On-Chip Electrochemical Sensing with an Enhanced Detecting Signal Due to Concentration Polarization-Based Analyte Preconcentration.

Here, we integrated two key technologies within a microfluidic system, an electrokinetic preconcentration of analytes by ion Concentration Polarization (CP) and local electrochemical sensors to detect the analytes, which can synergistically act to significantly enhance the detection signal. This synergistic combination, offering both decoupled and coupled operation modes for continuous monitoring, was validated by the intensified fluorescent intensities of CP-preconcentrated analytes and the associated enhanced electrochemical response using differential pulse voltammetry and chronoamperometry. The system performance was evaluated by varying the location of the active electrochemical sensor, target analyte concentrations, and electrolyte concentration using fluorescein molecules as the model analyte and Homovanillic acid (HVA) as the target bioanalyte within both phosphate-buffered saline (PBS) and artificial sweat solution. The combination of on-chip electrochemical sensing with CP-based preconcentration renders this generic approach adaptable to various analytes. This advanced system shows remarkable promise for enhancing biosensing detection in practical applications while bridging the gap between fundamental research and practical implementation.

Microvalve-Based Tunability of Electrically Driven Ion Transport through a Microfluidic System with an Ion-Exchange Membrane.

Microfluidic channels with an embedded ion permselective medium under the application of electric current are commonly used for electrokinetic processes as on-chip ion concentration polarization (ICP) and bioparticle preconcentration to enhance biosensing. Herein, we demonstrate the ability to dynamically control the electrically driven ion transport by integrating individually addressable microvalves. The microvalves are located along a main microchannel that is uniformly coated with a thin layer of an ion-exchange membrane (IEM). The interplay of ionic transport between the solution within the microchannel and the thin IEM, under an applied electric current, can be locally tuned by the deformation of the microvalve. This tunability provides a robust and simple means of implementing new functionalities into lab-on-a-chip devices, e.g., dynamic control over multiple ICP layers and their associated preconcentrated molecule plugs, multiplex sensing, suppression of biofouling, and plug dispersion, while maintaining the well-known application of microvalves as steric filtration.

Tunable Nanochannels Connected in Series for Dynamic Control of Multiple Concentration-Polarization Layers and Preconcentrated Molecule Plugs.

Integration of ionic permselective medium (e.g., nanochannels, membranes) within microfluidic channels has been shown to enable on-chip desalination, sample purification, bioparticle sorting, and biomolecule concentration for enhanced detection sensitivity. However, the ion-permselective mediums are generally of fixed properties and cannot be dynamically tuned. Here we study a microfluidic device consisting of an array of individually addressable elastic membranes connected in series on top of a single microfluidic channel that can be deformed to locally reduce the channel cross-section into a nanochannel. Dynamic tunability of the ion-permselective medium, as well as controllability of its location and ionic permselectivity, introduces a new functionality to microfluidics-based lab-on-a-chip devices, for example, dynamic localization of preconcentrated biomolecule plugs at different sensing regions for multiplex detection. Moreover, the ability to simultaneously form a series of preconcentrated plugs at desired locations increases parallelization of the system and the trapping efficiency of target analytes.

Electrothermal Active Control of Preconcentrated Biomolecule Plugs.

Concentration-polarization (CP)-based biomolecule preconcentration is highly effective in enhancing the detection sensitivity yet fails to precisely and dynamically control the location of the preconcentrated biomolecule plug to ensure overlap with the sensing region (e.g., immobilized molecular probes). Here, we used electrothermal (ET) stirring as a means of controlling the location of a preconcentrated biomolecule plug. The applied microfluidic device consisted of a Nafion membrane to induce the CP and an array of individually addressable microscale heaters for active local ET stirring. The experimental results demonstrated that such a novel platform enabled active control of the location of the preconcentrated plug of target biomolecules, ensuring its overlap with the functionalized microparticles and ultimately yielding enhanced detection sensitivity and binding kinetics. This was demonstrated using avidin-biotin particles as a simple bead-based bioassay model.

Distinct and independent dielectrophoretic behavior of the head and tail of sperm and its potential for the safe sorting and isolation of rare spermatozoa.

