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An integrated microfluidic biosensor is presented that combines sample pre-concentration and liposome-based signal amplification for the detection of enteric viruses present in environmental water samples. This microfluidic approach overcomes the challenges of long assay times of cell culture-based methods and the need to extensively process water samples to eliminate inhibitors for PCR-based methods. Here, viruses are detected using an immunoassay sandwich approach with the reporting antibodies tagged to liposomes. Described is the development of the integrated device for the detection of environmentally relevant viruses using feline calicivirus (FCV) as a model organism for human norovirus. In situ fabricated nanoporous membranes in glass microchannels were used in conjunction with electric fields to achieve pre-concentration of virus-liposome complexes and therefore enhance the antibody-virus binding efficiency. The concentrated complexes were eluted to a detection region downstream where captured liposomes were lysed to release fluorescent dye molecules that were then quantified using image processing. This system was compared to an optimized electrochemical liposome-based microfluidic biosensor without pre-concentration. The limit of detection of FCV of the integrated device was at 1.6 × 10(5) PFU/mL, an order of magnitude lower than that obtained using the microfluidic biosensor without pre-concentration. This significant improvement is a key step toward the goal of using this integrated device as an early screening system for viruses in environmental water samples.

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45- cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2-400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Drug-drug interactions (DDIs) with the HIV protease inhibitors (PIs) are complex, paradoxical (e.g., ritonavir/alprazolam), and involve multiple mechanisms. As part of a larger study to better understand these DDIs and to devise a framework for in vitro to in vivo prediction of these DDIs, we determined the inductive effect of ∼2 weeks of administration of two prototypic PIs, nelfinavir (NFV), ritonavir (RTV), and rifampin (RIF; induction positive control) on the cytochrome P450 enzymes CYP1A2, CYP2B6, CYP2C9, and CYP2D6 and the inductive or inductive plus inhibitory effect of NFV, RTV, or RIF on CYP3A and P-glycoprotein in healthy human volunteers. Statistically significant induction of CYP1A2 (2.1-, 2.9-, and 2.2-fold), CYP2B6 (1.8-, 2.4-, and 4-fold), and CYP2C9 (1.3-, 1.8-, and 2.6-fold) was observed after NFV, RTV, or RIF treatment, respectively (as expected, CYP2D6 was not induced). Moreover, we accurately predicted the in vivo induction of these enzymes by quantifying their induction by the PIs in human hepatocytes and by using RIF as an in vitro to in vivo scalar. On the basis of the modest in vivo induction of CYP1A2, CYP2B6, or CYP2C9, the in vivo paradoxical DDIs with the PIs are likely explained by mechanisms other than induction of these enzymes such as induction of other metabolic enzymes, transporters, or both.

We demonstrate that delta doping can be used to create a dimensionally confined region of metallic ferromagnetism in an antiferromagnetic (AFM) manganite host, without introducing any explicit disorder due to dopants or frustration of spins. Theoretical consideration of these additional carriers shows that they cause a local enhancement of ferromagnetic double exchange with respect to AFM superexchange, resulting in local canting of the AFM spins. This leads to a highly modulated magnetization, as measured by polarized neutron reflectometry. The spatial modulation of the canting is related to the spreading of charge from the doped layer and establishes a fundamental length scale for charge transfer, transformation of orbital occupancy, and magnetic order in these manganites. Furthermore, we confirm the existence of the canted, AFM state as was predicted by de Gennes [Phys. Rev. 118, 141 (1960)] but had remained elusive.

Using phase-sensitive streaming potential experiments in a vacuum chamber, we demonstrate that lowering the ambient pressure of the air surrounding a hydrophobic, Zeonor microfluidic substrate results in a decrease in the time scale required for equilibration of the electrokinetic potential. At ambient air pressures below 0.74 atm, the electrokinetic potential changes from ∼-84 mV to ∼-11 mV in 5 h, while the same decrease occurs in a period of over 200 h when the system is at 1 atm. Returning a sub-atmospheric system (where the electrokinetic potential had equilibrated to -11 mV) to atmospheric pressure did not result in any additional change in the electrokinetic potential. This can be described as a type of hysteresis of the electrokinetic potential with dissolved gas concentration. No time or pressure dependence was observed for the electrokinetic potential of hydrophilic (silica) substrates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Adipose progenitor cells (APCs) are widely investigated for soft tissue reconstruction following tumor resection; however, the long-term success of current approaches is still limited. In order to develop clinically relevant therapies, a better understanding of the role of cell-microenvironment interactions in adipose tissue regeneration is essential. In particular, the effect of extracellular matrix (ECM) mechanics on the regenerative capability of APCs remains to be clarified. We have used artificial ECMs based on photocrosslinkable RGD-alginate to investigate the adipogenic and pro-angiogenic potential of 3T3-L1 preadipocytes as a function of matrix stiffness. These hydrogels allowed us to decouple matrix stiffness from changes in adhesion peptide density or extracellular Ca(2+) concentration and provided a physiologically relevant 3D culture context. Our findings suggest that increased matrix rigidity promotes APC self-renewal and angiogenic capacity, whereas, it inhibits adipose differentiation. Collectively, this study advances our understanding of the role of ECM mechanics in adipose tissue formation and vascularization and will aid in the design of efficacious biomaterial scaffolds for adipose tissue engineering applications., (Copyright © 2011 Wiley Periodicals, Inc.)

