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
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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