A miniaturised semi-dynamic in-vitro model of human digestion.

Reliable in-vitro digestion models that are able to successfully replicate the conditions found in the human gastrointestinal tract are key to assess the fate and efficiency of new formulations aimed for oral consumption. However, current in-vitro models either lack the capability to replicate crucial dynamics of digestion or require large volumes of sample/reagents, which can be scarce when working with nanomaterials under development. Here, we propose a miniaturised digestion system, a digestion-chip, based on incubation chambers integrated on a polymethylmethacrylate device. The digestion-chip incorporates key dynamic features of human digestion, such as gradual acidification and gradual addition of enzymes and simulated fluids in the gastric phase, and controlled gastric emptying, while maintaining low complexity and using small volumes of sample and reagents. In addition, the new approach integrates real-time automated closed-loop control of two key parameters, pH and temperature, during the two main phases of digestion (gastric and intestinal) with an accuracy down to ± 0.1 °C and ± 0.2 pH points. The experimental results demonstrate that the digestion-chip successfully replicates the gold standard static digestion INFOGEST protocol and that the semi-dynamic digestion kinetics can be reliably fitted to a first kinetic order model. These devices can be easily adapted to dynamic features in an automated, sensorised, and inexpensive platform and will enable reliable, low-cost and efficient assessment of the bioaccessibility of new and expensive drugs, bioactive ingredients or nanoengineered materials aimed for oral consumption, thereby avoiding unnecessary animal testing.

Low-frequency electrokinetics in a periodic pillar array for particle separation.

Deterministic Lateral Displacement (DLD) exploits periodic arrays of pillars inside microfluidic channels for high-precision sorting of micro- and nano-particles. Previously we demonstrated how DLD separation can be significantly improved by the addition of AC electrokinetic forces, increasing the tunability of the technique and expanding the range of applications. At high frequencies of the electric field (>1 kHz) the behaviour of such systems is dominated by Dielectrophoresis (DEP), whereas at low frequencies the particle behaviour is much richer and more complex. In this article, we present a detailed numerical analysis of the mechanisms governing particle motion in a DLD micropillar array in the presence of a low-frequency AC electric field. We show how a combination of Electrophoresis (EP) and Concentration-Polarisation Electroosmosis (CPEO) driven wall-particle repulsion account for the observed experimental behaviour of particles, and demonstrate how this complete model can predict conditions that lead to electrically induced deviation of particles much smaller than the critical size of the DLD array.

Insulating traveling-wave electrophoresis.

Traveling-wave electrophoresis (TWE) is a method for transporting charged colloidal particles used in many microfluidic techniques for particle manipulation and fractionation. This method exploits the traveling-wave components of the electric field generated by an array of electrodes subjected to ac voltages with a phase delay between neighboring electrodes. In this article, we propose an alternative way of generating traveling-wave electric fields in microchannels. We apply a rotating electric field around a cylindrical insulating micropillar and the resulting traveling-wave modes induce particle drift around the cylinder. We term this phenomenon insulating traveling-wave electrophoresis (i-TWE) to distinguish it from standard TWE performed with arrays of microelectrodes. We characterized the particle drift experimentally and show a quantitative comparison of the particle velocity with theoretical predictions. Excellent agreement is found when the influence of electro-osmosis on the channel walls is also considered.

From mouth to gut: microfluidic in vitro simulation of human gastro-intestinal digestion and intestinal permeability.

Reproducible in vitro studies of bioaccessibility, intestinal absorption, and bioavailability are key to the successful development of novel food ingredients or drugs intended for oral administration. There is currently a lack of methods that offer the finesse required to study these parameters for valuable molecules typically found in small volumes - as is the case of nanomaterials, which are often used to carry and protect bioactives. Here, we describe a modular microfluidic-based platform for total simulation of the human gastro-intestinal tract. Digestion-chips and cell-based gut-chips were fabricated from PDMS by soft lithography. On-chip digestion was validated using a fluorescently labelled casein derivative, which followed typical Michaelis-Menten kinetics and showed temporal resolution and good agreement with well-established bench-top protocols. Irreversible inhibition of serine proteases using Pefabloc® SC and a 1 : 6 dilution was sufficient to mitigate the cytotoxicity of simulated digestion fluids. Caco-2/HT29-MTX co-cultures were grown on-chip under a continuous flow for 7 days to obtain a differentiated cell monolayer forming a 3D villi-like epithelium with clear tight junction formation, and with an apparent permeability (Papp) of Lucifer Yellow closely approximating values reported ex vivo (3.7 × 10-6 ± 1.4 × 10-6vs. 4.0 × 10-6 ± 2.2 × 10-6). Digesta from the digestion-chips were flowed through the gut-chip, demonstrating the capacity to study sample digestion and intestinal permeability in a single microfluidic platform holding great promise for use in pharmacokinetic studies.