Mice lose 20% to 25% of trabecular bone mineral content (BMC) during lactation and restore it after weaning through unknown mechanisms. We found that tibial Pthrp mRNA expression was upregulated fivefold by 7 days after weaning versus end of lactation in wild-type (WT) mice. To determine whether parathyroid hormone-related protein (PTHrP) stimulates bone formation after weaning, we studied a conditional knockout in which PTHrP is deleted from preosteoblasts and osteoblasts by collagen I promoter-driven Cre (Cre(ColI) ). These mice are osteopenic as adults but have normal serum calcium, calcitriol, and parathyroid hormone (PTH). Pairs of Pthrp(flox/flox) ;Cre(ColI) (null) and WT;Cre(ColI) (WT) females were mated and studied through pregnancy, lactation, and 3 weeks of postweaning recovery. By end of lactation, both genotypes lost lumbar spine BMC: WT declined by 20.6% ± 3.3%, and null decreased by 22.5% ± 3.5% (p < .0001 versus baseline; p = NS between genotypes). During postweaning recovery, both restored BMC to baseline: WT to -3.6% ± 3.7% and null to 0.3% ± 3.7% (p = NS versus baseline or between genotypes). Similar loss and full recovery of BMC were seen at the whole body and hind limb. Histomorphometry confirmed that nulls had lower bone mass at baseline and that this was equal to the value achieved after weaning. Osteocalcin, propeptide of type 1 collagen (P1NP), and deoxypyridinoline increased equally during recovery in WT and null mice; PTH decreased and calcitriol increased equally; serum calcium was unchanged. Urine calcium increased during recovery but remained no different between genotypes. Although osteoblast-derived PTHrP is required to maintain adult bone mass and Pthrp mRNA upregulates in bone after weaning, it is not required for recovery of bone mass after lactation. The factors that stimulate postweaning bone formation remain unknown., (Copyright © 2011 American Society for Bone and Mineral Research.)

Conflicting drug-drug interaction (DDI) studies with the HIV protease inhibitors (PIs) suggest net induction or inhibition of intestinal or hepatic CYP3A. As part of a larger DDI study in healthy volunteers, we determined the effect of extended administration of two PIs, ritonavir (RTV) or nelfinavir (NFV), or the induction-positive control rifampin on intestinal and hepatic CYP3A activity as measured by midazolam (MDZ) disposition after a 14-day treatment with the PI in either staggered (MDZ ∼12 h after PI) or simultaneous (MDZ and PI coadministered) manner. Oral and intravenous MDZ areas under the plasma concentration-time curves were significantly increased by RTV or NFV and were decreased by rifampin. Irrespective of method of administration, RTV decreased net intestinal and hepatic CYP3A activity, whereas NFV decreased hepatic but not intestinal CYP3A activity. The magnitude of these DDIs was more accurately predicted using PI CYP3A inactivation parameters generated in sandwich-cultured human hepatocytes rather than human liver microsomes.

We report the positive dielectrophoretic (pDEP) characterization of wild-type and ethambutol-treated Mycobacterium smegmatis populations via automated pDEP cell trapping experiments. The automated technique was validated by measurements of carboxylate-modified polystyrene microspheres and Escherichia coli . The characterization of M. smegmatis identifies a key frequency regime where the membrane-specific action of ethambutol leads to a change in the cellular dielectrophoretic response. This work represents the first such characterization of Mycobacteria and highlights the potential for DEP measurements to measure changes in mycobacterial membrane properties associated with chemical treatments or genetic mutation.

This article reviews existing methods for the isolation, fractionation, or capture of rare cells in microfluidic devices. Rare cell capture devices face the challenge of maintaining the efficiency standard of traditional bulk separation methods such as flow cytometers and immunomagnetic separators while requiring very high purity of the target cell population, which is typically already at very low starting concentrations. Two major classifications of rare cell capture approaches are covered: (1) non-electrokinetic methods (e.g., immobilization via antibody or aptamer chemistry, size-based sorting, and sheath flow and streamline sorting) are discussed for applications using blood cells, cancer cells, and other mammalian cells, and (2) electrokinetic (primarily dielectrophoretic) methods using both electrode-based and insulative geometries are presented with a view towards pathogen detection, blood fractionation, and cancer cell isolation. The included methods were evaluated based on performance criteria including cell type modeled and used, number of steps/stages, cell viability, and enrichment, efficiency, and/or purity. Major areas for improvement are increasing viability and capture efficiency/purity of directly processed biological samples, as a majority of current studies only process spiked cell lines or pre-diluted/lysed samples. Despite these current challenges, multiple advances have been made in the development of devices for rare cell capture and the subsequent elucidation of new biological phenomena; this article serves to highlight this progress as well as the electrokinetic and non-electrokinetic methods that can potentially be combined to improve performance in future studies.

