Publications

This manuscript presents portable and low cost electronic system for specific point-of-use dielectrophoresis applications. The system is composed of two main modules: a) a multiphase generator based on a Class E amplifier, which provides 4 sinusoidal signals (0°, 90°, 180°, 270°) at 1 MHz with variable output voltage up to 10 Vpp (Vm) and an output driving current of 1 A; and b) a dielectrophoresis-based microfluidic chip containing two interdigitated electrodes. The system has been validated by concentrating Escherichia Coli at 1 MHz while applying a continuous flow of 5 ?L/min. Device functionalities were verified under different conditions achieving a 83% trapping efficiency in the best case. © Springer International Publishing Switzerland 2014.

JTD Keywords: bacteria, emulsifier, enterobacter, exopolysaccharide, Bacteria, Bacteria concentrations, Biochemical engineering, Cell concentrator, Class e amplifier, Class-e amplifier, Device functionality, Dielectrophoresis, Electronic equipment, Electronics, Electrophoresis, Emulsifier, Enterobacter, Escherichia coli, Exopolysaccharide, Inter-digitated electrodes, Lab-on-a-chip (loc), Low cost, Low costs, Low-cost electronics, Medical computing, Monosaccharide, Portable device, Power amplifiers, Trapping efficiencies

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting

The motion of nanomotors triggered by light sources will provide new alternative routes to power nanoarchitectures without the need of chemical fuels. However, most light-driven nanomotors are triggered by UV-light, near infrared reflection, or laser sources. It is demonstrated that nanocap shaped Au/TiO2 nanomotors (175 nm in diameter) display increased Brownian motion in the presence of broad spectrum visible light. The motion results from the surface plasmon resonance effect leading to self-electrophoresis between the Au and TiO2 layers, a mechanism called plasmonic photocatalytic effect in the field of photocatalysis. This mechanism is experimentally characterized by electron energy loss spectroscopy, energy-filtered transmission electron microscopy, and optical video tracking. This mechanism is also studied in a more theoretical manner using numerical finite-difference time-domain simulations. The ability to power nanomaterials with visible light may result in entirely new applications for externally powered micro/nanomotors.

JTD Keywords: Enhanced Brownian motion, Fuel-free nanomotors, Nanomachines, Self-electrophoresis, Visible light

Background: Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Methods: Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Results: Histones have a high affinity for ssDNA in 0.15 M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (−) strand. Conclusions: At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. General significance: In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin.

JTD Keywords: Electrophoresis, Force spectroscopy, Histones, Magnetic tweezers, Nucleosome, Single-stranded DNA

Nanogap electrodes have attracted a lot of consideration as promising platform for molecular electronic and biomolecules detection. This is mainly for their higher aspect ratio, and because their electrical properties are easily accessed by current-voltage measurements. Nevertheless, application of standard current-voltages measurements used to characterize nanogap response, and/or to modify specific nanogap electrodes properties, represents an issue. Since the strength of electrical fields in nanoscaled devices can reach high values, even at low voltages. Here, we analyzed the effects induced by different methods of surface modification of nanogap electrodes, in test-voltage application, employed for the electrical detection of a desoxyribonucleic acid (DNA) target. Nanogap electrodes were functionalized with two antisymmetric oligo-probes designed to have 20 terminal bases complementary to the edges of the target, which after hybridization bridges the nanogap, closing the electrical circuit. Two methods of functionalization were studied for this purpose; a random self-assembling of a mixture of the two oligo-probes (OPs) used in the platform, and a selective method that controls the position of each OP at selected side of nanogap electrodes. We used for this aim, the electrophoretic effect induced on negatively charged probes by the application of an external direct current voltage. The results obtained with both functionalization methods where characterized and compared in terms of electrode surface covering, calculated by using voltammetry analysis. Moreover, we contrasted the electrical detection of a DNA target in the nanogap platform either in site-selective and in randomly assembled nanogap. According to our results, a denser, although not selective surface functionalization, is advantageous for such kind of applications.

JTD Keywords: Biosensor bioelectronics, DNA electrophoresis, Nanogap electrodes, Self-assembled monolayers, Site-selective deposition

Bacteria concentration and detection is time-consuming in regular microbiology procedures aimed to facilitate the detection and analysis of these cells at very low concentrations. Traditional methods are effective but often require several days to complete. This scenario results in low bioanalytical and diagnostic methodologies with associated increased costs and complexity. In recent years, the exploitation of the intrinsic electrical properties of cells has emerged as an appealing alternative approach for concentrating and detecting bacteria. The combination of dielectrophoresis (DEP) and impedance analysis (IA) in microfluidic on-chip platforms could be key to develop rapid, accurate, portable, simple-to-use and cost-effective microfluidic devices with a promising impact in medicine, public health, agricultural, food control and environmental areas. The present document reviews recent DEP and IA combined approaches and the latest relevant improvements focusing on bacteria concentration and detection, including selectivity, sensitivity, detection time, and conductivity variation enhancements. Furthermore, this review analyses future trends and challenges which need to be addressed in order to successfully commercialize these platforms resulting in an adequate social return of public-funded investments.

