Induction and suppression of cell lysis in an electrokinetic microfluidic system.

The ability to strategically induce or suppress cell lysis is critical for many cellular-level diagnostic and therapeutic applications conducted within electrokinetic microfluidic platforms. The chemical and structural integrity of sub-cellular components is important when inducing cell lysis. However, metal electrodes and electrolytes participate in undesirable electrochemical reactions that alter solution composition and potentially damage protein, RNA, and DNA integrity within device microenvironments. For many biomedical applications, cell viability must be maintained even when device-imposed cell-stressing stimuli (e.g., electrochemical reaction byproducts) are present. In this work, we explored a novel and tunable method to accurately induce or suppress device-imposed artifacts on human red blood cell (RBC) lysis in non-uniform AC electric fields. For precise tunability, a dielectric hafnium oxide (HfO2 ) layer was used to prevent electron transfer between the electrodes and the electric double layer and thus reduce harmful electrochemical reactions. Additionally, a low concentration of Triton X-100 surfactant was explored as a tool to stabilize cell membrane integrity. The extent of hemolysis was studied as a function of time, electrode configuration (T-shaped and star-shaped), cell position, applied non-uniform AC electric field, with uncoated and HfO2 coated electrodes (50 nm), and absence and presence of Triton X-100 (70 µM). Tangible outcomes include a parametric analysis relying upon literature and this work to design, tune, and operate electrokinetic microdevices to intentionally induce or suppress cellular lysis without altering intracellular components. Implications are that devices can be engineered to leverage or minimize device-imposed biological artefacts extending the versatility and utility of electrokinetic diagnostics.

Reaction-Free Concentration Gradient Generation in Spatially Nonuniform AC Electric Fields.

The ability to generate stable, spatiotemporally controllable concentration gradients is critical for both electrokinetic and biological applications such as directional wetting and chemotaxis. Electrochemical techniques for generating solution and surface gradients display benefits such as simplicity, controllability, and compatibility with automation. Here, we present an exploratory study for generating microscale spatiotemporally controllable gradients using a reaction-free electrokinetic technique in a microfluidic environment. Methanol solutions with ionic fluorescein isothiocyanate (FITC) molecules were used as an illustrative electrolyte. Spatially nonuniform alternating current (AC) electric fields were applied using hafnium dioxide (HfO2)-coated Ti/Au electrode pairs. Results from spatial and temporal analyses along with control experiments suggest that the FITC ion concentration gradient in bulk fluid (over 50 µm from the electrode) was established due to spatial variation of electric field density, and was independent of electrochemical reactions at the electrode surface. The established ion concentration gradients depended on both amplitudes and frequencies of the oscillating AC electric field. Overall, this work reports a novel approach for generating stable and spatiotemporally tunable gradients in a microfluidic chamber using a reaction-free electrochemical methodology.

Changes in Membrane Dielectric Properties of Porcine Kidney Cells Provide Insight into the Antiviral Activity of Glycine.

The ability to monitor the status and progression of viral infections is important for development and screening of new antiviral drugs. Previous research illustrated that the osmolyte glycine (Gly) reduced porcine parvovirus (PPV) infection in porcine kidney (PK-13) cells by stabilizing the capsid protein and preventing virus capsid assembly into viable virus particles. Dielectrophoresis (DEP) was examined herein as a noninvasive, electric field- and frequency-dependent tool for real-time monitoring of PK-13 cell responses to obtain information about membrane barrier functionality and polarization. DEP responses of PK-13 cells were compared to those of PPV-infected cells in the absence and presence of the osmolyte glycine. With infection progression, PK-13 DEP spectra shifted toward lower frequencies, reducing crossover frequencies (fCO). The spherical single-shell model was used to extract PK-13 cell dielectric properties. Upon PPV infection, specific membrane capacitance increased over the time progression of virus attachment, penetration, and capsid protein production and assembly. Following glycine treatment, the DEP spectra displayed attenuated fCO and specific membrane capacitance values shifted back toward uninfected PK-13 cell values. These results suggest that DEP can be used to noninvasively monitor the viral infection cycle and screen antiviral compounds. DEP can augment traditional tools by elucidating membrane polarization changes related to drug mechanisms that interrupt the virus infection cycle.

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient.

