Rapid, sensitive and real-time multiplexing platform for the analysis of protein and nucleic-acid biomarkers.

We describe a multiplexing technology, named Evalution, based on novel digitally encoded microparticles in microfluidic channels. Quantitative multiplexing is becoming increasingly important for research and routine clinical diagnostics, but fast, easy-to-use, flexible and highly reproducible technologies are needed to leverage the advantages of multiplexing. The presented technology has been tailored to ensure (i) short assay times and high reproducibility thanks to reaction-limited binding regime, (ii) dynamic control of assay conditions and real-time binding monitoring allowing optimization of multiple parameters within a single assay run, (iii) compatibility with various immunoassay formats such as coflowing the samples and detection antibodies simultaneously and hence simplifying workflows, (iv) analyte quantification based on initial binding rates leading to increased system dynamic range and (v) high sensitivity via enhanced fluorescence collection. These key features are demonstrated with assays for proteins and nucleic acids showing the versatility of this technology.

Continuous-flow electrical lysis device with integrated control by dielectrophoretic cell sorting.

We present a device capable of electrical cell lysis and evaluation of lysis efficiency in continuous flow using dielectrophoretic cell sorting. We use a combination of AC electrical fields and so-called liquid electrodes to avoid bubble creation at the electrode surface. The electrical field distribution is calculated in different electrode configurations by numerical simulations. Cell sorting shows high lysis efficiency, 99% of yeast cells sorted after lysis featuring dielectric properties similar to dead cells. A study of the potential device throughput is performed.

Continuous separation of cells by balanced dielectrophoretic forces at multiple frequencies.

We present a particle-sorting device based on the opposition of dielectrophoretic forces. The forces are generated by an array of electrode chambers located in both sidewalls of a main flow channel. Particles with different dielectric response perceive different force magnitudes and are therefore continuously focused to different streamlines in the flow channel. We relate the particles' dielectric response to their output position in the downstream channel. We demonstrate the performance of the device by separating a mixed yeast cell population into pure fractions of viable and nonviable cells. Finally, we use the device to enrich red blood cells infected with Babesia bovis, a major pathogen in cattle and simultaneously confirm the hypothesis that infection with B. bovis causes significant changes in the dielectric response of red blood cells.

Dielectrophoresis-based particle exchanger for the manipulation and surface functionalization of particles.

We present a microfluidic device where micro- and nanoparticles can be continuously functionalized in flow. This device relies on an element called "particle exchanger", which allows for particles to be taken from one medium and exposed to some reagent while minimizing mixing of the two liquids. In the exchanger, two liquids are brought in contact and particles are pushed from one to the other by the application of a dielectrophoretic force. We determined the maximum flow velocity at which all the particles are exchanged for a range of particle sizes. We also present a simple theory that accounts for the behaviour of the device when the particle size is scaled. Diffusion mixing in the exchanger is also evaluated. Finally, we demonstrate particle functionalization within the microfluidic device by coupling a fluorescent tag to avidin-modified 880 nm particles. The concept presented in this paper has been developed for synthesis of modified particles but is also applicable to on-chip bead-based chemistry or cellular biology.

Dielectrophoretic sorting on a microfabricated flow cytometer: label free separation of Babesia bovis infected erythrocytes.

Dielectrophoresis is a method that has demonstrated great potential in cell discrimination and isolation. In this study, the dielectrophoretic sorting of normal and Babesia bovis infected erythrocytes was performed using a microfabricated flow cytometer. Separation was possible through exploitation of the dielectric differences between normal and infected erythrocytes, essentially due to the higher ionic membrane permeability of B. bovis infected cells. Sorting experiments were performed inside a microchip made from Pt microelectrodes and SU-8 channels patterned on a glass substrate. Optimum cell separation was achieved at 4 MHz using an in vitro culture of B. bovis suspended in 63 mS/m phosphate buffer and applying a sinusoidal voltage of 15 V peak-to-peak. Normal erythrocytes experienced stronger positive dielectrophoresis (pDEP) than B. bovis infected cells, moving them closer to the microelectrodes. Under these conditions it was possible to enrich the fraction of infected cells from 7 to 50% without the need of extensive sample preparation or labelling. Throughout the experiments very few microliters of sample were used, suggesting that this system may be considered suitable for integration in a low-cost automated device to be used in the in situ diagnostic of babesiosis.