Often, in semen samples with minute amounts of sperm, even the single spermatozoon required to fertilize an oocyte cannot be found in the ejaculate. This is primarily because currently, sperm is generally searched for manually under a microscope. In this study, dielectrophoresis (DEP) was investigated as an alternative automated technique for sorting sperm cells. Using a quadrupolar electrode array it was shown that the head and tail of the sperm had independent and unique crossover frequencies corresponding to the transition of the DEP force from repulsive (negative) to attractive (positive). These surprising results were further analyzed, showing that the head and tail have their own distinct electrical properties. This significant result allows for the sperm's head, which contains the DNA, to be distanced from potentially damaging high electric fields using negative DEP while simultaneously manipulating and sorting the sperm using the positive DEP response of the tail. A proof of concept sorting chip was designed and tested. The low crossover frequency of the tail also allows for the use of a higher conductivity, and thus more physiological, medium than the conventional DEP solutions. Although more research is required to design and optimize an efficient, user-friendly, and high-throughput device, this research is a proof of concept that DEP has the potential to automate and improve the processing of semen samples, especially those containing only rare spermatozoa.

Dielectrophoretic characterization and isolation of jellyfish stinging capsules.

Jellyfish stinging capsules known as nematocysts are explosive, natural-injection systems with high potential as a natural drug-delivery system. These organelles consist of a capsule containing a highly folded thin needle-like tubule and a matrix highly concentrated with charged constituents that enable the tubule to fire and penetrate a target. For the purpose of using these nematocysts as drug delivery system it is first required to purify subpopulations from heterogeneous population of capsules and to investigate each subpopulation's distinct function and characteristics. Here, the nematocysts' dielectric properties were experimentally investigated using dielectrophoretic and electrorotational spectra with best fits derived from theoretical models. The dielectric characterization adds to our understanding of the nematocysts' structure and function and is necessary for the dielectrophoretic isolation and manipulation of populations. As expected, the effect of monovalent and divalent exchange cations resulted in higher inner conductivity for the NaCl treated capsules; this result stands in agreement with their relative higher osmotic pressure. In addition, an efficient dielectrophoretic isolation of different nematocyst subpopulations was demonstrated, paving the way to an understanding of nematocysts' functional diversity and the development of an efficient drug delivery platform.

Interplay between Nanochannel and Microchannel Resistances.

Current nanochannel system paradigm commonly neglects the role of the interfacing microchannels and assumes that the ohmic electrical response of a microchannel-nanochannel system is solely determined by the geometric properties of the nanochannel. In this work, we demonstrate that the overall response is determined by the interplay between the nanochannel resistance and various microchannel attributed resistances. Our experiments confirm a recent theoretical prediction that in contrast to what was previously assumed at very low concentrations the role of the interfacing microchannels on the overall resistance becomes increasingly important. We argue that the current nanochannel-dominated conductance paradigm can be replaced with a more correct and intuitive microchannel-nanochannel-resistance-model-based paradigm.

Dielectrophoretic characterization of cells in a stationary nanoliter droplet array with generated chemical gradients.

A novel design of reusable microfluidic platform that generates a stationary nanoliter droplet array (SNDA) for cell incubation and analysis, equipped with a complementary array of individually addressable electrodes for each microwell is studied. Various solute concentration gradients were generated between the wells where dielectrophoresis (DEP) was used to characterize the effect of the gradients on the cell's response. The feasibility of generating concentration gradients and observation of DEP responses was demonstrated using a gradient of salts in combination with microparticles and viable cells. L1210 Lymphoma cells were used as the model cells in these experiments. Lymphoma cells' cross-over frequency (COF) decreased with increasing stress conditions. Specifically, a linear decrease in the cell COF was measured as a function of solution tonicity and blebbistatin dose. Lymphoma cells were incubated under a gradient of the chemotherapeutic agent doxorubicin (DOX), which led to saturation in the cell-COF response at 30 nM DOX, demonstrating the potential of the platform in screening of label-free drugs.

Clinical relevance of serum ionized magnesium concentration in dogs with myxomatous mitral valve disease.