Electrokinetic deterministic lateral displacement for fractionation of vesicles and nano-particles.

We describe fractionation of sub-micron vesicles and particles suspended in high conductivity electrolytes using an electrokinetically biased Deterministic Lateral Displacement (DLD) device. An optimised, asymmetric array of micron-sized pillars and gaps, with an AC electric field applied orthogonal to the fluid flow gives an approximately ten-fold reduction in the intrinsic critical diameter (Dc) of the device. The asymmetry in the device maximises the throughput. Fractionation of populations of 100 nm and 400 nm extruded vesicles is achieved in 690 mS m-1 KCl, and 100 nm, 200 nm and 500 nm polystyrene particles in 105 mS m-1 KCl. The electrokinetically biased DLD may provide solutions for simple and rapid isolation of extracellular vesicles.

Partitioning of Small Hydrophobic Molecules into Polydimethylsiloxane in Microfluidic Analytical Devices.

Polydimethylsiloxane (PDMS) is ubiquitously used in microfluidics. However, PDMS is porous and hydrophobic, potentially leading to small molecule partitioning. Although many studies addressed this issue and suggested surface/bulk modifications to overcome it, most were not quantitative, did not address which variables besides hydrophobicity governed molecule absorption, and no modification has been shown to completely obviate it. We evaluated qualitatively (confocal microscopy) and quantitatively (fluorescence spectroscopy) the effects of solute/solvent pairings, concentration, and residence time on molecule partitioning into PDMS. Additionally, we tested previously reported surface/bulk modifications, aiming to determine whether reduced PDMS hydrophobicity was stable and hindered molecule partitioning. Partitioning was more significant at lower concentrations, with the relative concentration of rhodamine-B at 20 µM remaining around 90% vs. 10% at 1 µM. Solute/solvent pairings were demonstrated to be determinant by the dramatically higher partitioning of Nile-red in a PBS-based solvent as opposed to ethanol. A paraffin coating slightly decreased the partitioning of Nile-red, and a sol-gel modification hindered the rhodamine-B diffusion into the PDMS bulk. However, there was no direct correlation between reduced surface hydrophobicity and molecule partitioning. This work highlighted the need for pre-assessing the absorption of test molecules into the microfluidic substrates and considering alternative materials for fabrication.

Wall Repulsion of Charged Colloidal Particles during Electrophoresis in Microfluidic Channels.

Electrophoresis describes the motion of charged particles suspended in electrolytes when subjected to an external electric field. Previous experiments have shown that particles undergoing electrophoresis are repelled from nearby channel walls, contrary to the standard description of electrophoresis that predicts no hydrodynamic repulsion. Dielectrophoretic (DEP) repulsive forces have been commonly invoked as the cause of this wall repulsion. We show that DEP forces can only account for this wall repulsion at high frequencies of applied electric field. In the presence of a low-frequency field, quadrupolar electro-osmotic flows are observed around the particles. We experimentally demonstrate that these hydrodynamic flows are the cause of the widely observed particle-wall interaction. This hydrodynamic wall repulsion should be considered in the design and application of electric-field-driven manipulation of particles in microfluidic devices.

Short communication: A simple and accurate method of measuring the zeta-potential of microfluidic channels.

We describe an improved method for determining the electroosmotic mobility and zeta potential of surfaces based on a current-monitoring method. This technique eliminates the requirement for measurements of channel dimensions and sample conductivities, leading to a simple high precision measurement. The zeta potential of PDMS is measured for native surfaces and surfaces treated with a nonionic surfactant in low-conductivity electrolytes.

Concentration-Polarization Electroosmosis near Insulating Constrictions within Microfluidic Channels.

