Characterization of Nanoparticles in Diverse Mixtures Using Localized Surface Plasmon Resonance and Nanoparticle Tracking by Dark-Field Microscopy with Redox Magnetohydrodynamics Microfluidics.

Redox magnetohydrodynamics (RMHD) microfluidics is coupled with dark-field microscopy (DFM) to offer high-throughput single-nanoparticle (NP) differentiation in situ and operando in a flowing mixture by localized surface plasmon resonance (LSPR) and tracking of NPs. The color of the scattered light allows visualization of the NPs below the diffraction limit. Their Brownian motion in 1-D superimposed on and perpendicular to the RMHD trajectory yields their diffusion coefficients. LSPR and diffusion coefficients provide two orthogonal modalities for characterization where each depends on a particle's material composition, shape, size, and interactions with the surrounding medium. RMHD coupled with DFM was demonstrated on a mixture of 82 ± 9 nm silver and 140 ± 10 nm gold-coated silica nanospheres. The two populations of NPs in the mixture were identified by blue/green and orange/red LSPR and their scattering intensity, respectively, and their sizes were further evaluated based on their diffusion coefficients. RMHD microfluidics facilitates high-throughput analysis by moving the sample solution across the wide field of view absent of physical vibrations within the experimental cell. The well-controlled pumping allows for a continuous, reversible, and uniform flow for precise and simultaneous NP tracking of the Brownian motion. Additionally, the amounts of nanomaterials required for the analysis are minimized due to the elimination of an inlet and outlet. Several hundred individual NPs were differentiated from each other in the mixture flowing in forward and reverse directions. The ability to immediately reverse the flow direction also facilitates re-analysis of the NPs, enabling more precise sizing.

Dopamine, vocalization, and astrocytes.

Dopamine, the main catecholamine neurotransmitter in the brain, is predominately produced in the basal ganglia and released to various brain regions including the frontal cortex, midbrain and brainstem. Dopamine's effects are widespread and include modulation of a number of voluntary and innate behaviors. Vigilant regulation and modulation of dopamine levels throughout the brain is imperative for proper execution of motor behaviors, in particular speech and other types of vocalizations. While dopamine's role in motor circuitry is widely accepted, its unique function in normal and abnormal speech production is not fully understood. In this perspective, we first review the role of dopaminergic circuits in vocal production. We then discuss and propose the conceivable involvement of astrocytes, the numerous star-shaped glia cells of the brain, in the dopaminergic network modulating normal and abnormal vocal productions.

Miniaturized probe on polymer SU-8 with array of individually addressable microelectrodes for electrochemical analysis in neural and other biological tissues.

An SU-8 probe with an array of nine, individually addressable gold microband electrodes (100 µm long, 4 µm wide, separated by 4-µm gaps) was photolithographically fabricated and characterized for detection of low concentrations of chemicals in confined spaces and in vivo studies of biological tissues. The probe's shank (6 mm long, 100 µm wide, 100 µm thick) is flexible, but exhibits sufficient sharpness and rigidity to be inserted into soft tissue. Laser micromachining was used to define probe geometry by spatially revealing the underlying sacrificial aluminum layer, which was then etched to free the probes from a silicon wafer. Perfusion with fluorescent nanobeads showed that, like a carbon fiber electrode, the probe produced no noticeable damage when inserted into rat brain, in contrast to damage from an inserted microdialysis probe. The individual addressability of the electrodes allows single and multiple electrode activation. Redox cycling is possible, where adjacent electrodes serve as generators (that oxidize or reduce molecules) and collectors (that do the opposite) to amplify signals of small concentrations without background subtraction. Information about electrochemical mechanisms and kinetics may also be obtained. Detection limits for potassium ferricyanide in potassium chloride electrolyte of 2.19, 1.25, and 2.08 µM and for dopamine in artificial cerebral spinal fluid of 1.94, 1.08, and 5.66 µM for generators alone and for generators and collectors during redox cycling, respectively, were obtained.

Combining magnetic forces for contactless manipulation of fluids in microelectrode-microfluidic systems.

