Perfusion in microfluidic cross-flow: separation of white blood cells from whole blood and exchange of medium in a continuous flow

We describe a microfluidic technique for separation of particles and cells and a device that employs this technique to separate white blood cells (WBC) from whole human blood. The separation is performed in cross-flow in an array of microchannels with a deep main channel and large number of orthogonal, shallow side channels. As a suspension of particles advances through the main channel, a perfusion flow through the side channels gradually exchanges the medium of the suspension and washes away particles that are sufficiently small to enter the shallow side channels. The microfluidic device is tested with a suspension of polystyrene beads and is shown to efficaciously exchange the carrier medium while retaining all beads. In tests with whole human blood, the device is shown to reduce the content of red blood cells (RBC) by a factor of approximately 4000 with retention of 98% of WBCs. The ratio between WBCs and RBCs reached at an outlet of the device is 2.4 on average. The device is made of a single cast of poly(dimethylsiloxane) sealed with a cover glass and is simple to fabricate. The proposed technique of separation by perfusion in continuous cross-flow could be used to enrich rare populations of cells based on differences in size, shape, and deformability.     

Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick

This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and "spiked" standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.     

A high-efficiency cross-flow micronebulizer interface for capillary electrophoresis and inductively coupled plasma mass spectrometry.

A pneumatic nebulizer interface for capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICPMS) is reported. The interface is constructed using a high-efficiency cross-flow micronebulizer (HECFMN) and has the following features. (1) Makeup solutions can be fed to the interface by nebulizer self-aspiration and liquid gravity pressurization. (2) The liquid dead volume of the interface is approximately 65 nL, much smaller than those (200-2500 nL) reported for other interfaces. (3) The interface can be stably operated at a liquid flow rate down to 5 microL/min with a high analyte transport efficiency up to 95% to the plasma and (4) does not induce noticeable laminar flow in the CE capillary at typical nebulizer gas flow rates of 0.8-1.2 L/min. Because of these features, baseline resolution of 10 lanthanides with a CE-ICPMS system using the HECFMN interface is achieved, and detection limits and peak asymmetry are 0.05-1 microg/L and 0.93-1.23, respectively, improved significantly over those reported previously for a CE-ICPMS system using a high-efficiency nebulizer interface. Peak precision for the 10 lanthanides is in the range of 6.2-12.3% RSD (N = 5). Peak widths are from 9.1 s for 139La to 17.9 s for 175Lu. The effects of nebulizer gas flow rate, makeup solution flow rate, and spray chamber volume on CE-ICPMS signal intensity and separation are also evaluated for the HECFMN interface by the separation of Cr3+ and Cr2O7(2-).     

Biomimetic autoseparation of leukocytes from whole blood in a microfluidic device

Leukocytes comprise less than 1% of all blood cells. Enrichment of their number, starting from a sample of whole blood, is the required first step of many clinical and basic research assays. We created a microfluidic device that takes advantage of the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, to separate leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels designed to enhance lateral migration of leukocytes and their subsequent extraction from the erythrocyte-depleted region near the sidewalls. A single pass through the device produces a 34-fold enrichment of the leukocyte-to-erythrocyte ratio. It operates on microliter samples of whole blood, provides positive, continuous flow selection of leukocytes, and requires neither preliminary labeling of cells nor input of energy (except for a small pressure gradient to support the flow of blood). This effortless, efficient, and inexpensive technology can be used as a lab-on-a-chip component for initial whole blood sample preparation. Its integration into microanalytical devices that require leukocyte enrichment will enable accelerated transition of these devices into the field for point-of-care clinical testing.     

Nanoliter viscometer for analyzing blood plasma and other liquid samples

We have developed a microfabricated nanoliter capillary viscometer that quickly, easily, and inexpensively measures the viscosity of liquids. The measurement of viscosity is based on capillary pressure-driven flow inside microfluidic channels (depth approximately 30 microm and width approximately 300 microm). Accurate and precise viscosity measurements can be made in less than 100 s while using only 600 nL of liquid sample. The silicon-glass hybrid device (18 mm by 15 mm) contains on-chip components that measure the driving capillary pressure difference and the relevant geometrical parameters; these components make the nanoliter viscometer completely self-calibrating, robust, and easy to use. Several different microfabricated viscometers were tested using solutions with viscosities ranging from 1 to 5 cP, a range relevant to biological fluids (urine, blood, blood plasma, etc.). Blood plasma samples collected from patients with the symptoms of hyperviscosity syndrome were tested on the nanoliter capillary viscometer to an accuracy of 3%. Such self-calibrating nanoliter viscometers may have widespread applications in chemical, biological, and medical laboratories as well as in personal health care.     