Electric fields are commonly used to trap and separate micro- and nanoparticles near channel constrictions in microfluidic devices. The trapping mechanism is attributed to the electrical forces arising from the nonhomogeneous electric field caused by the constrictions, and the phenomenon is known as insulator-based-dielectrophoresis (iDEP). In this paper, we describe stationary electroosmotic flows of electrolytes around insulating constrictions induced by low frequency AC electric fields (below 10 kHz). Experimental characterization of the flows is described for two different channel heights (50 and 10 µm), together with numerical simulations based on an electrokinetic model that considers the modification of the local ionic concentration due to surface conductance on charged insulating walls. We term this phenomenon concentration-polarization electroosmosis (CPEO). The observed flow characteristics are in qualitative agreement with the predictions of this model. However, for shallow channels (10 µm), trapping of the particles on both sides of the constrictions is also observed. This particle and fluid behavior could play a major role in iDEP and could be easily misinterpreted as a dielectrophoretic force.

Electrokinetic biased deterministic lateral displacement: scaling analysis and simulations.

Deterministic Lateral Displacement (DLD) is a microfluidic technique where arrays of micropillars within a microchannel deflect particles leading to size-based segregation. We recently demonstrated that applying AC electric fields orthogonal to the fluid flow increases the separation capabilities of these devices with a deflection angle that depends on the electric field magnitude and frequency. Particle deviation occurs in two distinct regimes depending on frequency. At high frequencies particles deviate due to negative dielectrophoresis (DEP). At low frequencies (below 1 kHz) particles oscillate perpendicular to the flow direction due to electrophoresis and are also deflected within the device. Significantly, the threshold electric field magnitude for the low frequency deviation is much lower than for deflection at high frequencies by DEP. In order to characterize the enhanced separation at low frequencies, the induced deviation was compared between the two frequency ranges. For high frequencies, we develop both theoretically and experimentally scaling laws for the dependence of particle deviation on several parameters, namely the amplitude of the applied voltage, particle size and liquid velocity where DEP forces compete with viscous drag. A novel theoretical framework is presented that enables simulation of particle trajectories subjected to DEP forces in DLD devices. Deviation angles predicted by simulations are in very good agreement with experimental data. At low frequencies (below 1 kHz), particles follow the same scaling law, but with much lower voltages. This indicates that electrokinetic phenomena other than DEP play an important role in driving particle behaviour. Experiments show that at low frequencies, particle motion is affected by quadrupolar electrohydrodynamic flows around the insulating pillars of the DLD array. We quantify the difference between the two frequency regimes and show that an electrokinetic model based only on DEP forces is limited to frequencies of 1 kHz and above.

Combining DC and AC electric fields with deterministic lateral displacement for micro- and nano-particle separation.

This paper describes the behavior of particles in a deterministic lateral displacement (DLD) separation device with DC and AC electric fields applied orthogonal to the fluid flow. As proof of principle, we demonstrate tunable microparticle and nanoparticle separation and fractionation depending on both particle size and zeta potential. DLD is a microfluidic technique that performs size-based binary separation of particles in a continuous flow. Here, we explore how the application of both DC and AC electric fields (separate or together) can be used to improve separation in a DLD device. We show that particles significantly smaller than the critical diameter of the device can be efficiently separated by applying orthogonal electric fields. Following the application of a DC voltage, Faradaic processes at the electrodes cause local changes in medium conductivity. This conductivity change creates an electric field gradient across the channel that results in a nonuniform electrophoretic velocity orthogonal to the primary flow direction. This phenomenon causes particles to focus on tight bands as they flow along the channel countering the effect of particle diffusion. It is shown that the final lateral displacement of particles depends on both particle size and zeta potential. Experiments with six different types of negatively charged particles and five different sizes (from 100 nm to 3 µm) and different zeta potential demonstrate how a DC electric field combined with AC electric fields (that causes negative-dielectrophoresis particle deviation) could be used for fractionation of particles on the nanoscale in microscale devices.

AC electrokinetic biased deterministic lateral displacement for tunable particle separation.

We describe a novel particle separation technique that combines deterministic lateral displacement (DLD) with orthogonal electrokinetic forces. DLD is a microfluidic technique for continuous flow particle separation based on size. We describe new tunable devices that use a combination of AC electric fields with DLD to separate particles below the critical diameter. Planar electrodes were integrated into a classical DLD device to produce a force orthogonal to the fluid flow direction. Experiments with 3.0 µm, 1.0 µm and 500 nm diameter microspheres show that at low frequencies (up to 500 Hz) particles oscillate in the direction of the field due to electrophoretic (EP)/electroosmotic (EO) forces. As the frequency of the field increases, the amplitude of these oscillations vanishes and, eventually dielectrophoresis (DEP) becomes the dominant electrokinetic force on the particles (DEP arises from electric field inhomogeneities caused by the presence of the DLD posts). Both mechanisms alter the paths of the particles inside the DLD devices leading to enhanced sorting of particles below the critical diameter of the device.