Objectives: To determine whether an "optimal" distortion product otoacoustic emission (DPOAE) protocol that (1) used optimal stimulus levels and primary-frequency ratios for each f2, (2) simultaneously measured 2f2 - f1 and 2f1 - f2 distortion products, (3) controlled source contribution, (4) implemented improved calibration techniques, (5) accounted for the influence of middle ear reflectance, and (6) applied multivariate analyses to DPOAE data results in improved accuracy in differentiating between normal-hearing and hearing-impaired ears, compared with a standard clinical protocol., Design: Data were collected for f2 frequencies ranging from 0.75 to 8 kHz in 28 normal-hearing and 78 hearing-impaired subjects. The protocol included a control condition incorporating standard stimulus levels and primary-frequency ratios calibrated with a standard SPL method and three experimental conditions using optimized stimuli calibrated with an alternative forward pressure level method. The experimental conditions differed with respect to the level of the reflection-source suppressor tone and included conditions referred to as the null suppressor (i.e., no suppressor tone presented), low-level suppressor (i.e., suppressor tone presented at 58 dB SPL), and high-level suppressor (i.e., suppressor tone presented at 68 dB SPL) conditions. The area under receiver operating characteristic (A(ROC)) curves and sensitivities for fixed specificities (and vice versa) were estimated to evaluate test performance in each condition., Results: A(ROC) analyses indicated (1) improved test performance in all conditions using multivariate analyses, (2) improved performance in the null suppressor and low suppressor experimental conditions compared with the control condition, and (3) poorer performance below 4 kHz with the high-level suppressor. As expected from A(ROC), sensitivities for fixed specificities and specificities for fixed sensitivities were highest for the null suppressor and low suppressor conditions and lowest for standard clinical procedures. The influence of 2f2 - f1 and reflectance on test performance were negligible., Conclusions: Predictions of auditory status based on DPOAE measurements in clinical protocols may be improved by the inclusion of (1) optimized stimuli, (2) alternative calibration techniques, (3) low-level suppressors, and (4) multivariate analyses.

Methods for seeding high-viability (>85%) three-dimensional (3D) alginate-chondrocyte hydrogel scaffolds are presented that employ photocrosslinking of methacrylate-modified alginate with the photoinitiator VA-086. Comparison with results from several other photoinitiators, including Irgacure 2959, highlights the role of solvent, ultraviolet exposure, and photoinitiator cytotoxicity on process viability of bovine chondrocytes in two-dimensional culture. The radicals generated from VA-086 photodissociation are shown to be noncytotoxic at w/v concentrations up to 1.5%, enabling photocrosslinking without significant cell death. The applicability of these photoinitiators for generating 3D tissue-engineered constructs is evaluated by measuring cell viability in 3D constructs with aggregate moduli in the 10-20 kPa range. Hydrogels with encapsulated bovine chondrocytes were constructed with >85% viability using VA-086. While the commonly used Irgacure 2959 is noncytotoxic in its native state and crosslinks the alginate at weight fractions much lower than VA-086, the cytotoxicity of IRG2959's photogenerated radical leads to viabilities below 70% in the conditions tested.

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2⁻/⁻ mutants. However, the developmental ontogeny and cellular identity of these "thymic" PTH-expressing cells is unknown. We found that the lethality of aparathyroid Gcm2⁻/⁻ mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2⁻/⁻ mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant "thymic PTH.", Competing Interests: The authors have declared that no competing interests exist.

We simulate electrothermally induced flow in polymeric, insulator-based dielectrophoresis (iDEP) systems with DC-offset, AC electric fields at finite thermal Péclet number, and we identify key regimes where electrothermal (ET) effects enhance particle deflection and trapping. We study a single, two-dimensional constriction in channel depth with parametric variations in electric field, channel geometry, fluid conductivity, particle electrophoretic (EP) mobility, and channel electroosmotic (EO) mobility. We report the effects of increasing particle EP mobility, channel EO mobility, and AC and DC field magnitudes on the mean constriction temperature and particle behavior. Specifically, we quantify particle deflection and trapping, referring to the deviation of particles from their pathlines due to dielectrophoresis as they pass a constriction and the stagnation of particles due to negative dielectrophoresis near a constriction, respectively. This work includes the coupling between fluid, heat, and electromagnetic phenomena via temperature-dependent physical parameters. Results indicate that the temperature distribution depends strongly on the fluid conductivity and electric field magnitude, and particle deflection and trapping depend strongly on the channel geometry. Electrothermal (ET) effects perturb the EO flow field, creating vorticity near the channel constriction and enhancing the deflection and trapping effects. ET effects alter particle deflection and trapping responses in insulator-based dielectrophoresis devices, especially at intermediate device aspect ratios (2 ≤ r ≤ 7) in solutions of higher conductivity (σ m ≥ 1 × 10(-3)S/m). The impact of ET effects on particle deflection and trapping are diminished when particle EP mobility or channel EO mobility is high. In almost all cases, ET effects enhance negative dielectrophoretic particle deflection and trapping phenomena., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many mathematical models for in vitro to in vivo prediction of drug-drug interactions (DDIs) of orally administered victim drugs have been developed. However, to date, none of these models have been applicable to all intravenously administered victim drugs. We derived and conducted a sensitivity/error analysis of a modification to the existing multiple mode interaction prediction model such that it is applicable to all intravenously administered victim drugs. Using this model we showed that ignoring the hepatic extraction ratio (EH) (as low as 0.3) of intravenously administered victim drugs can result in 1) substantial underestimation of f(m, CYPi) (the fraction of hepatic clearance of the victim drug via a given enzymatic pathway) and 2) error in dissecting the contribution of intestinal and hepatic components of DDIs for orally administered drugs. Using this model we describe DDI boundaries (degree of inhibition or induction) at which ignoring the EH of commonly used victim drugs results in ≥30% error in the predicted area under the concentration-time curve (AUC) ratio or contribution of intestinal interaction to a DDI (CYP3A probes only). For the most widely used victim drug midazolam, these boundaries for AUC ratio are net inhibition (I/K(i) or λ/k(deg)) ≥1.3 or fold induction ≥2.1; for intestinal contribution the boundaries are 0.37 and 1.5, respectively. To accurately predict the intravenous AUC ratio, intestinal contribution, or f(m, CYPi) 1) for all induction DDIs irrespective of EH of the victim drug and 2) for modest to potent inhibition DDIs even when the EH is moderate (≥0.3), we propose that our model be used.