A novel method to drive and manipulate fluid in a contactless way in a microelectrode-microfluidic system is demonstrated by combining the Lorentz and magnetic field gradient forces. The method is based on the redox-reaction [Fe(CN)6]3-/[Fe(CN)6]4- performed in a magnetic field oriented perpendicular to the ionic current that crosses the gap between two arrays of oppositely polarized microelectrodes, generating a magnetohydrodynamic flow. Additionally, a movable magnetized CoFe micro-strip is placed at different positions beneath the gap. In this region, the magnetic flux density is changed locally and a strong magnetic field gradient is formed. The redox-reaction changes the magnetic susceptibility of the electrolyte near the electrodes, and the resulting magnetic field gradient exerts a force on the fluid, which leads to a deflection of the Lorentz force-driven main flow. Particle Image Velocity measurements and numerical simulations demonstrate that by combining the two magnetic forces, the flow is not only redirected, but also a local change of concentration of paramagnetic species is realized.

Redox-Magnetohydrodynamically Controlled Fluid Flow with Poly(3,4-ethylenedioxythiophene) Coupled to an Epitaxial Light Sheet Confocal Microscope for Image Cytometry Applications.

We present the merging of two technologies to perform continuous high-resolution fluorescence imaging of cellular suspensions in a deep microfluidics chamber with no moving parts. An epitaxial light sheet confocal microscope (e-LSCM) was used to image suspensions enabled by fluid transport via redox-magnetohydrodynamics (R-MHD). The e-LSCM features a linear solid state sensor, oriented perpendicular to the direction of flow, that can bin the emission across different numbers of pixels, yielding electronically adjustable optical sectioning. This, in addition to intensity thresholding, defines the axial resolution, which was validated with an optical phantom of polystyrene microspheres suspended in agarose. The linear fluid speed within the microfluidics chamber was uniform (0.16-2.9%) across the 0.5-1.0 mm lateral field of view (dependent upon the chosen magnification) with continuous acquisition. Also, the camera's linear exposure periods were controlled to ensure an accurate image aspect ratio across this span. Poly(3,4-ethylenedioxythiophene) (PEDOT) was electrodeposited as an immobilized redox film on electrodes of a chip for R-MHD, and the fluid flow was calibrated to specific linear speeds as a function of applied current. Images of leukocytes stained with acridine orange, a fluorescent, amphipathic vital dye that intercalates DNA, were acquired in the R-MHD microfluidics chamber with the e-LSCM to demonstrate imaging of biological samples. The combination of these technologies provides a miniaturizable platform for large sample volumes and high-throughput, image-based analysis without the requirement of moving parts, enabling development of robust, point-of-care image cytometry.

Application of Electrochemical Redox Cycling: Toward Differentiation of Dopamine and Norepinephrine.

The electrochemical redox cycling behavior of dopamine (DA), norepinephrine (NE), and their mixture was investigated using coplanar gold microband electrode arrays at four generator-collector gap conditions (4, 12, 20, and 28 µm). This method provides opportunity for differentiating the catecholamines in mixtures by monitoring the current at collector electrodes activated at different distances from generator electrodes. It takes advantage of the ECC' mechanism associated with the electrochemical oxidation of catecholamines, in which DA and NE have rate constants that differ by a factor of 7.5 for the first order intramolecular cyclization (C) following electron transfer (E). Collector electrodes activated at different distances from the generators were used to examine the process of the following chemistry at different time points, because spatial relationships are related to temporal ones through diffusion. Solutions of artificial cerebral spinal fluid containing 50 µM DA, 50 µM NE, and a DA-NE mixture of 50 µM of each were examined. The collection efficiency during redox cycling for NE had a greater dependence on gap width than DA, and the collector current of NE became silent at ∼20 µm. The collector current of the mixture approaches that of DA alone with increasing gap, suggesting that differentiation of DA and NE may be possible. The collector current of the mixture is further affected by the homogeneous reaction (C') between oxidized and cyclized products of DA and NE and drops below that of DA alone. This may be used for differentiation in more complicated chemical systems.

Poly(3,4-ethylenedioxythiophene)-Modified Electrodes for Microfluidics Pumping with Redox-Magnetohydrodynamics: Improving Compatibility for Broader Applications by Eliminating Addition of Redox Species to Solution.