BACKGROUND: Hypomagnesemia is associated with a poor prognosis in humans with congestive heart failure (CHF), but studies in veterinary medicine are limited. HYPOTHESIS: Serum ionized magnesium concentration [iMg2+ ] would decrease as CHF progresses compared with the initial diagnostic levels and that lower [iMg2+ ] would be negatively associated with prognosis in dogs with CHF. ANIMALS: A total of 181 client-owned dogs with myxomatous mitral valve disease (MMVD) were included. They were classified into the preclinical stage (NO-CHF, n = 108), stage C (n = 42), and stage D (n = 31) based on the American College of Veterinary Internal Medicine MMVD classification. METHODS: This is a retrospective study from 2 referral centers. The [iMg2+ ] was compared among the NO-CHF, stage C, and stage D groups. Kaplan-Meier curves and the log-rank test were used to compare the incidence of death between groups. Multivariable Cox regression analysis was used to estimate the association of hypomagnesemia with the death. RESULTS: In the stage D group, the [iMg2+ ] was lower than that in the NO-CHF (P < .0001) and stage C groups (P < .003). In the Kaplan-Meier survival analysis, the 1-year cumulative survival rate in hypomagnesemic dogs was 53% compared with 91.5% in normomagnesemic dogs (log-rank test, P < .0001). In the multivariable Cox analysis, lower concentration of [K+ ] and [iMg2+ ], along with higher Evel , were associated with negative prognoses. Specifically, hypomagnesemia was associated with an approximately 4-fold increased risk of death (hazard ratio = 4.015; 95% confidence interval, 1.537-10.488; P = .005). CONCLUSIONS AND CLINICAL IMPORTANCE: Assessing the [iMg2+ ] might serve as a potential marker for estimating the severity and prognosis indirectly in dogs with MMVD. Combining [iMg2+ ] measurement with other diagnostic methods, such as echocardiography, could improve the prognostic evaluation of MMVD in dogs.

Evaluation of the clinical usefulness of pancreatic alpha amylase as a novel biomarker in dogs with acute pancreatitis: a pilot study.

Pancreatic alpha amylase (P-AMY) is used as a biomarker of acute pancreatitis (AP) in human medicine. To our knowledge, there are no studies evaluating the usefulness of P-AMY in dogs with AP. In this study, we evaluated the diagnostic value of P-AMY, currently not verified in veterinary medicine. The AP group (n = 40) consisted of dogs with AP diagnosed using clinical signs and laboratory examinations, including abnormal canine pancreatic lipase (cPL) concentration, and compatible abdominal ultrasound examination at first presentation. Evaluation of the canine AP severity (CAPS) score was performed. The control group (n = 38) was composed of normal dogs without any abnormalities in clinical findings, blood exams or diagnostic imaging. The correlation of P-AMY with cPL was confirmed by Pearson's correlation analysis (r = 0.564, p < .001). The sensitivity and specificity for the most appropriate cut-off values of P-AMY were recorded similar to the values of DGGR. The dogs with AP and CAPS ≥11 had significantly higher serum P-AMY (p = .016) contrary to DGGR lipase and cPL. Furthermore, there was a significant difference in the median P-AMY dependent on the presence of systemic inflammatory response syndrome (p = .001). P-AMY showed similar level of diagnostic accuracy along with sensitivity and specificity compared to DGGR lipase. In addition, P-AMY showed a significant association with CAPS score, contrary to cPL and DGGR lipase. Along with other biomarkers associated with AP, P-AMY has the potential of usefulness as a supportive diagnostic and prognostic biomarker of AP in dogs.

Electrical impedance spectroscopy of microchannel-nanochannel interface devices.

We report experimental verification of the depression of the slope in the Warburg branch of the electrochemical impedance spectrum using a fabricated microchannel-nanochannel device. This was previously theoretically predicted to occur with increasing dc bias voltage as a result of nanochannel electro-osmotic flow and provides an example of the influence of net fluid flow on electrokinetic transport. The dominant influence of nanochannel polarization in the kHz range of the impedance response is also demonstrated experimentally. This latter effect may be significant in both fundamental electrokinetics of micronanochannel devices as well as in practical molecular sensing applications.

Case report: Successful medical management of adrenocortical carcinoma with metastasis in a Maltese dog.

Introduction: Adrenocortical carcinoma (ACC) with metastasis has a grave prognosis, and adrenalectomy is associated with a high perioperative mortality rate in dogs. A favorable outcome following trilostane treatment in patients with metastatic ACC confirmed by a decreased size of the adrenal tumor and metastatic lesions has not been reported in dogs. Case description: A 12-year-old neutered male Maltese dog was diagnosed with a right adrenal tumor and a hepatic mass. Adrenal-dependent hyperadrenocorticism (ADH) was diagnosed based on clinical signs and an adrenocorticotropic hormone stimulation test (ACTHST). In addition, tests for plasma metanephrine and normetanephrine ruled out a pheochromocytoma. Based on cytology and computed tomography, unresectable metastatic ACC was confirmed. The dog was managed with trilostane due to the presence of distant metastasis. Medical management improved the clinical signs and post-ACTHST cortisol concentrations. One year after the first presentation, the clinical signs and ACTHST test showed a favorable outcome. In addition, computed tomography revealed a decreased size of the right adrenal tumor and resolution of the hepatic mass. Conclusions: Trilostane could be considered as a treatment option for unresectable metastatic ACC. A decrease in tumor size following treatment with trilostane has not been reported in dogs. This case report is the first to demonstrate a favorable outcome of metastatic ACC following trilostane mono therapy for >1 year.