Microfluidic-based DNA purification in a two-stage, dual-phase microchip containing a reversed-phase and a photopolymerized monolith

In this report, we show that a novel capillary-based photopolymerized monolith offering unprecedented efficiency (approximately 80%) for DNA extraction from submicroliter volumes of whole blood (Wen, J.; Guillo, C.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78, 1673-1681) can be translated to microfluidic devices. However, owing to the large mass of protein present in blood, both DNA binding capacity and extraction efficiency were significantly decreased when extraction of DNA was carried out directly from whole blood (38+/-1%). To circumvent this, a novel two-stage microdevice was developed, consisting in a C18 reversed-phase column for protein capture (stage 1) in series with a monolithic column for DNA extraction (stage 2). The two-stage, dual-phase design improves the capability of the monolith for whole blood DNA extraction by approximately 100-fold. From a 10-microL load of whole blood containing 350 ng of DNA, 99% (340+/-10 ng) traverses the C18 phase while approximately 70% (1020+/-45 ug) of protein is retained. A total of 240+/-2 ng of DNA was eluted from the second-stage monolith, resulting in an overall extraction efficiency of 69+/-1%. This provided not only an improvement in extraction efficiency over other chip-based DNA extraction solid phases but also the highest extraction efficiency reported to-date for such sample volumes in a microfluidic device. As an added bonus, the two-stage, dual-phase microdevice allowed the 2-propanol wash step, typically required to remove proteins from the DNA extraction phase for successful PCR, to be completely eliminated, thus streamlining the process without affecting the PCR amplifiability of the extracted DNA.     

Optical characterization of a compact multilayer-mirror polarimeter in the extreme-ultraviolet range

A molybdenum-silicon (Mo/Si) multilayer-mirror (MLM) polarimeter has been constructed and used to analyze the extreme-ultraviolet (EUV) emission from excited HeI and HeII states following electron impact on He for wavelengths ranging from approximately 256 to 584 A. A ratio of reflectivities for s- and p-polarized light, R(s):R(p) approximately , and a resolving power of lambda/Dlambda approximately 6 at 304 A were obtained. These characteristics and the use of a VYNS (a copolymer material composed of 90% vinyl chloride and 10% vinyl acetate) spectral filter were sufficient to allow a detailed polarization study of the first two members of the Lyman series of He(+) at wavelengths of 304 A (HeII p --> s) and 256 A (HeII p --> s) for impact-electron energies ranging from threshold to 1500 eV. The MLM has also been used as a single flat-surface mirror polarimeter for the analysis of longer-wavelength radiation (517 to 584 A) from the (snp) (1) P (o) --> (1s(2)) (1) S series of neutral He with R(s)/R(p) approximately. Although MLM polarimeters were previously used for EUV measurements with bright photon sources such as those provided by synchrotron facilities, the results presented clearly demonstrate the feasibility of such devices with lower-intensity electron and ion impact sources. The compact design of the apparatus makes it suitable as a portable measurement and calibration device.     

Sample preparation using a miniaturized supported liquid membrane device connected on-line to packed capillary liquid chromatography

A miniaturized supported liquid membrane device has been developed for sample preparation and connected on-line to a packed capillary liquid chromatograph. The device consists of hydrophobic polypropylene hollow fiber, inserted and fastened in a cylindrical channel in a Kel-F piece. The pores of the fiber are filled with an organic solvent, in this study 6-undecanone, thus forming a liquid membrane. The sample is pumped on the outside of the hollow fiber (donor), and the analytes are selectively enriched and trapped in the fiber lumen (acceptor). With this approach, the volume of the acceptor solution can be kept as low as 1-2 μL. This stagnant acceptor solution is then transferred through capillaries attached to the fiber ends to the LC system. The system was tested with a secondary amine (bambuterol), as a model substance in aqueous standard solutions as well as in plasma. The best extraction efficiency in aqueous solution, with an acceptor volume of 1.9 μL, was 32.5% at a donor flow rate of 2.5 μL/min. At flow rates above 20 μL/min, the concentration enrichment per time unit was approximately constant, at 0.9 times/min, i.e., 9 times enrichment in about 10 min. The overall repeatability (RSD) for spiked plasma samples was ～4% (n = 12). Linear calibration curves of peak area versus bambuterol concentration were obtained for both aqueous standard solutions and spiked plasma samples. The detection limit for bambuterol in plasma, after 10 min of extraction at a flow rate of 24 μL/min, was 80 nM.     

