Fernanda Camacho-Alanis

Immunoglobulin G and bovine serum albumin streaming dielectrophoresis in a microfluidic device

Dielectrophoresis (DEP) has demonstrated to be a versatile tool to manipulate micro‐ and nanoparticles with applications for positioning, separation and fractionation. Recent developments of DEP have also shown that DEP can be used for the manipulation of biomolecules, such as DNA. Here, we focus on the manipulation of proteins using insulator‐based dielectrophoresis (iDEP). We designed suitable post arrays in a microfluidic channel and use numerical simulations to calculate the electric field distribution as well as concentration of proteins according to a convection–diffusion model for both negative and positive DEP. Experimentally, we find DEP trapping of mainly protein aggregates in phosphate buffer. However, when adding a charged zwitterionic detergent, we observed DEP streamlining of immunoglobulin G (IgG) and bovine serum albumin (BSA). Our experimental observations are in excellent agreement with numerical simulations and indicate positive DEP behavior of IgG and BSA under the employed experimental conditions. Our results demonstrate DEP streaming of proteins in an iDEP device for the first time and indicate the potential of protein DEP for separation and fractionation.

Dielectrophoresis of lambda-DNA using 3D carbon electrodes.

Carbon electrodes have recently been introduced as an alternative to metal electrodes and insulator structures for dielectrophoretic applications. Here, an experimental and theoretical study employing an array of 3D carbon electrodes contained in a microfluidic channel for the dielectrophoretic manipulation of DNA is presented. First evidence that carbon‐electrode DEP can be used for the manipulation and trapping of biomolecules such as DNA is reported. In particular, the dielectrophoretic response of λ‐DNA (48.5 kbp) under various frequencies and flow conditions necessary for retention of λ‐DNA are studied. Negative DEP is observed at frequencies above 75 kHz and positive DEP is present in the range below 75 kHz and down to 5 kHz. We further implement a theoretical model to capture the experimental findings in sufficient detail. Our theoretical considerations based on reported scaling laws for linear and supercoiled DNA further suggest that carbon‐electrode DEP devices could be employed in future analytical applications such as DNA preconcentration and fractionation.

Tuning direct current streaming dielectrophoresis of proteins

Dielectrophoresis (DEP) of biomolecules has large potential to serve as a novel selectivity parameter for bioanalytical methods such as (pre)concentration, fractionation, and separation. However, in contrast to well-characterized biological cells and (nano)particles, the mechanism of protein DEP is poorly understood, limiting bioanalytical applications for proteins. Here, we demonstrate a detailed investigation of factors influencing DEP of diagnostically relevant immunoglobulin G (IgG) molecules using insulator-based DEP (iDEP) under DC conditions. We found that the pH range in which concentration of IgG due to streaming iDEP occurs without aggregate formation matches the pH range suitable for immunoreactions. Numerical simulations of the electrokinetic factors pertaining to DEP streaming in this range further suggested that the protein charge and electroosmotic flow significantly influence iDEP streaming. These predictions are in accordance with the experimentally observed pH-dependent iDEP streaming profiles as well as the determined IgG molecular properties. Moreover, we observed a transition in the streaming behavior caused by a change from positive to negative DEP induced through micelle formation for the first time experimentally, which is in excellent qualitative agreement with numerical simulations. Our study thus relates molecular immunoglobulin properties to observed iDEP, which will be useful for the future development of protein (pre)concentration or separation methods based on DEP.

Quantification of pH gradients and implications in insulator-based dielectrophoresis of biomolecules.