Evaluation of plasma prealbumin as a novel inflammatory biomarker in dogs: a pilot study.

Introduction: Prealbumin (PAB) is a plasma protein synthesized in the hepatic parenchymal cells. PAB has a short half-life (~2 days), and its concentration is affected by changes in transcapillary escape. Measurement of PAB is widely used in hospitalized patients in human medicine due to its decreasing concentration in states of inflammation and malnutrition. However, only a few studies are available in dogs. The aim of this study is to determine whether the plasma PAB concentration decreases in dogs with inflammation and to evaluate the relationship between the plasma PAB concentration and inflammation-related parameters in dogs. Methods: A total of 94 dogs were divided into healthy (n = 33) and diseased (n = 61) groups. These were further divided into group A (n = 24) and group B (n = 37) according to plasma C-reactive protein (CRP) levels. Group A included dogs with a plasma CRP < 10 mg/L, and group B consisted of dogs with a plasma CRP ≥ 10 mg/L. Patient signalment, history, physical examination findings, hematologic and biochemical parameters, various inflammatory markers, and plasma PAB levels were investigated and compared between groups. Results: The plasma PAB concentration was found to be lower in group B than in the other groups (p < 0.001), but no statistical difference was found when comparing the control group and group A (p > 0.05). A plasma PAB < 6.3 mg/dL predicted an increased CRP level (10 mg/L or greater) with a sensitivity of 89.5% and a specificity of 86.5%. Receiver operating characteristic curve analysis revealed that the area under the curve for PAB was higher than that for the white blood cell count, neutrophil count, albumin level, lactate level, neutrophil-to-lymphocyte ratio, and neutrophil percentage-to-albumin ratio. In addition, the PAB concentration was significantly negatively correlated with the CRP concentration (r = -0.670, p < 0.001). Conclusion: In conclusion, this is the first study to demonstrate the clinical usefulness of the plasma PAB concentration as an inflammatory marker in dogs. These findings suggest that measuring the plasma PAB concentration along with the CRP concentration may be more useful for evaluating inflammation than measuring CRP alone in canine patients.

Digital microfluidics-like manipulation of electrokinetically preconcentrated bioparticle plugs in continuous-flow.

Herein, we demonstrate digital microfluidics-like manipulations of preconcentrated biomolecule plugs within a continuous flow that is different from the commonly known digital microfluidics involving discrete (i.e. droplets) media. This is realized using one- and two-dimensional arrays of individually addressable ion-permselective membranes with interconnecting microfluidic channels. The location of powered electrodes, dictates which of the membranes are active and generates either enrichment/depletion diffusion layers, which, in turn, control the location of the preconcentrated plug. An array of such powered membranes enables formation of multiple preconcentrated plugs of the same biosample as well as of preconcentrated plugs of multiple biosample types introduced via different inlets in a selective manner. Moreover, digital-microfluidics operations such as up-down and left-right translation, merging, and splitting, can be realized, but on preconcentrated biomolecule plugs instead of on discrete droplets. This technology, based on nanoscale electrokinetics of ion transport through permselective medium, opens future opportunities for smart and programmable digital-like manipulations of preconcentrated biological particle plugs for various on-chip biological applications.

Dielectrophoretic Characterization of Dynamic Microcapsules and Their Magnetophoretic Manipulation.

In this work, we present dielectrophoresis (DEP) and in situ electrorotation (ROT) characterization of reversibly stimuli-responsive "dynamic" microcapsules that change the physicochemical properties of their shells under varying pH conditions and can encapsulate and release (macro)molecular cargo on demand. Specifically, these capsules are engineered to open (close) their shell under high (low) pH conditions and thus to release (retain) their encapsulated load or to capture and trap (macro)molecular samples from their environment. We show that the steady-state DEP and ROT spectra of these capsules can be modeled using a single-shell model and that the conductivity of their shells is influenced most by the pH. Furthermore, we measured the transient response of the angular velocity of the capsules under rotating electric field conditions, which allows us to directly determine the characteristic time scales of the underlying physical processes. In addition, we demonstrate the magnetic manipulation of microcapsules with embedded magnetic nanoparticles for lab-on-chip tasks such as encapsulation and release at designated locations and the in situ determination of their physicochemical state using on-chip ROT. The insight gained will enable the advanced design and operation of these dynamic drug delivery and smart lab-on-chip transport systems.

Enhanced cargo loading of electrically powered metallo-dielectric pollen bearing multiple dielectrophoretic traps.