Due to low charge carrier mobilities in polymer-based solar cells, device performance is dictated by the nanoscale morphology of the active layer components. However, their morphological details are notoriously difficult to distinguish due to the low electron contrast difference between the components. Phase-sensitive neutron reflectivity (PSNR) is uniquely suited to characterize these systems due to the large, natural scattering length density difference between two common device materials, poly(3-hexylthiophene) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM). Using PSNR we find a high concentration of PCBM at the substrate and near but not at the air interface. Herein we discuss the method of applying PSNR to polymer-based solar cells, the results obtained, and an evaluation of its effectiveness.

Geometrically enhanced differential immunocapture (GEDI) and an antibody for prostate-specific membrane antigen (PSMA) are used for high-efficiency and high-purity capture of prostate circulating tumor cells from peripheral whole blood samples of castrate-resistant prostate cancer patients.

We utilize time-resolved electrokinetic measurements in order to study the electrokinetic properties of silica and TOPAS microfluidic channels as a function of the time history of the fluid-solid interface. In pressure-driven flow through TOPAS microchannels, the zeta-potential as inferred from streaming potential measurements decays exponentially by a factor of 1.5 with a characteristic decay time of 3 h after the initial formation of the fluid-solid interface. A similar exponential decay is observed immediately after water is exchanged for ethanol as the solvent in the system. In electroosmotically driven flow through TOPAS microchannels, the zeta-potential as inferred through current monitoring experiments was constant in time. No electrokinetic transients were observed in silica microchannels under these flow conditions.

We report the antiferromagnetic (AFM) interlayer exchange coupling between Ga0.97Mn0.03As ferromagnetic semiconductor layers separated by Be-doped GaAs spacers. Polarized neutron reflectivity reveals a characteristic splitting at the wave vector corresponding to twice the multilayer period, indicating that the coupling between the ferromagnetic layers is AFM. When the applied field is increased to above the saturation field, this AFM coupling is suppressed. This behavior is not observed when the spacers are undoped, suggesting that the observed AFM coupling is mediated by doped charge carriers.

Digoxin, a commonly prescribed cardiac glycoside with a narrow therapeutic window, is routinely used in pharmacokinetic studies to assess the in vivo activity of the drug efflux pump P-glycoprotein. To minimize adverse events, a sub-therapeutic dose of digoxin is usually administered, producing low plasma concentrations requiring a sensitive detection technique. Commonly available immunoassay techniques do not provide the required sensitivity to measure these low plasma concentrations and are potentially non-specific in certain subject populations. Previously published mass spectrometric techniques require either large plasma volumes or a tandem mass spectrometer. To overcome these challenges we have developed a sensitive and specific LC-MS method for the quantification of digoxin in small volumes of human plasma and urine. Plasma (1 mL) was extracted with methyl t-butyl ether under basic conditions followed by LC-MS detection of the sodium adducts of digoxin (803.4 m/z) and digitoxin (787.4 m/z, internal standard). Linearity and accuracy were demonstrated across a wide range of digoxin plasma concentration (0.05-1.5 ng/mL). This specific, sensitive, validated digoxin LC-MS assay can be used to quantify sub-therapeutic digoxin plasma concentrations in men and women (pregnant and non-pregnant).

Spin echo scattering angle measurement (SESAME) is a sensitive interference technique for measuring neutron diffraction. The method uses waveplates or birefringent prisms to produce a phase separation (the Larmor phase) between the "up" and "down" spin components of a neutron wavefunction that is initially prepared in a state that is a linear combination of in-phase up and down components. For neutrons, uniformly birefringent optical elements can be constructed from closed solenoids with appropriately shaped cross sections. Such elements are inconvenient in practice, however, both because of the precision they demand in the control of magnetic fields outside the elements and because of the amount of material required in the neutron beam. In this paper, we explore a different option in which triangular-cross-section solenoids used to create magnetic fields for SESAME have gaps in one face, allowing the lines of magnetic flux to "leak out" of the solenoid. Although the resulting field inhomogeneity produces aberrations in the Larmor phase, the symmetry of the solenoid gaps causes the aberrations produced by neighboring pairs of triangular solenoids to cancel to a significant extent. The overall symmetry of the SESAME apparatus leads to further cancellations of aberrations, providing an architecture that is easy to construct and robust in performance.

We discuss the structure of water at hydrophobic interfaces from the standpoint of its impact on electrokinetic phenomena in microfluidic devices fabricated from hydrophobic polymers such as Teflon or Zeonor. Water structuring at hydrophobic interfaces has been described as a source of interfacial charge (see Part 1, this issue), and dewetting phenomena, whether via depletion layers or nanobubbles, contribute to slip and enhanced apparent electrokinetic potentials. Issues concerning the impact of hydrodynamic slip and the role of diffuse interfacial structures are discussed. These issues are coupled with each other and with interfacial charge concerns, providing challenges for measurements of individual parameters.

