Dynamic radial positioning of a hydrodynamically focused particle stream enabled by a three-dimensional microfluidic nozzle.

The ability to confine flows and focus particle streams has become an integral component of the design of microfluidic systems for the analysis of a wide range of samples. Presented here is the implementation of a 3D microfluidic nozzle capable of both focusing particles as well as dynamically positioning those particles in selected flow lamina within the downstream analysis channel. Through the independent adjustment of the three sheath inlet flows, the nozzle controlled the size of a focused stream for 6, 10, and 15 µm polystyrene microparticles. Additional flow adjustment allowed the nozzle to dynamically position the focused particle stream to a specific area within the downstream channel. This unique ability provides additional capability and sample flexibility to the system. In order to gain insight into the fluidic behavior of the system, experimental conditions and results were duplicated within 4.75 µm using a COMSOL Multiphysics(®) model to elucidate the structure, direction, proportion, and fate of fluid lamina throughout the nozzle region. The COMSOL Multiphysics model showed that the position and distribution of particles upon entering the nozzle have negligible influence over its focusing ability, extending the experimental results into a wider range of particle sizes and system flow rates. These results are promising for the application of this design to allow for a relatively simple, fast, fully fluidically controlled nozzle for selective particle focusing and positioning for further particle analysis and sorting.

Optical chromatographic sample separation of hydrodynamically focused mixtures.

Optical chromatography relies on the balance between the opposing optical and fluid drag forces acting on a particle. A typical configuration involves a loosely focused laser directly counter to the flow of particle-laden fluid passing through a microfluidic device. This equilibrium depends on the intrinsic properties of the particle, including size, shape, and refractive index. As such, uniquely fine separations are possible using this technique. Here, we demonstrate how matching the diameter of a microfluidic flow channel to that of the focusing laser in concert with a unique microfluidic platform can be used as a method to fractionate closely related particles in a mixed sample. This microfluidic network allows for a monodisperse sample of both polystyrene and poly(methyl methacrylate) spheres to be injected, hydrodynamically focused, and completely separated. To test the limit of separation, a mixed polystyrene sample containing two particles varying in diameter by less than 0.5 µm was run in the system. The analysis of the resulting separation sets the framework for continued work to perform ultra-fine separations.

Note: pneumatically modulated liquid delivery with feedback control.

We present the design and characterization of a pneumatically driven liquid delivery system using an embedded microcontroller with feedback control capable of maintaining a stable, constant flow rate over several hours of operation. Flow rates with relative standard deviations less than 1% were achieved and compared to a typical laboratory syringe pump.

On-the-fly cross flow laser guided separation of aerosol particles based on size, refractive index and density-theoretical analysis.

Laser separation of particles is achieved using forces resulting from the momentum exchange between particles and photons constituting the laser radiation. Particles can experience different optical forces depending on their size and/or optical properties, such as refractive index. Thus, particles can move at different speeds in the presence of an optical force, leading to spatial separations. In this paper, we present a theoretical analysis on laser separation of non-absorbing aerosol particles moving at speeds (1-10 cm/sec) which are several orders of magnitude greater than typical particle speeds used in previous studies in liquid medium. The calculations are presented for particle deflection by a loosely focused Gaussian 1064 nm laser, which simultaneously holds and deflects particles entrained in flow perpendicular to their direction of travel. The gradient force holds the particles against the viscous drag for a short period of time. The scattering force simultaneously pushes the particles, perpendicular to the flow, during this period. Our calculations show particle deflections of over 2500 µm for 15 µm aerosol particles, and a separation of over 1500 µm between 5 µm and 10 µm particles when the laser is operated at 10 W. We show that a separation of about 421 µm can be achieved between two particles of the same size (10 µm) but having a refractive index difference of 0.1. Density based separations are also possible. Two 10 µm particles with a density difference of 600 kg/m3 can be separated by 193 µm. Examples are shown for separation distances between polystyrene, poly(methylmethacrylate), silica and water particles. These large laser guided deflections represent a novel achievement for optical separation in the gas phase.

Enhanced optical chromatography in a PDMS microfluidic system.

The purely refractive index driven separation of uniformly sized polystyrene, n = 1.59 and poly(methylmethacrylate), n = 1.49 in an optical chromatography system has been enhanced through the incorporation of a custom poly(dimethysiloxane) (PDMS) microfluidic system. A customized channel geometry was used to create separate regions with different linear flow velocities tailored to the specific application. These separate flow regions were then used to expose the entities in the separation to different linear flow velocities thus enhancing their separation relative to the same separation in a constant velocity flow environment. A microbiological sample containing spores of the biological warfare agent, Bacillus anthracis, and a common environmental interferent, mulberry pollen, was investigated to test the use of tailored velocity regions. These very different samples were analyzed simultaneously only through the use of tailored velocity regions.

Cloning and tissue distribution of two Na+/H+ exchangers from the Malpighian tubules of Aedes aegypti.