Isoelectric focusing (IEF) is a powerful tool for amphoteric protein separations because of high sensitivity, bio-compatibility, and reduced complexity compared to chromatography or mechanical separation techniques. IEF miniaturization is attractive because it enables rapid analysis, easier adaptation to point of care applications, and smaller sample demands. However, existing small-scale IEF tools have not yet been able to analyze single protein spots from array libraries, which are ubiquitous in many pharmaceutical discovery and screening protocols. Thus, we introduce an in situ, novel, miniaturized protein analysis approach that we have termed surface isoelectric focusing (sIEF). Low volume printed sIEF gels can be run at length scales of ∼300 µm, utilize ∼0.9 ng of protein with voltages below 10 V. Further, the sIEF device platform is so simple that it can be integrated with protein library arrays to reduce cost; devices demonstrate reusability above 50 uses. An acrylamide monomer solution containing broad-range carrier ampholytes was microprinted with a Nano eNablerTM between micropatterned gold electrodes spaced 300 µm apart on a glass slide. The acrylamide gel was polymerized in situ followed by protein loading via printed diffusional exchange. A pH gradient formed via carrier ampholyte stacking when electrodes were energized; the gradient was verified using ratiometric pH-sensitive FITC/TRITC dyes. Green fluorescent protein (GFP) and R-phycoerythrin (R-PE) were utilized both as pI markers and to test sIEF performance as a function of electric field strength and ampholyte concentration. Factors hampering sIEF included cathodic drift and pH gradient compression, but were reduced by co-printing non-ionic Synperonic® F-108 surfactant to reduce protein-gel interactions. sIEF gels achieved protein separations in <10 min yielding bands < 50 µm wide with peak capacities of ∼8 and minimum pI differences from 0.12 to 0.14. This new sIEF technique demonstrated comparable focusing at ∼100 times smaller dimensions than any previous IEF. Further, sample volumes required were reduced four orders of magnitude from 20 µL for slab gel IEF to 0.002 µL for sIEF. In summary, sIEF advantages include smaller volumes, reduced power consumption, and microchip surface accessibility to focused bands along with equivalent separation resolutions to prior IEF tools. These attributes position this new technology for rapid, in situ protein library analysis in clinical and pharmaceutical settings.

Determination of water-soluble and fat-soluble vitamins in tears and blood serum of infants and parents by liquid chromatography/mass spectrometry.

Tears serve as a viable diagnostic fluid with advantages including less invasive sample to collect and less complex to prepare for analysis. Several water-soluble and fat-soluble vitamins were detected and quantified in human tears and compared with blood serum levels. Samples from 15 family pairs, each pair consisting of a four-month-old infant and one parent were analyzed; vitamin concentrations were compared between tears and blood serum for individual subjects, between infants and parents, and against self-reported dietary intakes. Water-soluble vitamins B1, B2, B3 (nicotinamide), B5, B9 and fat-soluble vitamin E (α-tocopherol) were routinely detected in tears and blood serum while fat-soluble vitamin A (retinol) was detected only in blood serum. Water-soluble vitamin concentrations measured in tears and blood serum of single subjects were comparable, while higher concentrations were measured in infants compared to their parents. Fat-soluble vitamin E concentrations were lower in tears than blood serum with no significant difference between infants and parents. Serum vitamin A concentrations were higher in parents than infants. Population trends were compiled and quantified using a cross correlation factor. Strong positive correlations were found between tear and blood serum concentrations of vitamin E from infants and parents and vitamin B3 concentrations from parents, while slight positive correlations were detected for infants B3 and parents B1 and B2 concentrations. Correlations between infants and parents were found for the concentrations of B1, B2, B3, and E in tears, and the concentrations of B2, A, and E in blood serum. Stronger vitamin concentration correlations were found between infants and parents for the breast-fed infants, while no significant difference was observed between breast-fed and bottle-fed infants. This work is the first to demonstrate simultaneous vitamin A, B, and E detection and to quantify correlations between vitamin concentrations in tears and blood serum. Our results suggest that tears are a viable biofluid to monitor nutritional health because they sufficiently mirror blood serum data and may enhance the speed of deficiency diagnoses.

Platinum electrode modification: Unique surface carbonization approach to improve performance and sensitivity.

