution), differential hydrostatic pressure (created by adjusting liquid levels in the four reservoirs) was used to control cell flow exclusively through the channel crossing. Single cell loading into the separation channel was achieved by electrophoretic means by applying a set of potentials at the four reservoirs, counteracting the hydrostatic flow. A special docking (adhering) procedure for the loaded cell was applied before lysis by repeatedly connecting and disconnecting a set of low potentials, allowing precise positioning of the cell within the separation channel. Cell lysis was then effected within 40 ms under an applied CE separation voltage of 1.4 kV (280 V cm(-1)) within the working electrolyte (pH 9.2 borate buffer) without additional lysates. The docked lysing approach reduced dispersion of released intracellular constituents, and significantly improved the reproducibility of CE separations. Glutathione (GSH) was used as a model intracellular component in single human erythrocyte cells. NDA derivatized GSH was detected using LIF. A throughput of 15 samples h(-1), a retention time precision of 2.4% RSD was obtained for 14 consecutively injected cells. The average cellular concentration of GSH in human erythrocytes was found to be 7.2 [times] 10(-4)+/- 3.3 x 10(-4) M (63 +/- 29 amol per cell). The average separation efficiency for GSH in lysed cells was 2.13 x 10(6)+/- 0.4 x 10(6) plates per m, and was about a factor of 5 higher than those obtained with GSH standards using pinched injection.

Electrophoresis, Capillary↗