Exosomes are a specific subpopulation of extracellular vesicles that have gained interest because of their many potential biomedical applications. However, exosome isolation and characterization are the first steps toward designing novel applications. This work presents a direct current−insulator-based dielectrophoretic (DC-iDEP) approach to simultaneously capture and separate exosomes by size. To do so, a microdevice consisting of a channel with two electrically insulating post sections was designed. Each section was tailored to generate different nonuniform spatial distributions of the electric field and, therefore, different dielectrophoretic forces acting on exosomes suspended in solution. Side channels were placed adjacent to each section to allow sample recovery. By applying an electric potential difference of 2000 V across the length of the main channel, dielectrophoretic size-based separation of exosomes was observed in the device. Analysis of particle size in each recovered fraction served to assess exosome separation efficiency. These findings show that iDEP can represent a first step toward designing a high-throughput, fast, and robust microdevice capable of capturing and discriminating different subpopulations of exosomes based on their size.

Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjugates and the unreacted native protein. From a ribonuclease A (RNase A) PEGylation reaction, mono-PEGylated RNase A (mono-PEG RNase A) has proven therapeutic effects against cancer, reason for which there is an interest in isolating it from the rest of the reaction products. Experimental trapping of PEGylated RNase A inside an electrokinetically driven microfluidic device has been previously demonstrated. Now, from a theoretical point of view, we have studied the electrokinetic phenomena involved in the dielectrophoretic streaming of the native RNase A protein and the trapping of the mono-PEG RNase A inside a microfluidic channel. To accomplish this, we used two 3D computational models, a sphere and an ellipse, adapted to each protein. The effect of temperature on parameters related to trapping was also studied. A temperature increase showed to rise the electric and thermal conductivities of the suspending solution, hindering dielectrophoretic trapping. In contrast, the dynamic viscosity of the suspending solution decreased as the temperature rose, favoring the dielectrophoretic manipulation of the proteins. Also, our models were able to predict the magnitude and direction of the velocity of both proteins indicating trapping for the PEGylated conjugate or no trapping for the native protein. In addition, a parametric sweep study revealed the effect of the protein zeta potential on the electrokinetic response of the protein. We believe this work will serve as a tool to improve the design of electrokinetically driven microfluidic channels for the separation and recovery of PEGylated proteins in one single step. Published by AIP Publishing. [http://dx

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BackgroundThe identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry.Methodology and Principal FindingsWe developed a 1-minute covalent pre-gel staining protocol for proteins, which does not require de-staining before the mass spectrometry analysis. We investigated the electrophoretic properties of derivatized proteins and peptides and studied their behavior in mass spectrometry. Further, we elucidated the preferred reaction of proteins with Uniblue A and demonstrate the integration of the peptide derivatization into typical informatics tools.Conclusions and SignificanceThe Uniblue A staining method drastically speeds up the sample preparation for the mass spectrometry based identification of proteins. The application of this chemo-proteomic strategy will be advantageous for routine quality control of proteins and for time-critical tasks in protein analysis.

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Ribonuclease A (RNase A) has proven potential as a therapeutic agent, especially in its PEGylated form. Grafting of PEG molecules to this protein yields mono-PEGylated (mono-PEG) and di-PEGylated (di-PEG) RNase A conjugates, and the unreacted protein. Mono-PEG RNase A is of great interest. The use of electrokinetic forces in microdevices represents a novel alternative to chromatographic methods to separate this specie. This work describes the dielectrophoretic behavior of the main protein products of the RNase A PEGylation inside a microchannel with insulators under direct current electric fields. This approach represents the first step in route to design micro-bioprocesses to separate PEGylated RNase A from unreacted native protein. The three proteins exhibited different dielectrophoretic behaviors. All of them experienced a marked streaming pattern at 3000 V consistent with positive dielectrophoresis. Native protein was not captured at any of the conditions tested, while mono-PEG RNase A and di-PEG RNase A were captured presumably due to positive dielectrophoresis at 4000 and 2500 V, respectively. Concentration of mono-PEG RNase A with a maximal enrichment efficiency of ≈9.6 times the feed concentration was achieved in few seconds. These findings open the possibility of designing novel devices for rapid separation, concentration, and recovery of PEGylated RNase A in a one-step operation.

Gallic acid production in a batch bioreactor was evaluated using as catalytic material the mouldy polyurethane solids (MPS) obtained from a solid-state fermentation (SSF) bioprocess carried out for tannase production by Aspergillus niger GH1 on polyurethane foam powder (PUF) with 5 % (v/w) of tannic acid as inducer. Fungal biomass, tannic acid consumption and tannase production were kinetically monitored. SSF was stopped when tannase activity reached its maximum level. Effects of washing with distilled water and drying on the tannase activity of MPS were determined. Better results were obtained with dried and washed MPS retaining 84 % of the tannase activity. Maximum tannase activity produced through SSF after 24 h of incubation was equivalent to 130 U/gS with a specific activity of 36 U/mg. The methylgallate was hydrolysed (45 %) in an easy, cheap and fast bioprocess (30 min). Kinetic parameters of tannase self-immobilized on polyurethane particles were calculated to be 5 mM and 04.1 × 10(-2) mM/min for K M and V max, respectively. Results demonstrated that the MPS, with tannase activity, can be successfully used for the production of the antioxidant gallic acid from methyl-gallate substrate. Direct use of PMS to produce gallic acid can be advantageous as no previous extraction of enzyme is required, thus reducing production costs.

Tannase is an inducible enzyme with important applications in the food and pharmaceutical industries. This enzyme was produced by the fungus Aspergillus niger GH1 under solid-state fermentation using polyurethane foam as solid support and tannic acid as sole carbon source and tannase inducer. Physicochemical properties of A. niger tannase were characterized, and the kinetic and thermodynamics parameters on methyl gallate hydrolysis were evaluated. The enzyme was stable in a pH range of 2-8 and a functional temperature range of 25-65 °C. The highest k(cat) value was 2,611.10 s(-1) at 65 °C. Tannase had more affinity for methyl gallate at 45 °C with a K(M) value of 1.82 mM and an efficiency of hydrolysis (k(cat)/K(M)) of 330.01 s(-1) mM(-1). The lowest E(a) value was found to be 21.38 kJ/mol at 4.4 mM of methyl gallate. The lowest free energy of Gibbs (ΔG) and enthalpy (ΔH) were found to be 64.86 and 18.56 kJ/mol, respectively. Entropy (ΔS) was -0.22 kJ/mol K. Results suggest that the A. niger GH1 tannase is an attractive enzyme for industrial applications due its catalytic and thermodynamical properties.