Many microfluidic devices, also known as lab-on-a-chip devices, employ electrochemical detection methods using microelectrodes. Miniaturizing electrodes inevitably reduces electrode sensitivity and decreases the S/N, which limits applications within microfluidic devices. However, microelectrode surface modification can increase the surface area and sensitivity. In the present work, we report substantial improvement in platinum electrode performance and sensitivity by coating with carbon from red blood cells. The larger goal of this work was to measure DC electrical resistances of red blood cell suspensions in a microchannel for hematocrit determination. It was observed that as current responses of red blood cell suspensions were measured, the platinum electrode performance (reproducibility and S/N) improved with time. The platinum electrode electrocatalytic activity for red blood cell current measurements improved by 140%. Systematic experimentation revealed that red blood cells adsorb and carbonize the platinum electrode surfaces. The electrode surfaces before and after performance improvements were analyzed by field emission scanning electron microscopy, energy dispersive spectrometry, and Raman spectrometry. The formed carbon layers on the electrode surfaces were found to be proteomic and increased surface area with a porous three-dimensional structure, thus improving performance and stabilizing currents.

Electrochemical hematocrit determination in a direct current microfluidic device.

Hematocrit (HCT) tests are widely performed to screen blood donors and to diagnose medical conditions. Current HCT test methods include conventional microhematocrit, Coulter counter, CuSO4 specific gravity, and conductivity-based point-of-care (POC) HCT devices, which can be either expensive, environmentally inadvisable, or complicated. In the present work, we introduce a new and simple microfluidic system for a POC HCT determination. HCT was determined by measuring current responses of blood under 100 V DC for 1 min in a microfluidic device containing a single microchannel with dimensions of 180 µm by 70 µm and 10 mm long. Current responses of red blood cell (RBC) suspensions in PBS or separately plasma at HCT concentrations of 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 70 vol% were measured to show feasibility of the microfluidic system for HCT determination. Key parameters affecting current responses included electrolysis bubbles and irreversible RBC adsorption; parameters were optimized via addition of nonionic surfactant Triton X-100 into sample solution and carbonizing electrode surfaces. The linear trend line of current responses over a range of RBC concentrations were obtained in both PBS and plasma. This work suggested that a simple microfluidic device could be a promising platform for a new POC HCT device.

Size and medium conductivity dependence on dielectrophoretic behaviors of gas core poly-L-lysine shell nanoparticles.

Dynamic (dis)assembly of biocompatible nanoparticles into 3D, packed structures would benefit drug delivery, films, and diagnostics. Dielectrophoretic (DEP) microdevices can rapidly assemble and manipulate polarizable particles within nonuniform electric fields. DEP has primarily discerned micrometer particles since nanoparticles experience smaller forces. This work examines conductivity and size DEP dependencies of previously unexplored spherical core-shell nanoparticle (CSnp) into 3D particle assemblies. Poly-L-lysine shell material was custom synthesized around a gas core to form CSnps. DEP frequencies from 1 kHz to 80 MHz at fixed 5 volts peak-to-peak and medium conductivities of 10(-5) and 10(-3) S/m were tested. DEP responses of ∼220 and ∼400 nm poly-L-lysine CSnps were quantified via video intensity densitometry at the microdevice's quadrapole electrode center for negative DEP (nDEP) and adjacent to electrodes for positive DEP. Intensity densitometry was then translated into a relative DEP response curve. An unusual nDEP peak occurred at ∼57 MHz with 25-80 times greater apparent nDEP force. All electrical circuit components were then impedance matched, which changed the observed response to weak positive DEP at low frequencies and consistently weak nDEP from ∼100 kHz to 80 MHz. This impedance-matched behavior agrees with conventional Clausius-Mossotti DEP signatures taking into account the gas core's contributions to the polarization mechanisms. This work describes a potential pitfall when conducting DEP at higher frequencies in microdevices and concurrently demonstrates nDEP behavior for a chemically and structurally distinct particle system. This work provides insight into organic shell material properties in nanostructures and strategies to facilitate dynamic nanoparticle assemblies.

Improving electrokinetic microdevice stability by controlling electrolysis bubbles.