A new approach using electrodes modified with poly(3,4-ethylenedioxythiophene) (PEDOT) was implemented to perform redox-magnetohydrodynamics (MHD) microfluidics and eliminate the need to add redox species to solution, thus removing interferences with detection, sample, and reagents for lab-on-a-chip applications. This accomplishment not only retains the unique properties of redox-MHD pumping (i.e., programmable fluid speeds and flow patterns without the need for side walls, horizontal flat flow profiles, looping flow, no electrode corrosion, and no bubble formation), but also achieves a wider sustainable voltage range and currents that can be as much as 7+ times higher (and therefore correspondingly higher velocities) than in past studies involving unmodified electrodes and redox species in solution. PEDOT, a conducting polymer that has been shown to exhibit low cytotoxicity, was electropolymerized on microband gold electrodes (25 mm long ×103 µm wide). A cell (325 µL) with distant side walls was formed by placing a 620 µm thick poly(dimethylsiloxane), PDMS, gasket with an opening of 3.2 cm × 1.5 cm on the chip, and a glass slide lid prevented evaporation. A 0.37 T magnet under the chip generated a magnetic field perpendicular to the chip surface. The cell was filled with 0.095 M NaCl electrolyte containing 10 µm polystyrene beads to visualize and quantify fluid flow using optical video microscopy. Fluid speeds of 590 µm s(-1) were observed immediately after applying a potential step. A linear relationship between applied electronic current and fluid velocity was shown. Vertical flow profiles under applied current conditions were curved, with a weak parabolic fit.

Redox cycling behavior of individual and binary mixtures of catecholamines at gold microband electrode arrays.

The electrochemical redox cycling (RC) behavior of individual and binary mixtures of three catecholamines are investigated using gold microelectrode arrays in vitro. Catecholamines showed reversible or irreversible responses during RC depending on their oxidation products' cyclization rate. The RC behavior of the binary mixtures supports the disproportionation reaction of catecholamines, which has been previously reported, but not under RC conditions or with mixtures. This fundamental study provides insights on the effects of complicated mechanisms and kinetics on RC and sets the foundation for future applications of RC for in vivo multi-neurotransmitter analysis.

Redox-magnetohydrodynamics, flat flow profile-guided enzyme assay detection: toward multiple, parallel analyses.

A proof-of-concept superparamagnetic microbead-enzyme complex was integrated with microfluidics pumped by redox-magneto-hydrodynamics (MHD) to take advantage of the magnet (0.56 T) beneath the chip and the uniform flat flow profile, as a first step toward developing multiple, parallel chemical analyses on a chip without the need for independent channels. The superparamagnetic beads were derivatized with alkaline phosphatase (a common enzyme label for biochemical assays) and magnetically immobilized at three different locations on the chip with one directly on the path to the detector and the other two locations adjacent to, but off the path, by a distance >5 times the detector diameter. Electroactive p-aminophenol, enzymatically generated at the bead-enzyme complex from its electroinactive precursor p-aminophenyl phosphate in a solution containing a redox species [Ru(NH3)6](3+/2+) for pumping and Tris buffer, was transported by redox-MHD and detected with square wave voltammetry at a 312 µm diameter gold microdisk stationed 2 mm downstream from the bead-complex on the flow path. Oppositely biased pumping electrodes, consisting of 2.5 cm long gold bands and separated by 5.6 mm, flanked the active flow region containing the bead-enzyme complex and detection site. The signal from adjacent paths was only 20% of that for the direct path and ≤8% when pumping electrodes were inactive.

3D imaging of flow patterns in an internally-pumped microfluidic device: redox magnetohydrodynamics and electrochemically-generated density gradients.