This paper combines new experimental data for electrokinetic characterization of hydrophobic polymers with a detailed discussion of the putative origins of charge at water-hydrophobe interfaces. Complexities in determining the origin of charge are discussed in the context of design and modeling challenges for electrokinetic actuation in hydrophobic microfluidic devices with aqueous working fluids. Measurements of interfacial charge are complicated by slip and interfacial water structuring phenomena (see Part 2, this issue). Despite these complexities, it is shown that (i) several hydrophobic materials, such as Teflon and Zeonor, have predictable electrokinetic properties and (ii) electrokinetic data for hydrophobic microfluidic systems is most consistent with the postulate that hydroxyl ion adsorption is the origin of charge.

We demonstrate microfluidic devices for terahertz spectroscopy of biomolecules in aqueous solutions. The devices are fabricated out of a plastic material that is both mechanically rigid and optically transparent with near-zero dispersion in the terahertz frequency range. Using a lowpower terahertz time-domain spectrometer, we experimentally measure the absorption spectra of the vibrational modes of bovine serum albumin from 0.5 - 2.5 THz and find good agreement with previously reported data obtained using large-volume solutions and a high-power free-electron laser. Our results demonstrate the feasibility of performing high sensitivity terahertz spectroscopy of biomolecules in aqueous solutions with detectable molecular quantities as small as 10 picomoles using microfluidic devices.

Glyburide (glibenclamide, INN), a second generation sulfonylurea is widely used in the treatment of gestational diabetes mellitus (GDM). None of the previously reported analytical methods provide adequate sensitivity for the expected sub-nanogram/mL maternal and umbilical cord plasma concentrations of glyburide during pregnancy. We developed and validated a sensitive and low sample volume liquid chromatographic-mass spectrometric (LC-MS) method for simultaneous determination of glyburide (GLY) and its metabolite, 4-transhydroxy glyburide (M1) in human plasma (0.5 mL) or urine (0.1 mL). The limits of quantitation (LOQ) for GLY and M1 in plasma were 0.25 and 0.40 ng/mL, respectively whereas it was 1.06 ng/mL for M1 in urine. As measured by quality control samples, precision (% coefficient of variation) of the assay was <15% whereas the accuracy (% deviation from expected) ranged from -10.1 to 14.3%. We found that the GLY metabolite, M1 is excreted in the urine as the glucuronide-conjugate.

We present the development of a continuous-flow, "dielectrophoretic spectrometer" based on insulative DEP techniques and three-dimensional geometric design. Hot-embossed thermoplastic devices allow for high-throughput analysis and geometric control of electric fields via ridged microstructures patterned in a high width-to-depth aspect ratio (250:1) channel. We manipulate particles with dc-biased, ac electric fields and generate continuous-output streams of particles with a transverse outlet position specified by linear and nonlinear particle mobilities. We show, with simulation and experiment, that characteristic shape factors can be defined that capture the effects of constrictions in channel depth and that modulating the angle of these constrictions changes the resulting local DEP force. Microdevices are fabricated with an insulative constriction in channel depth, whose angle of incidence with the direction of flow varies continuously across the channel width. The resulting electric field gradients enable demonstration of a dielectrophoretic spectrometer that separates particles and controls their transverse channel position.

Numerous reports have documented drug interactions with grapefruit juice (GFJ) that occur via inhibition of CYP3A enzymes. As reported by Glaeser et al. in the March 2007 issue of this journal, there is increasing recognition that GFJ may also affect the activity of influx (e.g., OATPs) and efflux (e.g., P-glycoprotein) transporters. This commentary focuses on these interactions between GFJ and drug transporters.

Biconcave cylindrical lenses are used to focus beams of x rays or neutrons using the refractive properties of matter. In the case of neutrons, the refractive properties of magnetic induction can similarly focus and simultaneously polarize the neutron beam without the concomitant attenuation of matter. This concept of a magnetic refractive lens was tested using a compound lens consisting of 99 pairs of cylindrical permanent magnets. The assembly successfully focused the intensity of a white beam of cold neutrons of one spin state at the detector, while defocusing the other. This experiment confirmed that a lens of this nature may boost the intensity locally by almost an order of magnitude and create a polarized beam. An estimate of the performance of a more practically dimensioned device suitable for incorporation in reflectometers and slit-geometry small angle scattering instruments is given.

We show that rubidium vapor can be produced within the core of a photonic band-gap fiber yielding an optical depth in excess of 2,000. Our technique for producing the vapor is based on coating the inner walls of the fiber core with organosilane and using light-induced atomic desorption to release Rb atoms into the core. As an initial demonstration of the potential of this system for supporting ultralow-level nonlinear optical interactions, we perform electromagnetically induced transparency with control-field powers in the nanowatt regime, which represents more than a 1,000-fold reduction from the power required for bulk, focused geometries.

Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.