The voltage-operating window for many electrokinetic microdevices is limited by electrolysis gas bubbles that destabilize microfluidic system causing noise and irreproducible responses above ∼3 V DC and less than ∼1 kHz AC at 3 Vpp. Surfactant additives, SDS and Triton X-100, and an integrated semipermeable SnakeSkin® membrane were employed to control and assess electrolysis bubbles from platinum electrodes in a 180 by 70 µm, 10 mm long microchannel. Stabilized current responses at 100 V DC were observed with surfactant additives or SnakeSkin® barriers. Electrolysis bubble behaviors, visualized via video microscopy at the electrode surface and in the microchannels, were found to be influenced by surfactant function and SnakeSkin® barriers. Both SDS and Triton X-100 surfactants promoted smaller bubble diameters and faster bubble detachment from electrode surfaces via increasing gas solubility. In contrast, SnakeSkin® membranes enhanced natural convection and blocked bubbles from entering the microchannels and thus reduced current disturbances in the electric field. This data illustrated that electrode surface behaviors had substantially greater impacts on current stability than microbubbles within microchannels. Thus, physically blocking bubbles from microchannels is less effective than electrode functionalization approaches to stabilize electrokinetic microfluidic systems.

Theoretical and experimental examination of particle-particle interaction effects on induced dipole moments and dielectrophoretic responses of multiple particle chains.

Dielectrophoresis (DEP), an electrokinetic phenomenon based on particle polarizations in nonuniform electric fields, is increasingly employed for particle and cell characterizations and manipulations in microdevices. However, particle number densities are rarely varied and particle-particle interactions are largely overlooked, but both affect particle's effective polarizations by changing the local electric field, which directly impacts particle assembly into chains. This work examines theoretical and experimental particle-particle interactions and dielectrophoretic responses in nonuniform electric fields, then presents individual and chain velocities of spherical polystyrene microparticles and red blood cells (RBCs) under DEP forces in a modified quadruple electrode microdevice. Velocities are independently compared between 1, 2, 3, and 4 polystyrene beads and RBCs assembled into chains aligned with the electric field. Simulations compared induced dipole moments for particles experiencing the same (single point) and changing (multiple points) electric fields. Experiments and simulations are compared by plotting DEP velocities versus applied signal frequency from 1 kHz to 80 MHz. Simulations indicate differences in the DEP force exerted on each particle according to chain position. Simulations and experiments show excellent qualitative agreement; chains with more particles experienced a decrease in the DEP response for both polystyrene beads and RBCs. These results advance understanding of the extent that induced dipole polarizations with multiple particle chains affect observed behaviors in electrokinetic cellular diagnostic systems.

Direct current insulator-based dielectrophoretic characterization of erythrocytes: ABO-Rh human blood typing.

A microfluidic platform developed for quantifying the dependence of erythrocyte (red blood cell, RBC) responses by ABO-Rh blood type via direct current insulator dielectrophoresis (DC-iDEP) is presented. The PDMS DC-iDEP device utilized a 400 x 170 µm² rectangular insulating obstacle embedded in a 1.46-cm long, 200-µm wide inlet channel to create spatial non-uniformities in direct current (DC) electric field density realized by separation into four outlet channels. The DC-iDEP flow behaviors were investigated for all eight blood types (A+, A-, B+, B-, AB+, AB-, O+, O-) in the human ABO-Rh blood typing system. Three independent donors of each blood type, same donor reproducibility, different conductivity buffers (0.52-9.1 mS/cm), and DC electric fields (17.1-68.5 V/cm) were tested to investigate separation dependencies. The data analysis was conducted from image intensity profiles across inlet and outlet channels in the device. Individual channel fractions suggest that the dielectrophoretic force experienced by the cells is dependent on erythrocyte antigen expression. Two different statistical analysis methods were conducted to determine how distinguishable a single blood type was from the others. Results indicate that channel fraction distributions differ by ABO-Rh blood types suggesting that antigens present on the erythrocyte membrane polarize differently in DC-iDEP fields. Under optimized conductivity and field conditions, certain blind blood samples could be sorted with low misclassification rates.

Explorations of ABO-Rh antigen expressions on erythrocyte dielectrophoresis: changes in cross-over frequency.