High-efficiency single-cell entrapment and fluorescence in situ hybridization analysis using a poly(dimethylsiloxane) microfluidic device integrated with a black poly(ethylene terephthalate) micromesh

Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 microm in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. This microfluidic device has been employed for the detection of beta-actin mRNA expression in individual Raji cells. Differences in the levels of beta-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.     

Aptamer-enabled efficient isolation of cancer cells from whole blood using a microfluidic device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.     

Stopped-flow enzyme assays on a chip using a microfabricated mixer

This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in < 1 s. The objective of the work was to perform kinetically based enzyme assays in the stopped-flow mode using a system of roughly 6 nL volume. Beta-galactosidase (beta-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-beta-D-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 +/- 13 microM and 44 +/- 3 s(-1) were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 +/- 6 microM and kcat was 47 +/- 4 s(-1). Enzyme inhibition assays with phenylethyl-beta-D-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.     

New mass transfer performance data of a cross-flow liquid desiccant dehumidification system

This paper presents new mass transfer performance data of a cross-flow liquid desiccant dehumidification system using a structured packed tower. The structured packing consists of cross-corrugated cellulose paper sheets with a surface area per unit volume ratio of 608 m² m^?3. The liquid desiccant, viz. calcium chloride, flows through the pad from top to bottom, while the air flows horizontally making it a cross-flow configuration. The experimental dehumidification effectiveness from the present study was compared with the widely used Chung's correlation (although developed for counter flow arrangement, as opposed to cross-flow in the current study) and Liu et al.'s correlation. A new empirical correlation was developed for the dehumidification effectiveness, which fitted the experimental data to within ±10%. The effect of varying air and solution inlet conditions and flow rates on the system performance was also quantified in the paper.     

Gravitational sedimentation induced blood delamination for continuous plasma separation on a microfluidics chip

Continuous plasma separation will be greatly helpful for dynamic metabolite monitoring in kinetics research and drug development. In this work, we proposed a continuous on-chip plasma separation method based on the natural aggregating and sedimentation behavior of red blood cells at low shear rate. In this approach, a glass capillary was first used to realize quick and obvious delamination of blood cells from plasma. A novel "dual-elbow" connector was designed to change the direction of delamination. The blood was finally separated by laminar flow and bifurcation on the microchip. Results demonstrated that the present device can efficiently and stably separate plasma from blood in a continuous means, e.g., in a 4 h separation we did not observe clogging or a trend of clogging. In addition, the present approach can avoid the damage to cells which usually occurs in separation with high shear rate in a microchannel and possible contaminants to plasma. The proposed microchip device is robust, simple, and inexpensive for long time plasma separation with high plasma recovery and less sample consumption. The present work provides an effective tool for metabolite monitoring in pharmacokinetics research and drug development.     

Flow injection analysis in a microfluidic format

A microfluidic flow injection analysis system has been designed and evaluated. The system incorporates within a single two-layer poly(dimethylsiloxane) monolith multiple pneumatically driven peristaltic pumps, an injection loop, a mixing column, and a transparent window for fluorescence detection. Central to this device is an injection system that mimics the operation of a standard six-port, two-way valve used in conventional liquid chromatography and flow injection experiments. Analyte and carrier solutions continuously flow through this injection system allowing for measurements and sample changes to be performed rapidly and simultaneously. Injection volumes of 1.25 nL generated peak area reproducibility of better than 3% relative standard deviation. The flow injection device was evaluated with fluorescent dyes and demonstrated a detection limit of 400 zmol for fluorescein. A rudimentary sample selection system allowed calibration curves to be rapidly produced, often in less than 10 min. The hydrolysis of fluorescein diphosphate by alkaline phosphatase demonstrates that chemical assays can be carried out with this device in a manner characterized by short analysis times and low sample consumption.     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.     