While cyclo-olefin polymer microchannels have the potential to improve both the optical detection sensitivity and the chemical resistance of polymer microanalytical systems, their surface properties are to date not thoroughly characterized. These surface properties dictate, among other things, electrokinetic effects when electric fields are present. Here, we report the measurement of the zeta potential of cyclo-olefin polymers (injection-molded and hot-embossed Zeonor 1060R and 1020R) microchannels as a function of pH, counter-ion concentration, storage conditions, and chemical treatment in aqueous solutions both with and without EOF-suppressing additives. In contrast with previous reports, significant surface charge is measured, consistent with titration of charged sites with pK(a) = 4.8. Storage in air, acetonitrile, or aqueous solutions has relatively minor effects. While the source of the surface charge is unclear, chemical functionalization has shown that carboxylic acid groups are not present at the surface, consistent with the chemical structure of Zeonor. EOF-suppressing additives (hydroxypropylmethylcellulose) and conditioning in perchloric acid allow the surface charge to be suppressed. We demonstrate dielectrophoretic particle trapping devices in Zeonor 1060R substrates that show reduced trapping voltage thresholds as compared to previous implementations in glass.

This paper presents the first systematic engineering study of the impact of chemical formulation and surface functionalization on the performace of free-standing microfluidic polymer elements used for high-pressure fluid control in glass microsystems. System design, chemical wet-etch processes, and laser-induced polymerization techniques are described, and parametric studies illustrate the effects of polymer formulation, glass surface modification, and geometric constraints on system performance parameters. In particular, this study shows that highly crosslinked and fluorinated polymers can overcome deficiencies in previously-reported microvalve architectures, particularly limited solvent compatibility. Substrate surface modification is shown effective in reducing the friction of the polymer-glass interface and thereby facilitating valve actuation. A microchip one-way valve constructed using this architecture shows a 2 x 10(8) ratio of forward and backward flow rates at 7 MPa. This valve architecture is integrated on chip with minimal dead volumes (70 pl), and should be applicable to systems (including chromatography and chemical synthesis devices) requiring high pressures and solvents of varying polarity.

A high-pressure (> 3 MPa) on-chip injector has been developed for microchip applications including HPLC. The mechanical injector is implemented using in situ photopolymerization of fluorinated acrylates inside wet-etched silica microchips. The injector allows reproducible injections as small as 180 pL with < 250 ms duration. The injector operated robustly over 60 days and over 1000 injections. The injector is unique among polymer-based valves as it functions in aqueous, acetonitrile, and mixed buffers at high pressures without detectable leakage., (Copyright 2004 American Chemical Society)

Laser-patterning of nanoporous membranes at the junction of a cross channel in a microchip is used to integrate protein concentration with an electrokinetic injection scheme. Upon application of voltage, linear electrophoretic concentration of charged proteins is achieved at the membrane surface because buffer ions can easily pass through the membrane while proteins larger than the molecular weight cutoff of the membrane (>5700) are retained. Simple buffer systems can be used, and the concentration results constitute outward evidence that the uniformity of buffer ion concentration is maintained throughout the process. Local and spatially averaged concentration are increased by 4 and 2 orders of magnitude, respectively, upon injection with moderate voltages (70-150 V) and concentration times (100 s). The degree of concentration is limited only by the solubility limit of the proteins. The porous polymer membrane can be used repeatedly as long as care is taken to avoid protein precipitation.

Chip-level integration of microdialysis membranes is described using a novel method for in situ photopatterning of porous polymer features. Rapid and inexpensive fabrication of nanoporous microdialysis membranes in microchips is achieved using a phase separation polymerization technique with a shaped UV laser beam. By controlling the phase separation process, the molecular weight cutoffs of the membranes can be engineered for different applications. Counterflow dialysis is used to demonstrate extraction of low molecular weight analytes from a sample stream, using two different molecular weight cutoff (MWCO) membranes; the first one with MWCO below 5700 for desalting protein samples, and the second one with a higher MWCO for size-based fractionation of proteins. Modeling based on a simple control volume analysis on the microdialysis system is consistent with measured concentration profiles, indicating both that membrane properties are uniform, well-defined, and reproducible and that diffusion of subcutoff analytes through the membrane is rapid.

This paper summarizes theory, experimental techniques, and the reported data pertaining to the zeta potential of silica and silicon with attention to use as microfluidic substrate materials, particularly for microchip chemical separations. Dependence on cation concentration, buffer and cation type, pH, cation valency, and temperature are discussed. The Debye-Hückel limit, which is often correctly treated as a good approximation for describing the ion concentration in the double layer, can lead to serious errors if it is extended to predict the dependence of zeta potential on the counterion concentration. For indifferent univalent electrolytes (e.g., sodium and potassium), two simple scalings for the dependence of zeta potential on counterion concentration can be derived in high- and low-zeta limits of the nonlinear Poisson-Boltzman equation solution in the double layer. It is shown that for most situations relevant to microchip separations, the high-zeta limit is most applicable, leading to the conclusion that the zeta potential on silica substrates is approximately proportional to the logarithm of the molar counterion concentration. The zeta vs. pH dependence measurements from several experiments are compared by normalizing the zeta based on concentration.