A Na(+)/H(+) exchanger (NHE) on the apical membrane of mosquito Malpighian tubule (MT) cells is believed to participate in the blood-feeding mosquito's vital secretion of Na(+) and fluid. This study presents the molecular cloning, primary structure, and tissue distribution of two cDNAs encoding Aedes aegypti mosquito MT NHEs. The cDNA sequences were obtained from mosquito MT total RNA using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE). The two sequences encode proteins of 678 and 1,179 amino acids with calculated molecular weights of 74,473 and 130,276, respectively. When comparing the 678 amino acid protein to the first 678 amino acids of the other protein, the two clones show 98% identity to each other. They also exhibit high identity to Drosophila melanogaster NHEs. Hydropathy analysis reveals that while both clones have 10-13 transmembrane segments, the 1,179 amino acid protein has an extensive carboxy terminus while the 678 amino acid protein has an extremely short carboxy terminus. RT-PCR analysis shows that both clones are expressed in the mosquito Malpighian tubules at the larval and pupal stages, in addition to the adult stage before and after blood-feeding. Expression of both clones was also detected in adult mosquito ovaries, midguts, and hindguts.

Domestic violence: legal, practice, and educational issues.

Domestic violence is a recognized and growing public health concern in the United States. Health care professionals have a duty to improve the identification of victims of domestic violence, intervene effectively, and advocate for better education to break the cycle of abuse.

Complete separation of urinary metabolites of paracetamol and substituted paracetamols by reversed-phase ion-pair high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic procedure has been developed for the separation of thirteen urinary metabolites of the analgesic drug paracetamol. The method involved the use of radially compressed columns packed with octadecylsilica with a particle diameter of 5 micron. Metabolites were chromatographed by linear gradient elution using an ion-pair solvent system composed of tetrabutylammonium hydroxide and Tris buffered to pH 5.0 with phosphoric acid, and acetonitrile as the organic solvent. Analyses can be performed at the rate of three per hour. This method enables the direct identification of sulphate and glucuronide conjugates of 3-thiomethylparacetamol and 3-thiomethylparacetamol sulphoxide which have only previously been detected following enzyme hydrolysis of urine samples. The application of this fully optimised separation to the study of the metabolism of substituted paracetamols is also discussed.

Anticoagulant activity in cell-free peritoneal fluid of an experimental pancreatic ascites tumor.

The ascitic form of a chemically-induced pancreatic ductal adenocarcinoma in the Syrian golden hamster was very bloody and indistinguishable from blood macroscopically. Unlike blood, the bloody fluid remained unclotted at room temperature. To explore the possibility of presence of anticoagulants, we mixed 40% cell-free fluid with 60% normal human plasma and tested the clottability of the mixture with standard techniques. Plasma containing the fluid showed markedly prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and recalcification time (RCT), and normal prothrombin time (PT) and reptilase time (RT). Comparing the prolongation of APTT of samples containing the fluid to those containing a commercial heparin, the fluid contained an anticoagulant activity equivalent to 0.436 +/- 0.03 unit heparin per ml (mean +/- SEM, n = 14). In addition to prolonging the APTT, TT and RCT, the fluid also inhibited the clotting and amidolytic activities of thrombin. "Heparsorb" had nearly completely neutralized the anticoagulant activity in fluid samples, while protamine sulfate was only partially effective. Incubation of fluid with pronase or phospholipase did not affect its anticoagulant activity; incubation with heparinase had only a minimal effect. Electrophoresis of an alkali digested fluid on cellulose acetate revealed the presence of heparan sulfate. The native ascitic fluid also contained other hemostatic components including platelets, fibrinogen and antithrombin III, but their concentrations were much lower than in blood. Apparently, heparan sulfate in the neoplastic effusion is largely responsible for the bloody ascites tumor remaining unclotted.

Improved high-performance liquid chromatographic separation of urinary paracetamol metabolites using radially compressed columns.

Methods have been adapted for the high-performance liquid chromatographic (HPLC) analysis of urinary paracetamol metabolites on radial compression columns. Enhanced resolution and decreased analysis time were two major advances. Various modifications to existing methods were made to counter the effect of the different C18 surface. Thus in ion suppression HPLC the addition of triethylamine at pH 3.0 (phosphate buffer) was necessary to block residual hydroxyl sites, while in ion-pair HPLC a higher tetrabutyl-ammonium hydroxide concentration of 0.01 M at pH 5.0 was used to enhance selectivity. The methods were successfully applied to the study of the metabolism of paracetamol, its glutathione conjugate and 3-thiomethylparacetamol in Sprague-Dawley rats. 3-Thiomethyl-paracetamol sulphoxide and its glucuronide and sulphate conjugates were shown to be metabolites of both 3-thiomethylparacetamol and paracetamol. 3-Thiomethylparacetamol sulphate was unresolved from the sulphates of paracetamol and 3-methoxyparacetamol in ion-pair HPLC. This raises a previously unrecognised problem in which the peak normally attributed to paracetamol sulphate contains metabolites arising from an oxidative metabolic pathway. Elevated levels of 3-methoxyparacetamol conjugates were found in human overdose urine and to some extent in analgesic nephropathy.