Determination of low molecular weight silicones in plasma and blood of women after exposure to silicone breast implants by GC/MS

A sensitive, one-step sample preparation method for detection of volatile, low molecular weight (LMW) cyclic silicones hexamethylcyclotrisiloxane (D3), octamethyl-cyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) in plasma and blood using gas chromatography coupled with mass spectrometry (GC/MS, SIM mode) is presented. In spiked experiments, extraction efficiencies for these siloxanes (100-20 000 ng/mL) were approximately 90% for plasma and approximately 80% for blood; only in the case of D3 was the recovery very low. Plasma and blood of women who are or were exposed to silicone gel-filled implants and of control subjects were analyzed for low molecular weight silicones. D3-D6 were not detectable in control plasma or blood. Although the investigated numbers of patients samples are very limited, and thus, no statistical analysis is possible, our data clearly show a general increase in the amount of LMW cyclic siloxanes in the bodies of women with silicone implants. In particular, several years after ruptured silicone implants were removed, siloxanes could still be found in blood samples from several women. Siloxane compound D3 varied between 6 and 12 ng/mL (plasma) and between 20 and 28 ng/mL (blood), whereas the concentration range of D4 was 14-50 ng/mL (plasma) and 79-92 ng/mL (blood). D5 and D6, with one exception, could not be detected.     

Differences in the Method by Which Plasma Is Separated from Whole Blood Influences Amphotericin B Plasma Recovery and Distribution Following Amphotericin B Lipid Complex Incubation Within Whole Blood

A previous investigation suggested that the use of plasma as the biological fluid for measurement of amphotericin B (AmpB) concentrations greatly underestimates the concentrations of AmpB in the total blood circulation following amphotericin B lipid complex (ABLC) administration to humans. The purpose of this study was to determine if differences in the method used to obtain plasma from whole blood influences the percentage of AmpB recovered in plasma following ABLC incubation in whole blood. ABLC (5 μg AmpB/ml; peak blood concentration observed in rabbits following intravenous bolus of ABLC at a dose of 1 mg/kg) was incubated in whole blood for 5 min at 25°C. These conditions were used to mimic the sample retrieval conditions used when blood is obtained from animals and human patients. Following incubation, plasma was obtained from whole blood using five different methods: (A) Whole blood was centrifuged for 5 min at 23°C, and the plasma was separated; (B) whole blood was stored at 4°C for 18 h, and the plasma was separated by gravity; (C) whole blood was stored at 23°C for 18 h, and the plasma was separated by gravity; (D) whole blood was stored at 37°C for 18 h in a water bath, and the plasma was separated by gravity; and (E) whole blood was stored at 30°C for 18 h in a water bath, and the plasma was separated by gravity. All samples were protected from light throughout the duration of the experiment. AmpB concentration in each plasma sample was determined by high-performance liquid chromatography (HPLC) using an external calibration curve. The whole blood : plasma Amp B concentration ratio and the percentage of AmpB partitioned into plasma following incubation of ABLC in whole blood for each plasma separation procedure was as follows: (A) 6.5 : 1 blood : plasma AmpB concentration ratio, 15.4% ± 1.6% AmpB in plasma; (B) 2.98 : 1 blood : plasma AmpB concentration ratio, 33.6% ± 7.7% AmpB in plasma; (C) 1.5 : 1 blood : plasma AmpB concentration ratio, 67.6% ± 10.3% AmpB in plasma; (D) 1.5 : 1 blood : plasma concentration ratio, 68.1% ± 1.1% AmpB in plasma; and (E) 1.2 : 1 blood : plasma AmpB concentration ratio; 83.4% ± 5.5% AmpB in plasma. These findings suggest that when measurement of AmpB in plasma is required following ABLC administration, incubation of whole blood at 30°C for 18 h appears to be the most effective method.     

Differences in the Method by Which Plasma Is Separated from Whole Blood Influences Amphotericin B Plasma Recovery and Distribution Following Amphotericin B Lipid Complex Incubation Within Whole Blood

