pH-gradient chromatofocusing of proteins on a chip

We present a novel microfluidic system for the pH-gradient focusing of proteins with the integration of 16 parallel micro-mixers, a micro-column, and a multiplexer. In this work we successfully achieved the creation of 16 non-linear gradients and the generation of a solid-phase micro-column for the realization of anion exchange chromatography on a single chip. With the device we demonstrated the separation of a protein mixture of R-phycoerythrin and FITC-BSA based on pH-gradient chromatofocusing

Parallel probing of drug uptake of single cancer cells on a microfluidic device

Drug resistance is frequently developing during treatment of cancer patients. Intracellular drug uptake is one of the important characteristics to understand mechanism of drug resistance. However, the heterogeneity of cancer cells requires the investigation of drug uptake at the single cell level. Here, we developed a microfluidic device for parallel probing of drug uptake. We combined a v-type valve and peristaltic pumping to select individual cells from a pool of prostate cancer cells (PC3) and place them successively in separate cell chambers in which they were exposed to the drug. Six different concentrations of doxorubicin, a naturally fluorescent anti-cancer drug, were created in loop-shaped reactors and exposed to the cell in closed 2 nL volume chambers. Monitoring every single cell over time in 18 parallel chambers revealed increased intracellular fluorescence intensity according to the dose of doxorubicin, as well as nuclear localization of the fluorescent drug after 2 h of incubation. The herein proposed technology demonstrated a first series of proof of concept experiments and it shows high potential to use for probing drug sensitivity of single cancer cell

Evaluation of peristaltic micromixers for highly integrated microfluidic systems

Microfluidic devices based on the multilayer soft lithography allow accurate manipulation of liquids, handling reagents at the sub-nanoliter level, and performing multiple reactions in parallel processors by adapting micromixers. Here, we have experimentally evaluated and compared several designs of micromixers and operating conditions to find design guidelines for the micromixers. We tested circular, triangular, and rectangular mixing loops and measured mixing performance according to the position and the width of the valves that drive nanoliters of fluids in the micrometer scale mixing loop. We found that the rectangular mixer is best for the applications of highly integrated microfluidic platforms in terms of the mixing performance and the space utilization. This study provides an improved understanding of the flow behaviors inside micromixers and design guidelines for micromixers that are critical to build higher order fluidic systems for the complicated parallel bio/chemical processes on a chip

Systematic Investigation of Insulin Fibrillation on a Chip

A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagents, stepwise concentration gradient generation, and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was selected to test the device. The effect of insulin and sodium chloride (NaCl) concentration on the formation of fibrillar structures was studied by observing the growth rate of partially folded protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of different NaCl and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in the formation of insulin fibrils and spherulites. The chip-system provides a bird’s eye view on protein aggregation, including an overview of the factors that affect the process and their interactions. This microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth

Microfluidic collagen patterning for tendon regeneration

We present a microfluidic approach to align collagen fibers for tendon regeneration. Collagen fibers with a specific orientation were patterned in a microfluidic channel by introducing collagen solution through integrated microstructures. The fluid flow in the pillar array was evaluated by computational modeling, and the aligned collagen fibers were analyzed quantitatively. Then, primary rat tenocytes were cultured on oriented and not-oriented collagen micropatterns, and their phenotypical commitment was evaluated. We believe that such a platform would be useful to replicate in vivo microenvironment for the study of regenerative processes

Understanding blood oxygenation in a microfluidic meander double side membrane contactor

Lung disease is one of the most important causes of high morbidity in preterm infants. In this work, we study a simple and easy to fabricate microfluidic device that demonstrates a great potential for blood oxygenation. A meander type architecture with double side vertical membrane arrangement has been selected as reference model to investigate the oxygenation process. The design criteria for the fabricated devices has been to maximize the oxygen saturation level while ensuring the physiological blood flow in order to avoid thrombus formation and channel blockage during operation. A mathematical model for the oxygen transfer has been developed and validated by the experimental study. The obtained results demonstrate that blood was successfully oxygenated up to approximately 98% of O-2 saturation and that the oxygen transfer rate at 1 mL/min blood flow rate was approximately 92 mL/minm(2). Finally, a sensitivity analysis of the key parameters, i.e. size of the channel, oxygen concentration in the gas phase and oxygen permeation properties of the membrane, is carried out to discuss the performance limits and to settle the guidelines for future developments.The authors would like to acknowledge the financial support from the Government of Aragón and the Education, Audiovisual and Culture Executive Agency (EU-EACEA) within the EUDIME - 'Erasmus Mundus Doctorate in Membrane Engineering' program (FPA 2011-0014, SGA 2012-1719, http://eudime.unical.it). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund. Authors acknowledge the LMA-INA for offering access to their instruments and expertise

On the improvement of alveolar-like microfluidic devices for efficient blood oxygenation

In this work, we study alveolar-like microfluidic devices with a horizontal membrane arrangement that demonstrate a great potential as small-scale blood oxygenator. The design criteria for the fabricated devices were to maximize the oxygen saturation level and minimize liquid chamber volume while ensuring the physiological blood flow in order to avoid thrombus formation and channel blockage during operation. The liquid chamber architecture was iteratively modified upon analysis of the fluid dynamics by computer modelling. Accordingly, two alveolar type architectures were fabricated, Alveolar Design 1 (AD1) and Alveolar Design 2 (AD2), and evaluated for oxygenation of sheep blood. The attained O2 transfer rate at 1 mL/min of blood flow rate for both devices was rather similar: 123 mL·min-1 ·m-2 and 127 mL·min-1 ·m-2 for AD1 and AD2 microfluidic devices, respectively. Among the studied, AD2 type geometry would lead to the lowest pressure drop and shear stress value upon implementation in a scaled microfluidic artificial lung (µAL) to satisfy oxygenation requirements of a 2.0 kg neonate.Government of Aragon and the Education, Audiovisual and Culture Executive Agency (EU-EACEA) within the EUDIME – ‘Erasmus Mundus Doctorate in Membrane Engineering’ program (FPA 2011-0014, SGA 2012-1719, http://eudime.unical.it). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund

Protein crystallization in a microfluidic contactor with nafion®117 membranes

UIDB/50006/2020 UIDP/50006/2020 “Erasmus Mundus Doctorate in Membrane Engineering”–EUDIME (FPA 2011–2014, http://www.eudime.unical.itProtein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent microcontactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen EggWhite Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.publishersversionpublishe

Standardized, Modular Parallelization Platform for Microfluidic Large-Scale Integration Cell Culturing Chips

Standardized high-throughput devices for microfluidic cell cultures are necessary to translate discoveries made in academia to applications in pharmaceutical industry. Here we present a platform with integrated pneumatic valves for standardized parallelization of multichamber chips (SPARC). In total, 192 chambers divided over three microfluidic building blocks (MFBBs) can be filled and purged with spatial and temporal independence. The dimensions of both the MFBB and the platform are standardized and thus compatible with common lab equipment. We characterize the valves at different pumping and gate pressures and show that the MFBBs are suitable for culturing human umbilical vein endothelial cells (HUVECs)