Redox magnetohydrodynamics (MHD) is a promising technique for developing new electrochemical-based microfluidic flow devices with unique capabilities, such as easily switching flow direction and adjusting flow speeds and flow patterns as well as avoiding bubble formation. However, a detailed description of all the forces involved and predicting flow patterns in confined geometries is lacking. In addition to redox-MHD, density gradients caused by the redox reactions also play important roles. Flow in these devices with small fluid volumes has mainly been characterized by following microbead motion by optical microscopy either by particle tracking velocimetry (PTV) or by processing the microbead images by particle image velocimetry (PIV) software. This approach has limitations in spatial resolution and dimensionality. Here we use fluorescence correlation spectroscopy (FCS) to quantitatively and accurately measure flow speeds and patterns in the ~5-50 µm/s range in redox-MHD-based microfluidic devices, from which 3D flow maps are obtained with a spatial resolution down to 2 µm. The 2 µm spatial resolution flow speeds map revealed detailed flow profiles during redox-MHD in which the velocity increases linearly from above the electrode and reaches a plateau across the center of the cell. By combining FCS and video-microscopy (with PTV and PIV processing approaches), we are able to quantify a vertical flow of ~10 µm/s above the electrodes as a result of density gradients caused by the redox reactions and follow convection flow patterns. Overall, combining FCS, PIV, and PTV analysis of redox-MHD is a powerful combination to more thoroughly characterize the underlying forces in these promising microfluidic devices.

Detection of dopamine in the presence of excess ascorbic acid at physiological concentrations through redox cycling at an unmodified microelectrode array.

The electrochemical behavior of dopamine was examined under redox cycling conditions in the presence and absence of a high concentration of the interferent ascorbic acid at a coplanar, microelectrode array where the area of the generator electrodes was larger than that of the collector electrodes. Redox cycling converts a redox species between its oxidized and reduced forms by application of suitable potentials on a set of closely located generator and collector electrodes. It allows signal amplification and discrimination between species that undergo reversible and irreversible electron transfer. Microfabrication was used to produce 18 individually addressable, 4-µm-wide gold band electrodes, 2 mm long, contained in an array having an interelectrode spacing of 4 µm. Because the array electrodes are individually addressable, each can be selectively biased to produce an overall optimal electrochemical response. Four adjacent microbands were shorted together to serve as the collector, and were flanked on each side by seven microbands shorted as the generator (a ratio of 1:3.5 of electroactive area, respectively). This configuration achieved a detection limit of 0.454 ± 0.026 µM dopamine at the collector in the presence of 100 µM ascorbic acid in artificial cerebrospinal fluid buffer, concentrations that are consistent with physiological levels. Enhancement by surface modification of the microelectrode array to achieve this detection limit was unnecessary. The results suggest that the redox cycling method may be suitable for in vivo quantification of transients and basal levels of dopamine in the brain without background subtraction.

Maximizing flow velocities in redox-magnetohydrodynamic microfluidics using the transient faradaic current.

There is a need for a microfluidic pumping technique that is simple to fabricate, yet robust, compatible with a variety of solvents, and which has easily controlled fluid flow. Redox-magnetohydrodynamics (MHD) offers these advantages. However, the presence of high concentrations of redox species, important for inducing sufficient convection at low magnetic fields for hand-held devices, can limit the use of redox-MHD pumping for analytical applications. A new method for redox-MHD pumping is investigated that takes advantage of the large amplitude of the transient portion of the faradaic current response that occurs upon stepping the potential sufficiently past the standard electrode potential, E°, of the pumping redox species at an electrode. This approach increases the velocity of the fluid for a given redox concentration. An electronic switch was implemented between the potentiostat and electrochemical cell to alternately turn on and off different electrodes along the length of the flow path to maximize this transient electronic current and, as a result, the flow speed. Velocities were determined by tracking microbeads in a solution containing electroactive potassium ferrocyanide and potassium ferricyanide, and supporting electrolyte, potassium chloride, in the presence of a magnetic field. Fluid velocities with slight pulsation were obtained with the switch that were 70% faster than the smooth velocities without the switch. This indicates that redox species concentrations can be lowered by a similar amount to achieve a given speed, thereby diminishing interference of the redox species with detection of the analyte in applications of redox-MHD microfluidics for chemical analysis.

Evaluation of heart tissue viability under redox-magnetohydrodynamics conditions: toward fine-tuning flow in biological microfluidics applications.