A quadrupole dielectrophoretic microdevice was utilized to examine the ABO-Rh dependencies on erythrocyte polarizations. This important step toward medical microdevice technology would transform key clinical blood tests from the laboratory into the field. Previous work in dielectrophoretic microdevices demonstrated that the large number of ABO antigens on erythrocyte membranes impacts their dielectrophoretic signature at 1 MHz. This work explores the dielectrophoretic behavior of native human erythrocytes categorized by their ABO-Rh blood types and directly compares these responses to the same erythrocyte sample modified to remove the A and B antigens. A ß(1-3)-galactosidase enzyme was utilized to cleave the ABO polysaccharide backbone at the galactosidase bonds. The enzymatic reaction was optimized by comparing agglutination of the native and modified blood cells in addition to UV-Vis and HPLC analysis of the reaction effluent for saccharide residues. Next, the dielectrophoretic behaviors of the native and modified erythrocytes were visually verified in a quadrupole electrode microdevice over a frequency range from 100 kHz to 80 MHz. The lower cross-over frequency (COF), which transitions from negative to positive dielectrophoresis, for ABO blood types tested (A+, A-, B+, B-, AB+, O+ and O-) differed over the range from 17 to 47 MHz. The COFs of the corresponding enzyme-modified erythrocytes were also determined and the range narrowed to 29-41 MHz. A second COF in the 70-80 MHz range was observed and was reduced in the presence of the transmembrane Rhesus factor. These results suggest that antigen expression on erythrocyte membrane surfaces influence cell polarizations in nonuniform AC fields.

Quantification of pH gradients and implications in insulator-based dielectrophoresis of biomolecules.

Direct current (DC) insulator-based dielectrophoretic (iDEP) microdevices have the potential to replace traditional alternating current dielectrophoretic devices for many cellular and biomolecular separation applications. The use of large DC fields suggest that electrode reactions and ion transport mechanisms can become important and impact ion distributions in the nanoliters of fluid in iDEP microchannels. This work tracked natural pH gradient formation in a 100 µm wide, 1 cm-long microchannel under applicable iDEP protein manipulation conditions. Using fluorescence microscopy with the pH-sensitive dye FITC Isomer I and the pH-insensitive dye TRITC as a reference, pH was observed to drop drastically in the microchannels within 1 min in a 3000 V/cm electric field; pH drops were observed in the range of 6-10 min within a 100 V/cm electric field and varied based on the buffer conductivity. To address concerns of dye transport impacting intensity data, electrokinetic mobilities of FITC were carefully examined and found to be (i) toward the anode and (ii) 1 to 2 orders of magnitude smaller than H⁺ transport which is responsible for pH drops from the anode toward the cathode. COMSOL simulations of ion transport showed qualitative agreement with experimental results. The results indicate that pH changes are severe enough and rapid enough to influence the net charge of a protein or cause aggregation during iDEP experiments. The results also elucidate reasonable time periods over which the phosphate buffering capacity can counter increases in H⁺ and OH⁻ for unperturbed iDEP manipulations.

DC insulator dielectrophoretic applications in microdevice technology: a review.

Dielectrophoresis is a noninvasive, nondestructive, inexpensive, and fast technique for the manipulation of bioparticles. Recent advances in the field of dielectrophoresis (DEP) have resulted in new approaches for characterizing the behavior of particles and cells using direct current (DC) electric fields. In such approaches, spatial nonuniformities are created in the channel by embedding insulating obstacles in the channel or flow field in order to perform separation or trapping. This emerging field of dielectrophoresis is commonly termed DC insulator dielectrophoresis (DC-iDEP), insulator-based dielectrophoresis (iDEP), or electrodeless dielectrophoresis (eDEP). In many microdevices, this form of dielectrophoresis has advantages over traditional AC-DEP, including single material microfabrication, remotely positioned electrodes, and reduced fouling of the test region. DC-iDEP applications have included disease detection, separation of cancerous cells from normal cells, and separation of live from dead bacteria. However, there is a need for a critical report to integrate these important research findings. The aim of this review is to provide an overview of the current state-of-art technology in the field of DC-iDEP for the separation and trapping of inert particles and cells. In this article, a review of the concepts and theory leading to the manipulation of particles via DC-iDEP is given, and insulating obstacle geometry designs and the characterization of device performance are discussed. This review compiles and compares the significant findings obtained by researchers in handling and manipulating particles.

Dynamic pH and Thermal Analysis of Paper-Based Microchip Electrophoresis.