Zeta potential data are reviewed for a variety of polymeric microfluidic substrate materials. Many of these materials currently used for microchip fabrication have only recently been employed for generation of electroosmotic flow. Despite their recent history, polymeric microfluidic substrates are currently used extensively for microchip separations and other techniques, and understanding of the surface zeta potential is crucial for experimental design. This paper proposes the use of pC (the negative logarithm of the counterion concentration) as a useful normalization for the zeta potential on polymer substrates in contact with indifferent univalent counterions. Normalizing zeta by pC facilitates comparison of results from many investigators. The sparseness of available data for polymeric substrates prevents complete and rigorous justification for this normalization; however, it is consistent with double layer and adsorption theory. For buffers with indifferent univalent cations, normalization with the logarithm of the counterion concentration in general collapses data onto a single zeta/pC vs. pH curve, and (with the exception of PMMA) the repeatability of the data is quite encouraging. Normalization techniques should allow improved ability to predict zeta potential performance on microfluidic substrates and compare results observed with different parameters.

The work reported here is a feasibility study of the extraction of material parameters from measurements of the linear x-ray attenuation coefficient of low atomic number absorbers. Computed tomography (CT) scans of small samples containing several liquids and solids were carried out with synchrotron radiation at the Australian National Beamline Facility (BL 20B) in Japan. Average values of the x-ray linear attenuation coefficient were extracted for each material for x-ray energies ranging from 11 keV to 20.5 keV. The electron density was estimated by applying results derived from a parametrization of the x-ray linear attenuation coefficient first developed by Jackson and Hawkes and extended for this work. Average estimates for the electron density of triethanolamine and acetic acid were made to within +5.3% of the actual value. Other materials examined included furfuraldehyde, perspex and teflon, for which average estimates of the electron density were less than 10% in excess of the calculated value.

Ammonioalkyl sulfonate internal salts are explored owing to their potential for improving electrokinetic pumps used to perform miniaturized HPLC separations. The internal salts investigated can be added at high molarity since they are net-neutral, and furthermore show potential for increasing electroosmotic pumping owing to their large positive dielectric increment. Streaming potential measurements of buffered aqueous systems with varying concentrations of ammonioalkyl sulfonate internal salts have been used to measure these dielectric increments, which increase with the length of the alkyl linker. Due to their positive dielectric increments and their decremental effect on solution conductivity, all of the measured species are predicted to improve the pressure generation (up to 85%) and efficiency performance (up to 140%) of electrokinetic pumps when added at 1 M concentration. RP-HPLC separations with an ammonioalkyl sulfonate (TMAPS) have been performed and indicate that separation performance is essentially unaffected by these species. These results indicate the potential for a variety of ammonioalkyl sulfonates to be used to improve electrokinetic pump performance for miniaturized HPLC.

Spatial patterning of thin polyacrylamide films bonded to self-assembled monolayers on silica microchannels is described as a means for manipulating cell-adhesion and electroosmotic properties in microchips. Streaming potential measurements indicate that the zeta potential is reduced by at least two orders of magnitude at biological pH, and the adhesion of several kinds of cells is reduced by 80-100%. Results are shown for cover slides and in wet-etched silica microchannels. Because the polyacrylamide film is thin and transparent, this film is consistent with optical manipulation of cells and detection of cell contents. The spatial patterning technique is straightforward and has the potential to aid on-chip analysis of single adherent cells.

We present a microchip-based, voltage-addressable on/off valve architecture that is fundamentally consistent with the pressures and solvents employed for high-pressure liquid chromatography. Laser photopatterning of polymer monoliths inside glass microchannels is used to fabricate mobile fluid control elements, which are opened and closed by electrokinetic pressures. The glass substrates and crosslinked polymer monoliths operate in water-acetonitrile mixtures and have been shown to hold off pressures as high as 350 bar (5000 p.s.i.). Open/closed flow ratios of 10(4) to 10(6) have been demonstrated over the pressure range 1.5-70 bar (20-1000 p.s.i.), and the pressure-leak relationship shows the potential for valving control of flow through packed or monolithic chromatography columns. We expect that this valve platform will enable multiplexing of multiple chromatographic separations on single microchips.

A detailed discussion of linear excitation schemes for IR planar-induced fluorescence (PLIF) imaging of CO and CO2 is presented. These excitation schemes are designed to avoid laser scattering, absorption interferences, and background luminosity while an easily interpreted PLIF signal is generated. The output of a tunable optical parametric amplifier excites combination or overtone transitions in these species, and InSb IR cameras collect fluorescence from fundamental transitions. An analysis of the dynamics of pulsed laser excitation demonstrates that rotational energy transfer is prominent; hence the excitation remains in the linear regime, and standard PLIF postprocessing techniques may be used to correct for laser sheet inhomogeneities. Analysis of the vibrational energy-transfer processes for CO show that microsecond-scale integration times effectively freeze the vibrational populations, and the fluorescence quantum yield following nanosecond-pulse excitation can be made nearly independent of the collisional environment. Sensitivity calculations show that the single-shot imaging of nascent CO in flames is possible. Signal interpretation for CO2 is more complicated, owing to strongly temperature-dependent absorption cross sections and strongly collider-dependent fluorescence quantum yield. These complications limit linear CO2 IR PLIF imaging schemes to qualitative visualization but indicate that increased signal level and improved quantitative accuracy can be achieved through consideration of laser-saturated excitation schemes.

We present new vibrational (infrared) planar laser-induced fluorescence (PLIF) imaging techniques for CO(2) that use a simple, inexpensive, high-pulse-energy transversely excited atmospheric CO(2) laser to saturate a CO(2) absorption transition at 10.6 mum. Strong excitation by use of a CO(2) laser provides increased signal levels at flame temperatures and simplifies image interpretation. Because rotational energy transfer and intramodal vibrational energy transfer are fast, vibrational distributions can be approximated by use of a simple three-temperature model. Imaging results from a 425 K unsteady transverse CO(2) jet and a laminar coflowing CO/H(2) diffusion flame with temperatures near 1500 K are presented. If needed, temperature-insensitive signal levels can be generated with a two-laser technique. These results illustrate the potential for saturated infrared PLIF in a variety of flows.