A microfluidic system containing a chamber for heart tissue biopsies, perfused with Krebs-Henseleit buffer containing glucose and antibiotic (KHGB) using peristaltic pumps and continuously stimulated, was used to evaluate tissue viability under redox-magnetohydrodynamics (redox-MHD) conditions. Redox-MHD possesses unique capabilities to control fluid flow using ionic current from oxidation and reduction processes at electrodes in a magnetic field, making it attractive to fine-tune fluid flow around tissues for "tissue-on-a-chip" applications. The manuscript describes a parallel setup to study two tissue samples simultaneously, and 6-min static incubation with Triton X100. Tissue viability was subsequently determined by assaying perfusate for lactate dehydrogenase (LDH) activity, where LDH serves as an injury marker. Incubation with KHGB containing 5 mM hexaammineruthenium(III) (ruhex) redox species with and without a pair of NdFeB magnets (∼ 0.39 T, placed parallel to the chamber) exhibited no additional tissue insult. MHD fluid flow, viewed by tracking microbeads with microscopy, occurred only when the magnet was present and stimulating electrodes were activated. Pulsating MHD flow with a frequency similar to the stimulating waveform was superimposed over thermal convection (from a hotplate) for Triton-KHGB, but fluid speed was up to twice as fast for ruhex-Triton-KHGB. A large transient ionic current, achieved when switching on the stimulating electrodes, generates MHD perturbations visible over varying peristaltic flow. The well-controlled flow methodology of redox-MHD is applicable to any tissue type, being useful in various drug uptake and toxicity studies, and can be combined equally with on- or off-device analysis modalities.

Low-temperature co-fired ceramic microchannels with individually addressable screen-printed gold electrodes on four walls for self-contained electrochemical immunoassays.

Microchannel devices were constructed from low-temperature co-fired ceramic (LTCC) materials with screen-printed gold (SPG) electrodes in three dimensions--on all four walls--for self-contained enzyme-linked immunosorbant assays with electrochemical detection. The microchannel confines the solution to a small volume, allowing concentration of electroactive enzymatically generated product and nearby electrodes provide high-speed and high-sensitivity detection: it also facilitates future integration with microfluidics. LTCC materials allow easy construction of three-dimensional structures compared with more traditional materials such as glass and polymer materials. Parallel processing of LTCC layers is more amenable to mass production and fast prototyping, compared with sequential processing for integrating multiple features into a single device. LTCC and SPG have not been reported previously as the basis for microchannel immunoassays, nor with integrated, individually addressable electrodes in three dimensions. A demonstration assay for mouse IgG at 5.0 ng/mL (3.3 × 10(-11) M) with electrochemical detection was achieved within a 1.8 cm long × 290 µm high × 130 µm wide microchannel (approximately 680 nL). Two of four SPG electrodes span the top and bottom walls and serve as the auxiliary electrode and the assay site, respectively. The other two (0.7 cm long × 97 µm wide) are centered lengthwise on the sidewalls of the channel. One serves as the working and the other as the pseudoreference electrode. The immunoassay components were immobilized at the bottom SPG region. Enzymatically generated p-aminophenol was detected at the internal working electrode within 15 s of introducing the enzyme substrate p-aminophenyl phosphate. A series of buffer rinses avoided nonspecific adsorption and false-positive signals.

Redox-magnetohydrodynamic microfluidics without channels and compatible with electrochemical detection under immunoassay conditions.

A unique capability of redox-magnetohydrodynamics (redox-MHD) for handling liquids on a small scale was demonstrated. A 1.2 muL solution plug was pumped from an injection site to a detector without the need for a channel to direct the flow. The redox pumping species did not interfere with enzymatic activity in a solution compatible with enzyme-linked immunoassays. Alkaline phosphatase (AP), a common enzyme label, converted p-aminophenyl phosphate (PAPP) to p-aminophenol (PAP(R)) in the presence of 2.5 mM Ru(NH(3))(6)Cl(2) and 2.5 mM Ru(NH(3))(6) Cl(3), in 0.1 M Tris buffer (pH = 9). A solution plug containing PAPP (no AP) was pumped through the surrounding solution containing AP (no PAPP), and the enzymatically generated PAP(R) was easily detected and distinguishable electrochemically from the pumping species with square wave voltammetry down to 0.1 mM concentrations. The test device consisted of a silicon chip containing individually addressable microband electrodes, placed on a 0.5 T NdFeB permanent magnet with the field oriented perpendicular to the chip. A 8.0 mm wide x 15.5 mm long x 1.5 mm high volume of solution was contained by a poly(dimethylsiloxane) gasket and capped with a glass slide. A steady-state fluid velocity of approximately 30 mum/s was generated in a reinforcing flow configuration between oppositely polarized sets of pumping electrodes with approximately 2.1 muA.