Paper-based microchip electrophoresis has the potential to bring laboratory electrophoresis tests to the point of need. However, high electric potential and current values induce pH and temperature shifts, which may affect biomolecule electrophoretic mobility thus decrease test reproducibility and accuracy of paper-based microfluidic electrophoresis. We have previously developed a microchip electrophoresis system, HemeChip, which has the capability of providing low-cost, rapid, reproducible, and accurate point-of-care (POC) electrophoresis tests for hemoglobin analysis. Here, we report the methodologies we implemented for characterizing HemeChip system pH and temperature during the development process, including utilizing commercially available universal pH indicator and digital camera pH shift characterization, and infrared camera characterizing temperature shift characterization. The characterization results demonstrated that pH shifts up to 1.1 units, a pH gradient up to 0.11 units/mm, temperature shifts up to 40 °C, and a temperature gradient up to 0.5 °C/mm existed in the system. Finally, we report an acid pre-treatment of the separation media, a cellulose acetate paper, mitigated both pH and temperature shifts and provided a stable environment for reproducible HemeChip hemoglobin electrophoresis separation.

Impacts of low concentration surfactant on red blood cell dielectrophoretic responses.

Cell dielectrophoretic responses have been extensively studied for biomarker expression, blood typing, sepsis, circulating tumor cell separations, and others. Surfactants are often added to the analytical buffer in electrokinetic cellular microfluidic systems to lower surface/interfacial tensions. In nonelectrokinetic systems, surfactants influence cell size, shape, and agglomeration; this has not been systematically documented in electrokinetic systems. In the present work, the impacts of the Triton X-100 surfactant on human red blood cells (RBCs) were explored via ultraviolet-visible spectroscopy (UV-Vis) and dielectrophoresis (DEP) to compare nonelectrokinetic and electrokinetic responses, respectively. The UV-Vis spectra of Triton X-100 treated RBCs were dramatically different from that of native RBCs. DEP responses of RBCs were compared to RBCs treated with low concentrations of Triton X-100 (0.07-0.17 mM) to ascertain surfactant effects on dielectric properties. A star-shaped electrode design was used to quantify RBC dielectric properties by fitting a single-shell oblate cell model to experimentally-derived DEP spectra. The presence of 0.07 and 0.11 mM of Triton X-100 shifted the RBC's DEP spectra yielding lower crossover frequencies ( f C O ) . The single-shell oblate model revealed that cell radius and membrane permittivity are the dominant influencers of DEP spectral shifts. The trends observed were similar for 0.11 mM and 0.07 mM Triton X-100 treated cells. However, a further increase of Triton X-100 to 0.17 mM caused cells to only exhibit negative DEP. The magnitude of the DEP force increased with Triton X-100 concentration. This work indicates that dynamic surfactant interactions with cell membranes alter cell dielectric responses and properties.

Dielectrophoretic characterization of erythrocytes: positive ABO blood types.

Dielectrophoretic manipulation of erythrocytes/red blood cells is investigated as a tool to identify blood type for medical diagnostic applications. Positive blood types of the ABO typing system (A+, B+, AB+ and O+) were tested and cell responses quantified. The dielectrophoretic response of each blood type was observed in a platinum electrode microdevice, delivering a field of 0.025V(pp)/microm at 1 MHz. Responses were recorded via video microscopy for 120 s and erythrocyte positions were tabulated at 20-30 s intervals. Both vertical and horizontal motions of erythrocytes were quantified via image object recognition, object tracking in MATLAB, binning into appropriate electric field contoured regions (wedges) and statistical analysis. Cells of O+ type showed relatively attenuated response to the dielectrophoretic field and were distinguished with greater than 95% confidence from all the other three blood types. AB+ cell responses differed from A+ and B+ blood types likely because AB+ erythrocytes express both the A and B glycoforms on their membrane. This research suggests that dielectrophoresis of untreated erythrocytes beyond simple dilution depends on blood type and could be used in portable blood typing devices.

Data representing two separate LC-MS methods for detection and quantification of water-soluble and fat-soluble vitamins in tears and blood serum.