The influence of gender, growth, and maturation on peak O(2) consumption (VO(2 peak)) in 11-13 yr olds were examined by using multilevel regression modeling. Subjects were 119 boys and 115 girls, aged 11.2 +/- 0.4 (SD) yr at the onset of the study. Sexual maturation was classified according to Tanner's indexes of pubic hair. VO(2 peak) was determined annually for 3 yr. The initial model identified body mass and stature as significant explanatory variables, with an additional positive effect for age and incremental effects for stage of maturation. A significant gender difference was apparent with lower values for girls, and an age-by-gender interaction indicated a progressive divergence in boys' and girls' VO(2 peak). Subsequent incorporation of the sum of two skinfold thicknesses into the model negated stature effects, reduced the gender term, and explained much of the observed maturity effects. The body mass exponent almost doubled, but the age-by-gender interaction term was consistent with the initial model.

The aim of this study was to examine the maturation responses of young people to submaximal treadmill exercise. Body mass was controlled using both the conventional ratio standard and allometric modelling. Ninety-seven boys and 97 girls with a mean age of 12.2 years completed a discontinuous, incremental exercise test to voluntary exhaustion. We measured peak oxygen uptake (VO2peak) and VO2 when running at 8, 9 and 10 km x h(-1). Sexual maturation was assessed visually using Tanner's indices of pubic hair. Peak VO2 was significantly higher in boys (P<0.001); this was still the case when the influence of body mass was covaried out. During submaximal exercise, no significant differences in absolute VO2 were observed between the sexes (P>0.05); however, values of VO2, expressed both in ratio with body mass and adjusted for body mass using allometry, were significantly greater in boys than in girls (P<0.001). For absolute VO2, significant main effects (P<0.05) were seen for maturity at each exercise stage. With the influence of body mass controlled using either the ratio standard or allometry, no significant main effects (P>0.05) for maturity were observed. Our results indicate that boys are less economical than girls while running at 8-10 km x h(-1) and that, independently of body mass, maturation does not influence the VO2 response to submaximal exercise.

An analysis of the distribution of trace metals in the kidney cortex in normal and brindled male mice has been carried out with a scanning proton microprobe. Enzyme histochemical staining techniques were used to distinguish between proximal and distal tubules. Average copper levels were increased in brindled kidney tissue sections, with the above-normal Cu accumulation found to occur entirely within the proximal tubules. Therefore, the proximal tubule is now regarded as the location where the defect in Cu transport in brindled mice is manifested the most clearly. The distribution of Fe was found to be non-uniform with some tubule cross-sections exhibiting high concentrations in both genotypes. The distribution of Zn was found to be uniform, and the concentration was similar for each genotype.

Recent determinations of the temperature dependence of acetone fluorescence have permitted the application of acetone planar laser-induced fluorescence imaging, which was already popular for mapping concentration, to the measurement of temperature. With a view toward developing temperature-imaging diagnostics, we present atmospheric-pressure fluorescence and absorption results acquired with excitation at eight wavelengths across the absorption feature of acetone and at temperatures from 300 to 1000 K. Modeling of the fluorescence yield of acetone is shown to be useful in explaining both these results and the variation of acetone fluorescence with pressure and composition that was observed in several studies. The model results in conjunction with the photophysics data provide guidance for the application of temperature diagnostics over a range of conditions while also suggesting useful multiparameter imaging approaches.

Study Objectives: To examine the ventilatory responses of prepubescent children to submaximal and peak exercise using appropriate allometric modeling to control for differences in body size., Design: Cross-sectional study of a representative sample of children., Setting: Middle schools (8 to 11 years) in Exeter, UK., Participants: We studied 101 boys and 76 girls aged 11.1 (0.4) years and classified Tanner stage 1 for pubic hair (no true pubic hair)., Measurements: At rest: stature, mass, sum of skinfolds, hemoglobin concentration, FVC, and FEV1. During treadmill exercise at 7, 8, 9, and 10 km/h, and at peak exercise: oxygen uptake (VO2), minute ventilation (VE), tidal volume (VT), and respiratory frequency (Rf)., Results: At peak exercise, boys' VO2, VE, and VT were significantly (p<0.01) higher than girls' values and remained so even when the influence of body size was controlled using allometric principles. There were no significant (p>0.05) sex differences in Rf or the ratios VT/FVC or VE/VO2. When data were compared at the same relative exercise intensity (ie, 70 to 75% or 80 to 85% peak VO2), no significant (p>0.05) sex differences in Rf, VT/FVC, or VE/VO2 were detected. Boys' higher (p<0.001) VO2 values were reflected by their higher VE which remained higher than values for girls at both submaximal levels even when the influence of body size was covaried out., Conclusions: Prepubescent boys demonstrate higher peak VO2 than girls and this is supported by a higher VE and VT, even when the influence of body size is accounted for using allometry. Other ventilatory responses to both peak exercise and exercise at the same relative intensity are remarkably similar in both boys and girls.