Magnetic fields for fluid motion.

Three forces induced by magnetic fields offer unique control of fluid motion and new opportunities in microfluidics. This article describes magnetoconvective phenomena in terms of the theory and controversy, tuning by redox processes at electrodes, early-stage applications in analytical chemistry, mature applications in disciplines far afield, and future directions for micro total analysis systems. (To listen to a podcast about this article, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html .).

Investigations of redox magnetohydrodynamic fluid flow at microelectrode arrays using microbeads.

Microbeads are used to track fluid flow over microband electrode arrays to investigate fundamentals of redox magnetohydrodynamics (redox-MHD) in a confined solution. The results may lead toward the design of micro total analysis systems with microfluidics based on the redox-MHD concept. Ion flux was generated by reduction and oxidation of electroactive potassium ferri- and ferrocyanide at selected individually addressable microelectrodes in the array. An external magnetic field was produced by a small, permanent magnet (0.38 T) placed directly below the array with its field perpendicular to the plane of the array. The cross product of ion flux and magnetic field produces a magnetic force (a portion of the Lorentz force equation) that causes the fluid to rotate around the active electrodes. Velocities up to 1.4 mm/s are demonstrated here. The effects on velocities were obtained for different concentrations of redox species, widths of electrodes, gaps between electrodes, and combinations of anodically- and cathodically polarized electrodes. The microbeads allowed mapping of flow patterns and velocities, both parallel and perpendicular to the array chip. The influence of counteracting shear forces, drag along the walls, and reinforcing flow are discussed. A significant result is the fairly flat flow profile across 650 microm, attained between electrodes that are oppositely biased.

Signal amplification in a microchannel from redox cycling with varied electroactive configurations of an individually addressable microband electrode array.

Amperometric detection at microelectrodes in lab-on-a-chip (LOAC) devices lose advantages in signal-to-background ratio, reduced ohmic iR drop, and steady-state signal when volumes are so small that diffusion fields reach the walls before flux becomes fully radial. Redox cycling of electroactive species between multiple, closely spaced microelectrodes offsets that limitation and provides amplification capabilities. A device that integrates a microchannel with an individually addressable microband electrode array has been used to study effects of signal amplification due to redox cycling in a confined, static solution with different configurations and numbers of active generators and collectors. The microfabricated device consists of a 22 microm high, 600 microm wide microchannel containing an array of 50 microm wide, 600 microm long gold microbands, separated by 25 microm gaps, interspersed with an 800 microm wide counter electrode and 400 microm wide passive conductor, with a distant but on-chip 400 microm wide pseudoreference electrode. Investigations involve solutions of potassium chloride electrolyte containing potassium ferrocyanide. Amplification factors were as high as 7.60, even with these microelectrodes of fairly large dimensions (which are generally less expensive, easier, and more reproducible to fabricate), because of the significant role that passive and active (instrumentally induced) redox cycling plays in confined volumes of enclosed microchannels. The studies are useful in optimizing designs for LOAC devices.

Dynamics of saxitoxin binding to saxiphilin c-lobe reveals conformational change.

Thermodynamic parameters (DeltaG, DeltaH, DeltaS, DeltaC(p)) have been determined to evaluate the dynamics of binding of saxitoxin to the c-lobe of saxiphilin. We have developed an improved method to rapidly express and purify recombinant saxiphilin c-lobe, and fully characterized it by mass spectrometry for the first time. Surface plasmon resonance (SPR) was used to characterize the interaction between saxitoxin and immobilized c-lobe. At 298 K, c-lobe binds saxitoxin with K(D)=1.2 nM, DeltaH degrees =-11.7+/-0.8 kcal/mol, and DeltaS degrees =1.17+/-0.07 cal/molK. Analysis of DeltaC(p) of toxin association at several temperatures suggests that hydrophobic forces contribute to the binding event. Additionally, changes in 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence upon binding to c-lobe in the presence and absence of saxitoxin support a conformational change in c-lobe upon saxitoxin binding.