A previous investigation suggested that the use of plasma as the biological fluid for measurement of amphotericin B (AmpB) concentrations greatly underestimates the concentrations of AmpB in the total blood circulation following amphotericin B lipid complex (ABLC) administration to humans. The purpose of this study was to determine if differences in the method used to obtain plasma from whole blood influences the percentage of AmpB recovered in plasma following ABLC incubation in whole blood. ABLC (5 μg AmpB/ml; peak blood concentration observed in rabbits following intravenous bolus of ABLC at a dose of 1 mg/kg) was incubated in whole blood for 5 min at 25°C. These conditions were used to mimic the sample retrieval conditions used when blood is obtained from animals and human patients. Following incubation, plasma was obtained from whole blood using five different methods: (A) Whole blood was centrifuged for 5 min at 23°C, and the plasma was separated; (B) whole blood was stored at 4°C for 18 h, and the plasma was separated by gravity; (C) whole blood was stored at 23°C for 18 h, and the plasma was separated by gravity; (D) whole blood was stored at 37°C for 18 h in a water bath, and the plasma was separated by gravity; and (E) whole blood was stored at 30°C for 18 h in a water bath, and the plasma was separated by gravity. All samples were protected from light throughout the duration of the experiment. AmpB concentration in each plasma sample was determined by high-performance liquid chromatography (HPLC) using an external calibration curve. The whole blood : plasma Amp B concentration ratio and the percentage of AmpB partitioned into plasma following incubation of ABLC in whole blood for each plasma separation procedure was as follows: (A) 6.5 : 1 blood : plasma AmpB concentration ratio, 15.4% ± 1.6% AmpB in plasma; (B) 2.98 : 1 blood : plasma AmpB concentration ratio, 33.6% ± 7.7% AmpB in plasma; (C) 1.5 : 1 blood : plasma AmpB concentration ratio, 67.6% ± 10.3% AmpB in plasma; (D) 1.5 : 1 blood : plasma concentration ratio, 68.1% ± 1.1% AmpB in plasma; and (E) 1.2 : 1 blood : plasma AmpB concentration ratio; 83.4% ± 5.5% AmpB in plasma. These findings suggest that when measurement of AmpB in plasma is required following ABLC administration, incubation of whole blood at 30°C for 18 h appears to be the most effective method.     

Quantitative in vivo microsampling for pharmacokinetic studies based on an integrated solid-phase microextraction system

An integrated microsampling approach based on solid-phase microextraction (SPME) was developed to provide a complete solution to highly efficient and accurate pharmacokinetic studies. The microsampling system included SPME probes that are made of poly(ethylene glycol) (PEG) and C18-bonded silica, a fast and efficient sampling strategy with accurate kinetic calibration, and a high-throughput desorption device based on a modified 96-well plate. The sampling system greatly improved the quantitative capability of SPME in two ways. First, the use of the C18-bonded silica/PEG fibers minimized the competition effect from analogues of the target analytes in a complicated sample matrix such as blood or plasma samples, which is a common problem associated with solid coating SPME fibers for quantitative analysis. Moreover, the C18-bonded silica/PEG fibers provide high sensitivity and a large dynamic range that covers the possible sample concentration range during diazepam administration and elimination. Second, the kinetic calibration method offers more accurate quantitation than the calibration curve method for in vivo SPME, because it compensates for convection and matrix effects during sampling. Therefore, it is especially suitable as a fast sampling technique for pre-equilibrium SPME. Furthermore, with the high-throughput desorption device, the integrated system offers compactness and high efficiency. Its feasibility for in vivo sampling was demonstrated by monitoring diazepam pharmacokinetics and validated by conventional chemical assays and equilibrium SPME. In addition, we propose a simple method to determine the apparent distribution constant between an SPME fiber and a blood matix (Kfs) and the distribution constant between an SPME fiber and a pure PBS buffer sample matrix (Kfb). As a result, both total and free concentrations of the drug and its metabolites can be detected simultaneously. Accordingly, the binding constants to the blood matrix can be obtained, which are of special significance for clinical diagnosis and drug discovery.     

Improvement of a dual frequency sputtering device for higher deposition rates of protective zirconium oxide films on ceramics

Zirconium thin films have been applied as protective coating films on ceramics by dual frequency oxygen plasma sputtering [Y. Ohtsu et al., Surf Coat Technol, 196 (2005) 81], where they were certified to be effective in modifying the surface state of china and porcelain with the water-repellency and the stoichiometric value of atomic ratio O/Zr in films. However, the deposition rate with the former device was about 0.6 nm/min, lower compared with a conventional radio frequency magnetron plasma device. Improvement of the deposition rate has been investigated by optimization of the geometry in a dual frequency plasma-sputtering device using O₂ and Ar mixture gases. That is, the ratio of plasma volume to that of the vacuum chamber was changed from 8 to 44%. The high-deposition rate of about 7 nm/min was attained at O₂ gas concentration of 10%, under the optimization of the geometry. The films have also kept the high transparency of 90%. These results indicate that the advanced dual frequency plasma-sputtering device is an effective plasma source for producing protective layers for ceramics.