Two separate liquid chromatography (LC)-mass spectrometry (MS) methods were developed for determination and quantification of water-soluble and fat-soluble vitamins in human tear and blood serum samples. The water-soluble vitamin method was originally developed to detect vitamins B1, B2, B3 (nicotinamide), B5, B6 (pyridoxine), B7, B9 and B12 while the fat-soluble vitamin method detected vitamins A, D3, 25(OH)D3, E and K1. These methods were then validated with tear and blood serum samples. In this data in brief article, we provide details on the two LC-MS methods development, methods sensitivity, as well as precision and accuracy for determination of vitamins in human tears and blood serum. These methods were then used to determine the vitamin concentrations in infant and parent samples under a clinical study which were reported in "Determination of Water-Soluble and Fat-Soluble Vitamins in Tears and Blood Serum of Infants and Parents by Liquid Chromatography/Mass Spectrometry DOI:10.1016/j.exer.2016.12.007 [1]". This article provides more details on comparison of vitamin concentrations in the samples with the ranges reported in the literature along with the medically accepted normal ranges. The details on concentrations below the limits of detection (LOD) and limits of quantification (LOQ) are also discussed. Vitamin concentrations were also compared and cross-correlated with clinical data and nutritional information. Significant differences and strongly correlated data were reported in [1]. This article provides comprehensive details on the data with slight differences or slight correlations.

Spatially variant red blood cell crenation in alternating current non-uniform fields.

Alternating-current (AC) electrokinetics involve the movement and behaviors of particles or cells. Many applications, including dielectrophoretic manipulations, are dependent upon charge interactions between the cell or particle and the surrounding medium. Medium concentrations are traditionally treated as spatially uniform in both theoretical models and experiments. Human red blood cells (RBCs) are observed to crenate, or shrink due to changing osmotic pressure, over 10 min experiments in non-uniform AC electric fields. Cell crenation magnitude is examined as functions of frequency from 250 kHz to 1 MHz and potential from 10 Vpp to 17.5 Vpp over a 100 µm perpendicular electrode gap. Experimental results show higher peak to peak potential and lower frequency lead to greater cell volume crenation up to a maximum volume loss of 20%. A series of experiments are conducted to elucidate the physical mechanisms behind the red blood cell crenation. Non-uniform and uniform electrode systems as well as high and low ion concentration experiments are compared and illustrate that AC electroporation, system temperature, rapid temperature changes, medium pH, electrode reactions, and convection do not account for the crenation behaviors observed. AC electroosmotic was found to be negligible at these conditions and AC electrothermal fluid flows were found to reduce RBC crenation behaviors. These cell deformations were attributed to medium hypertonicity induced by ion concentration gradients in the spatially nonuniform AC electric fields.

Solution pH change in non-uniform alternating current electric fields at frequencies above the electrode charging frequency.

AC Faradaic reactions have been reported as a mechanism inducing non-ideal phenomena such as flow reversal and cell deformation in electrokinetic microfluidic systems. Prior published work described experiments in parallel electrode arrays below the electrode charging frequency (fc ), the frequency for electrical double layer charging at the electrode. However, 2D spatially non-uniform AC electric fields are required for applications such as in plane AC electroosmosis, AC electrothermal pumps, and dielectrophoresis. Many microscale experimental applications utilize AC frequencies around or above fc . In this work, a pH sensitive fluorescein sodium salt dye was used to detect [H(+)] as an indicator of Faradaic reactions in aqueous solutions within non-uniform AC electric fields. Comparison experiments with (a) parallel (2D uniform fields) electrodes and (b) organic media were employed to deduce the electrode charging mechanism at 5 kHz (1.5fc ). Time dependency analysis illustrated that Faradaic reactions exist above the theoretically predicted electrode charging frequency. Spatial analysis showed [H(+)] varied spatially due to electric field non-uniformities and local pH changed at length scales greater than 50 µm away from the electrode surface. Thus, non-uniform AC fields yielded spatially varied pH gradients as a direct consequence of ion path length differences while uniform fields did not yield pH gradients; the latter is consistent with prior published data. Frequency dependence was examined from 5 kHz to 12 kHz at 5.5 Vpp potential, and voltage dependency was explored from 3.5 to 7.5 Vpp at 5 kHz. Results suggest that Faradaic reactions can still proceed within electrochemical systems in the absence of well-established electrical double layers. This work also illustrates that in microfluidic systems, spatial medium variations must be considered as a function of experiment time, initial medium conditions, electric signal potential, frequency, and spatial position.