Direct current (DC) insulator‐based dielectrophoretic (iDEP) microdevices have the potential to replace traditional alternating current dielectrophoretic devices for many cellular and biomolecular separation applications. The use of large DC fields suggest that electrode reactions and ion transport mechanisms can become important and impact ion distributions in the nanoliters of fluid in iDEP microchannels. This work tracked natural pH gradient formation in a 100 μm wide, 1 cm‐long microchannel under applicable iDEP protein manipulation conditions. Using fluorescence microscopy with the pH‐sensitive dye FITC Isomer I and the pH‐insensitive dye TRITC as a reference, pH was observed to drop drastically in the microchannels within 1 min in a 3000 V/cm electric field; pH drops were observed in the range of 6–10 min within a 100 V/cm electric field and varied based on the buffer conductivity. To address concerns of dye transport impacting intensity data, electrokinetic mobilities of FITC were carefully examined and found to be (i) toward the anode and (ii) 1 to 2 orders of magnitude smaller than H+ transport which is responsible for pH drops from the anode toward the cathode. COMSOL simulations of ion transport showed qualitative agreement with experimental results. The results indicate that pH changes are severe enough and rapid enough to influence the net charge of a protein or cause aggregation during iDEP experiments. The results also elucidate reasonable time periods over which the phosphate buffering capacity can counter increases in H+ and OH− for unperturbed iDEP manipulations.

Six-helix bundle and triangle DNA origami insulator-based dielectrophoresis

Self-assembled DNA nanostructures have large potential for nanoelectronic circuitry, targeted drug delivery, and intelligent sensing. Their applications require suitable methods for manipulation and nanoscale assembly as well as adequate concentration, purification, and separation methods. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro- and nanometer-sized objects. In order to exploit iDEP for DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. Here, we explore the dielectrophoretic behavior of six-helix bundle and triangle DNA origamis with identical sequence but large topological difference and reveal a characteristic frequency range of iDEP trapping. Moreover, the confinement of triangle origami in the iDEP trap required larger applied electric fields. To elucidate the observed DEP migration and trapping, we discuss polarizability models for the two species according to their structural difference complemented by numerical simulations, revealing a contribution of the electrophoretic transport of the DNA origami species in the iDEP trapping regions. The numerical model showed reasonable agreement with experiments at lower frequency. However, the extension of the iDEP trapping regions observed experimentally deviated considerably at higher frequencies. Our study demonstrates for the first time that DNA origami species can be successfully trapped and manipulated by iDEP and reveals distinctive iDEP behavior of the two DNA origamis. The experimentally observed trapping regimes will facilitate future exploration of DNA origami manipulation and assembly at the nano- and microscale as well as other applications of these nanoassemblies with iDEP.

Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of the dependence of crystal quality on crystal size.

Protein dielectrophoresis and the link to dielectric properties

There is a growing interest in protein dielectrophoresis (DEP) for biotechnological and pharmaceutical applications. However, the DEP behavior of proteins is still not well understood which is important for successful protein manipulation. In this paper, we elucidate the information gained in dielectric spectroscopy (DS) and electrochemical impedance spectroscopy (EIS) and how these techniques may be of importance for future protein DEP manipulation. EIS and DS can be used to determine the dielectric properties of proteins predicting their DEP behavior. Basic principles of EIS and DS are discussed and related to protein DEP through examples from previous studies. Challenges of performing DS measurements as well as potential designs to incorporate EIS and DS measurements in DEP experiments are also discussed.

Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices.

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin-fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Polarizability of Six-Helix Bundle and Triangle DNA Origami and Their Escape Characteristics from a Dielectrophoretic Trap.

DNA nanoassemblies, such as DNA origamis, hold promise in biosensing, drug delivery, nanoelectronic circuits, and biological computing, which require suitable methods for migration and precision positioning. Insulator-based dielectrophoresis (iDEP) has been demonstrated as a powerful migration and trapping tool for μm- and nm-sized colloids as well as DNA origamis. However, little is known about the polarizability of origami species, which is responsible for their dielectrophoretic migration. Here, we report the experimentally determined polarizabilities of the six-helix bundle origami (6HxB) and triangle origami by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the origami according to an adapted Kramers rate model, allowing their polarizabilities to be determined. We found that the 6HxB polarizability is larger than that of the triangle origami, which correlates with the variations in charge density of both origamis. Further, we discuss the orientation of both origami species in the dielectrophoretic trap and discuss the influence of diffusion during the escape process. Our study provides detailed insight into the factors contributing to the migration through dielectrophoretic potential landscapes, which can be exploited for applications with DNA and other nanoassemblies based on dielectrophoresis.