3-thiomethylparacetamol sulphate and glucuronide: metabolites of paracetamol and N-hydroxyparacetamol.

1. Two new metabolites of paracetamol, 3-thiomethylparacetamol sulphate and glucuronide, have been isolated and identified. 2. The metabolites occurred in both rat and mouse urine after administration of either paracetamol of N-hydroxyparacetamol. The amount excreted increased proportionally with the dose.

Metabolism of paracetamol by the isolated perfused kidney of the homozygous Gunn rat.

1. Isolated kidneys from homozygous Gunn rats were perfused with paracetamol in concentrations lower and higher than Km for paracetamol oxidation in the albino rat kidney. 2. Glucuronylation of paracetamol was not detected at either concentration. 3. An increase in oxidative metabolism at the higher concentration, similar to that seen with the Sprague-Dawley rat kidney, did not occur with kidneys from homozygous Gunn rats. 4. This finding does not support the hypothesis that the enhanced nephrotoxicity of paracetamol observed in the homozygous Gunn rat in vivo is due to increased intrarenal formation of reactive metabolites.

The metabolism and toxicity of paracetamol in Sprague-Dawley and Wistar rats.

Urinary paracetamol metabolites from Sprague-Dawley and Wistar rats were analysed by reversed-phase HPLC. Variations in the metabolic profile were observed as a function of dose, age, sex, species and route of administration. In addition the effect of 3-methylcholanthrene as an inducer of cytochrome P450 mixed function oxidase on paracetamol metabolism was also studied. Increased oxidative metabolism which lead to the formation of 3-thiomethylparacetamol conjugates along with paracetamol mercapturic acid could be correlated with increased susceptibility to hepatic damage. Furthermore it appears that the length of time taken for excretion and the level of free drug excreted may be involved in the aetiology of chronic renal damage.

Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing.

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C18 columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.

Hepatotoxicity of phenacetin and paracetamol in the Gunn rat.

Both phenacetin and paracetamol produce acute centrilobular liver necrosis in the homozygous Gunn rat. Paracetamol is more hepatotoxic than phenacetin, and both are more hepatotoxic to the homozygous Gunn rat than to the heterozygous Gunn rat or to the albino rat. These findings have relevance to the role of the compounds in the clinical syndromes of paracetamol induced liver necrosis and analgesic nephropathy.

Induction of paracetamol metabolism in the isolated perfused kidney.

1. At a perfusate concn. of 3.5-4.0 mM, 59 plus or minus 9 nmol of paracetamol h per g wet wt. were oxidized by isolated rat kidney. 2. Approx. half the paracetamol undergoing oxidation was converted to a mercapturic acid metabolite and the remainder was covalently bound to kidney protein. 3. Addition of GSH to the perfusate decreased the level of covalent binding. Depletion of cellular GSH, by prior administration of diethyl maleate, significantly decreased formation of the mercapturic acid metabolite. 4. The metabolic pathways of glucoronylation, sulphation and mercapturic acid formation were induced either by 3-methylcholanthrene pretreatment or by prolonged feeding of aspirin or paracetamol; covalent binding of paracetamol to kidney protein was not increased.

N-hydroxyacetaminophen: a postulated toxic metabolite of acetaminophen.

The decomposition of N-hydroxyacetaminophen has been shown to occur via an initial first-order dehydration step to N-acetyl-p-benzoquinone imine with a rate constant at pH 7.6 of 8.66 x 10(-3) min-1 and a half-life of 80 min. This is followed by a complex reaction between the quinone imine and the N-hydroxy compound to ultimately yield p-nitrosophenol and acetaminophen. The glucuronide and sulfate conjugates of N-hydroxyacetaminophen have been observed as urinary metabolites of N-hydroxyacetaminophen. No N-hydroxylated metabolites were found among the metabolites of acetaminophen. These results have been interpreted to show that N-hydroxyacetaminophen is not a metabolite of acetaminophen. It is proposed that the hepatotoxicity and nephrotoxicity of acetaminophen are mediated by a direct oxidation of acetaminophen to the toxic reactive intermediate N-acetyl-p-benzoquinone imine by the cytochrome P450 dependent mixed-function oxidase system.

Paracetamol and the isolated perfused kidney: metabolism and functional effects.

1. Renal metabolism of paracetamol has been studied in the isolated perfused rat kidney. 2. The major metabolites of paracetamol normally observed in vivo were present in low concentrations in the urine of the isolated perfused rat kidney. 3. Paracetamol was bound covalently to kidney protein in a linear relationship to dose up to 31.9 mM paracetamol in the perfusate. 4. Using Michaelis-Menten kinetics, the apparent Km of 5.5 mM and Vmax of 139 nmol/h/g wet wt. for renal paracetamol oxidation were observed. 5. Concn. of paracetamol greater than 14.2 mM induced immediate diuresis and diminished sodium reabsorption. Lower concn. were without effect on function.