The use of active particles for cargo transport offers unique potential for applications ranging from targeted drug delivery to lab-on-a-particle systems. Previously, deployment of metallo-dielectric Janus spherical particles (JPs) as mobile microelectrodes for transport and dielectrophoretic manipulation of cargo has been shown to be singularly controlled via an applied electric field. Herein, we extended this to a metallo-dielectric pollen featuring multiple dielectrophoretic traps associated with its many spikes, and characterized its loading capacities for various cargo sizes and frequencies. When compared to spherical JPs, the active pollen exhibited a significantly enhanced cargo loading capacity due to its multiple dielectrophoretic traps. These findings open new opportunities for application of bio-hybrid particles with diverse and irregular shapes, such as pollen, as efficient cargo carriers, local electroporation and targeted drug delivery.

Micromotor-Based Biosensing Using Directed Transport of Functionalized Beads.

Previous micromotor-based biosensing studies used to functionalize the surface of the micromotor with specific molecular probes for binding of target analyte, thus limiting the use of the micromotor for the specific target. In contrast, here, we introduce a novel approach of using a nonfunctionalized micromotor as a generic cargo carrier being able to perform label-free and dynamic loading, transport, and release of functionalized beads. Hence, such an approach enables one to use the same micromotor system for sensing of varying targets via different commercially available functionalized beads, demonstrating the use of micromotors as a practical and versatile means for biosensing. We have also introduced a simplified microfluidic design that can be used for immunosensing or DNA binding tests without necessity for complicated fluid handling (buffer exchange, washing, etc.) steps. We expect this approach to open up new realizations of simplified and generic biosensing platforms.

Periodic concentration-polarization-based formation of a biomolecule preconcentrate for enhanced biosensing.

Ionic concentration-polarization (CP)-based biomolecule preconcentration is an established method for enhancing the detection sensitivity of target biomolecules. However, the formed preconcentrated biomolecule plug rapidly sweeps over the surface-immobilized antibodies, resulting in a short-term overlap between the capture agent and the analyte, and subsequently suboptimal binding. To overcome this, we designed a setup allowing for the periodic formation of a preconcentrated biomolecule plug by activating the CP for predetermined on/off intervals. This work demonstrated the feasibility of cyclic CP actuation and optimized the sweeping conditions required to obtain the maximum retention time of a preconcentrated plug over a desired sensing region and enhanced detection sensitivity. The ability of this method to efficiently preconcentrate different analytes and to successfully increase immunoassay sensitivity underscore its potential in immunoassays serving the clinical and food testing industries.

Combining dielectrophoresis and concentration polarization-based preconcentration to enhance bead-based immunoassay sensitivity.

Ionic concentration-polarization (CP)-based biomolecule preconcentration is an established method for enhancing the detection sensitivity of a target biomolecule immunoassay. However, its main drawback lies in its inability to directly control the spatial overlap between the preconcentrated plug of biomolecules and the surface immobilized antibodies. To overcome this, we simultaneously preconcentrated freely suspended, surface functionalized nanoparticles and target molecules along the edge of a depletion layer, thus, increasing the binding kinetics and avoiding the need to tune their relative locations to ensure their spatial overlap. After the desired incubation time, the nanoparticles were dielectrophoretically trapped for postprocessing analysis of the binding signal. This novel combination of CP-based preconcentration and dielectrophoresis (DEP) was demonstrated through binding of avidin and biotin-conjugated particles as a model bead-based immunoassay, wherein increased detection sensitivity was demonstrated compared to an immunoassay without CP-based preconcentration. The DEP trapping of the beads following binding is important not only for an enhanced detection signal due to the preconcentration of the beads at the electrode edges but also for controlling their location for future applications integrating localized sensors. In addition, DEP may be important also as a preprocessing step for controlling the number of beads participating in the immunoassay.

Novel Electrochemical Flow Sensor Based on Sensing the Convective-Diffusive Ionic Concentration Layer.

Presented is a novel flow sensor based on electrochemical sensing of the ionic concentration-polarization (CP) layer developed within a microchannel-ion permselective membrane device. To demonstrate the working principle of the electrochemical flow sensor, the effect of advection on the transient and steady-state ionic concentration-polarization (CP) phenomenon in microchannel-Nafion membrane systems is studied. In particular, we focused on the local impedance, measured using an array of electrode pairs embedded at the bottom of the microchannel, as well as the total current across the permselective medium, as two approaches for estimating the flow. We examined both a stepwise application of CP under steady-state flow and a stepwise application of flow under steady-state CP.