Characterization and optimization of liquid electrodes for lateral dielectrophoresis.

Using the concept of insulator-based "electrodeless" dielectrophoresis, we present a novel geometry for shaping electric fields to achieve lateral deviation of particles in liquid flows. The field is generated by lateral planar metal electrodes and is guided along access channels to the active area in the main channel. The equipotential surfaces at the apertures of the access channels behave as vertical "liquid" electrodes injecting the current into the main channel. The field between a pair of adjacent liquid electrodes generates the lateral dielectrophoretic force necessary for particle manipulation. We use this force for high-speed deviation of particles. By adding a second pair of liquid electrodes, we focus a particle stream. The position of the focused stream can be swept across the channel by adjusting the ratio of the voltages applied to the two pairs. Based on conformal mapping, we provide an analytical model for estimating the potential at the liquid electrodes and the field distribution in the main channel. We show that the simulated particle trajectories agree with observations. Finally, we show that the model can be used to optimize the device geometry in different applications.

A virtual valve for smooth contamination-free flow switching.

We present a channel geometry that allows for clean switching between different inlets of a microchip without any contamination of the inlets or the downstream flow. We drive this virtual valve with a pneumatic pressure setup that minimizes disturbance of the downstream flow during the switching procedure by simultaneous variation of the pressures applied to the different inlets. We assess the efficiency of the setup by spectroscopic measurement of downstream dye concentrations, and demonstrate its practical utility by sequentially constructing multiple layers of alginate hydrogel. The method is potentially useful for a whole series of further applications, such as changing perfusion liquids for cell culture and cell analysis, metering, chemical-reaction initiation and multi-sample chromatography, to name a few.

Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer.

Impedance spectroscopy is a powerful tool for label-free analysis and characterisation of living cells. In this work, we achieved the detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer. The cellular modifications caused by the intracellular parasite result in a shift in impedance which can be measured dielectrically. Thus, a rapid cell-by-cell detection with microliter amounts of reagents is possible. Unlike other diagnostic tests, this method does not depend on extensive sample pre-treatment or expensive chemicals and equipment.

Tracking and synchronization of the yeast cell cycle using dielectrophoretic opacity.

Cell cycle synchronization is an important tool for the study of the cell division stages and signalling. It provides homogeneous cell cultures that are of importance to develop and improve processes such as protein synthesis and drug screening. The main approach today is the use of metabolic agents that block the cell cycle at a particular phase and accumulate cells at this phase, disturbing the cell physiology. We provide here a non-invasive and label-free continuous cell sorting technique to analyze and synchronize yeast cell division. By balancing opposing dielectrophoretic forces at multiple frequencies, we maximize sensitivity to the characteristic shape and internal structure changes occurring during the yeast cell cycle, allowing us to synchronize the culture in late anaphase.

A miniaturized continuous dielectrophoretic cell sorter and its applications.

There is great interest in highly sensitive separation methods capable of quickly isolating a particular cell type within a single manipulation step prior to their analysis. We present a cell sorting device based on the opposition of dielectrophoretic forces that discriminates between cell types according to their dielectric properties, such as the membrane permittivity and the cytoplasm conductivity. The forces are generated by an array of electrodes located in both sidewalls of a main flow channel. Cells with different dielectric responses perceive different force magnitudes and are, therefore, continuously focused to different equilibrium positions in the flow channel, thus avoiding the need of a specific cell labeling as discriminating factor. We relate the cells' dielectric response to their output position in the downstream channel. Using this microfluidic platform that integrates a method of continuous-flow cell separation based on multiple frequency dielectrophoresis, we succeeded in sorting viable from nonviable yeast with nearly 100% purity. The method also allowed to increase the infection rate of a cell culture up to 50% of parasitemia percentage, which facilitates the study of the parasite cycle. Finally, we prove the versatility of our device by synchronizing a yeast cell culture at a particular phase of the cell cycle avoiding the use of metabolic agents interfering with the cells' physiology.