[Kennedy disease in Peru: first cases with molecular diagnosis]. / Enfermedad de Kennedy en el Perú: primeros casos con diagnóstico molecular.

Kennedy's disease is an X-linked recessive disorder with onset in adulthood, characterized by progressive degeneration of spinal motor neurons due to a dynamic mutation in the androgen receptor gene. We report three families (five cases) characterized by progressive weakness involving both limbs and bulbar muscles, atrophy, tremor, cramps and endocrinologic disturbances; the neurophysiological studies demonstrated second motor neuron impairment. The molecular analysis identified abnormal CAG repeats expansion in the androgen receptor gene (AR) in all cases. Clinical features were consistent with other previous reports. These are the first Peruvian cases of Kennedy's disease with confirmed molecular diagnosis.

Enfermedad de Kennedy en el Perú: Primeros casos con diagnóstico molecular / Kennedy disease in Peru: First cases with molecular diagnosis

La enfermedad de Kennedy es un trastorno neurodegenerativo de herencia recesiva ligada al cromosoma X, de inicio en la adultez, caracterizado por degeneración progresiva de las neuronas motoras espinales, debido a una mutación dinámica del gen del receptor de andrógeno. Se presentan tres familias (cinco casos) con temblor, calambres, debilidad muscular generalizada lentamente progresiva con atrofia, afectación de músculos bulbares y alteraciones endocrinas. El estudio neurofisiológico demostró compromiso de segunda motoneurona. El análisis molecular mostró una expansión anormal de tripletes citosina-adenina-guanina en el gen de receptor de andrógeno en todos los casos. Todos los pacientes cursaron con una presentación clínica típica de la enfermedad siendo los primeros casos de enfermedad de Kennedy con diagnóstico molecular realizado en el Perú.

Kennedy’s disease is an X-linked recessive disorder with onset in adulthood, characterized by progressive degeneration of spinal motor neurons due to a dynamic mutation in the androgen receptor gene. We report three families (five cases) characterized by progressive weakness involving both limbs and bulbar muscles, atrophy, tremor, cramps and endocrinologic disturbances; the neurophysiological studies demonstrated second motor neuron impairment. The molecular analysis identified abnormal CAG repeats expansion in the androgen receptor gene (AR) in all cases. Clinical features were consistent with other previous reports. These are the first Peruvian cases of Kennedy´s disease with confirmed molecular diagnosis.

Fibrilación auricular silente en pacientes con infarto cerebral agudo en el Departamento de Emergencia del Instituto Nacional de Ciencias Neurológicas Diciembre 2012 - Junio 2013 / Silent atrial fibrillation in patients with acute cerebral infarction in the Emergency Department at the National Institute of Neurological Sciences December 2012 - June 2013

INTRODUCCION: La fibrilación auricular (FA) es la causa principal en los infartos cerebrales cardioembólicos, sin embargo, se ha encontrado fibrilación auricular silente asociada a otros tipos de infarto cerebral, encontrándose hasta en 15 por ciento de infartos cerebrales lacunares. OBJETIVOS: Describir la frecuencia de fibrilación auricular en pacientes con infarto cerebral agudo, así como sus características clínicas e imagenológicas. METODOS: Estudio observacional, descriptivo realizado en pacientes admitidos en el Servicio de Emergencia del Instituto Nacional de Ciencias Neurológicas (INCN), con primer evento clínico de infarto cerebral agudo (7 puntos (p=0.047). El territorio vascular anterior izquierdo tiende a ser el más afectado entre los pacientes con FA (p=0.053). No se encontraron diferencias entre los grupos respecto a género, antecedentes cardiovasculares y extensión del infarto cerebral. CONCLUSIONES: La frecuencia de fibrilación auricular fue 23.1 por ciento, la edad >75 años y un puntaje en la escala NIHSS >7 puntos están asociados con presencia de FA en infarto cerebral agudo, en la población estudiada.