JTD Keywords: Bacteria, Dielectrophoresis, Impedance, Microfluidics, On-chip

The present paper reports a bacteria autonomous controlled concentrator prototype with a user-friendly interface for bench-top applications. It is based on a micro-fluidic lab-on-a-chip and its associated custom instrumentation, which consists in a dielectrophoretic actuator, to pre-concentrate the sample, and an impedance analyser, to measure concentrated bacteria levels. The system is composed by a single micro-fluidic chamber with interdigitated electrodes and a instrumentation with custom electronics. The prototype is supported by a real-time platform connected to a remote computer, which automatically controls the system and displays impedance data used to monitor the status of bacteria accumulation on-chip. The system automates the whole concentrating operation. Performance has been studied for controlled volumes of Escherichia coli (E. coli) samples injected into the micro-fluidic chip at constant flow rate of 10 μL/min. A media conductivity correcting protocol has been developed, as the preliminary results showed distortion of the impedance analyser measurement produced by bacterial media conductivity variations through time. With the correcting protocol, the measured impedance values were related to the quantity of bacteria concentrated with a correlation of 0.988 and a coefficient of variation of 3.1%. Feasibility of E. coli on-chip automated concentration, using the miniaturized system, has been demonstrated. Furthermore, the impedance monitoring protocol had been adjusted and optimized, to handle changes in the electrical properties of the bacteria media over time.

JTD Keywords: Autonomous Device, Bacteria Concentrator, Dielectrophoresis, Escherichia coli, Impedance Analysis

DEP manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed and recovered in a small-volume fraction which, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.

JTD Keywords: Dielectrophoresis, PCR, Saccharomyces, Yeast

We describe a novel continuous-flow cell concentrator micro-device based on dielectrophoresis (DEP), and its associated custom-made control unit. The performances of a classical interdigitated metal electrode-based DEP microfluidic device and this enhanced version, that includes insulator-based pole structures, were compared using the same setup. Escherichia coli (E. coli) samples were concentrated at several continuous flows and the device's trapping efficiencies were evaluated by exhaustive cell counts. Our results show that pole structures enhance the retention up to 12.6%, obtaining significant differences for flow rates up to 20 μl/min, when compared to an equivalent classical interdigitated electrodes setup. In addition, we performed a subsequent proteomic analysis to evaluate the viability of the biological samples after the long exposure to the actuating electrical field. No E. coli protein alteration in any of the two systems was observed.

JTD Keywords: Concentrator, Dielectrophoresis, Escherichia coli, Lab- on- a- chip

This manuscript presents a portable and low cost electronic system for specific point-of-use dielectrophoresis applications. The system is composed of two main modules: a) a multiphase generator based on a Class E amplifier, which provides 4 sinusoidal signals (0°, 90°, 180°, 270°) at 1 MHz with variable output voltage up to 10 Vpp (Vm) and an output driving current of 1 A; and b) a dielectrophoresis-based microfluidic chip containing two interdigitated electrodes. The system has been validated by concentrating Escherichia coli (E. coli) at 1 MHz while applying a continuous flow of 5 µL/min. The device functionalities were verified under different conditions, achieving an 83% trapping efficiency when counter-phased signals are used.

JTD Keywords: Cell Concentrator, Class E amplifier, Dielectrophoresis, Electronics, Lab-on-a-chip (LOC), Low cost, Portable device

This manuscript presents portable and low cost electronic system for specific point-of-use dielectrophoresis applications. The system is composed of two main modules: a) a multiphase generator based on a Class E amplifier, which provides 4 sinusoidal signals (0º, 90º, 180º, 270º) at 1 MHz with variable output voltage up to 10 Vpp (Vm) and an output driving current of 1 A; and b) a dielectrophoresis-based microfluidic chip containing two interdigitated electrodes. The system has been validated by concentrating Escherichia Coli at 1 MHz while applying a continuous flow of 5 μL/min. Device functionalities were verified under different conditions achieving a 83% trapping efficiency in the best case.

JTD Keywords: Cell Concentrator, Class E amplifier, Dielectrophoresis, Electronics, Lab-on-a-chip (LOC), Low cost, Portable device

Dielectrophoresis (DEP) represents a powerful approach to manipulate and study living cells. Hitherto, several approaches have used 2-D DEP chips. With the aim to increase sample volume, in this study we used a 3-D carbon-electrode DEP chip to trap and release bacterial cells. A continuous flow was used to plug an Escherichia coli cell suspension first, to retain cells by positive DEP, and thereafter to recover them by washing with peptone water washing solution. This approach allows one not only to analyze DEP behavior of living cells within the chip, but also to further recover fractions containing DEP-trapped cells. Bacterial concentration and flow rate appeared as critical parameters influencing the separation capacity of the chip. Evidence is presented demonstrating that the setup developed in this study can be used to separate different types of bacterial cells.

JTD Keywords: Bacteria, Carbon electrode, Dielectrophoresis, E. coli, Separation

The H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NST(EPEC)). Y. enterocolitica cells expressing H-NST(EPEC) showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NST(EPEC) expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.

JTD Keywords: Bacterial Proteins/biosynthesis/genetics/ physiology, DNA-Binding Proteins/biosynthesis/genetics/ physiology, Electrophoresis, Gel, Two-Dimensional, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Essential, Proteome/analysis, RNA, Bacterial/biosynthesis, RNA, Messenger/biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Sequence Deletion, Yersinia enterocolitica/chemistry/genetics/growth & development/ physiology