Separation of nucleic acids has long served as a central goal of analytical research. Innovations in this field may soon enable the development of rapid, on-site sequencing devices that significantly improve both the availability and accuracy of detailed bioinformatics. However, achieving efficient continuous-flow operation and size-based fractionation of DNA still presents considerable challenges. Current methods have not yet satisfied the need for rapid fractionation of size-heterogeneous nucleic acid samples into specific and narrow size distributions. Dielectrophoretic (DEP) mechanisms integrated in microfluidic devices offer unique advantages for such applications, including short processing times, microscale reaction volumes, and the potential for low cost and portability. To facilitate such developments, we have adapted a microfluidic constriction sorter device to separate a wide range of nucleic acid analytes into distinct microchannel outlets. This work demonstrates selective and tunable deflection of DNA using alternating current (AC) insulator-based dielectrophoresis. We report conditions for size-based DEP sorting of 1.0, 10.2, 19.5, and 48.5 kbp dsDNA analytes, including both plasmid and genomic DNA. Applied potentials range from 200 to 2400 Vpp with frequencies ranging from 50 Hz to 20 kHz. These conditions result in sorting efficiencies up to 92% with a strong dependence on applied potentials and frequencies. In low-frequency AC fields, long DNA molecules form macro-ion clusters. This behavior is associated with an apparent shift from positive to negative DEP sorting behavior. Using a finite element model, we characterize the dynamics of sorting in the microdevice and link differential sorting to differences in dielectrophoretic mobility. We propose the use of a continuous-flow sorting strategy to facilitate future coupling to next generation sequencing approaches.

Single-walled carbon nanotubes (SWNTs) offer unique electrical and optical properties. Common synthesis processes yield SWNTs with large length polydispersity (several tens of nanometers up to centimeters) and heterogeneous electrical and optical properties. Applications often require suitable selection and purification. Dielectrophoresis is one manipulation method for separating SWNTs based on dielectric properties and geometry. Here, we present a study of surfactant and single-stranded DNA-wrapped SWNTs suspended in aqueous solutions manipulated by insulator-based dielectrophoresis (iDEP). This method allows us to manipulate SWNTs with the help of arrays of insulating posts in a microfluidic device around which electric field gradients are created by the application of an electric potential to the extremities of the device. Semiconducting SWNTs were imaged during dielectrophoretic manipulation with fluorescence microscopy making use of their fluorescence emission in the near IR. We demonstrate SWNT trapping at low-frequency alternating current (AC) electric fields with applied potentials not exceeding 1000 V. Interestingly, suspended SWNTs showed both positive and negative dielectrophoresis, which we attribute to their ζ potential and the suspension properties. Such behavior agrees with common theoretical models for nanoparticle dielectrophoresis. We further show that the measured ζ potentials and suspension properties are in excellent agreement with a numerical model predicting the trapping locations in the iDEP device. This study is fundamental for the future application of low-frequency AC iDEP for technological applications of SWNTs.

Efficient separations of particles with micron and submicron dimensions are extremely useful in preparation and analysis of materials for nanotechnological and biological applications. Here, we demonstrate a nonintuitive, yet efficient, separation mechanism for μm and subμm colloidal particles and organelles, taking advantage of particle transport in a nonlinear post array in a microfluidic device under the periodic action of electrokinetic and dielectrophoretic forces. We reveal regimes in which deterministic particle migration opposite to the average applied force occurs for a larger particle, a typical signature of deterministic absolute negative mobility (dANM), whereas normal response is obtained for smaller particles. The coexistence of dANM and normal migration was characterized and optimized in numerical modeling and subsequently implemented in a microfluidic device demonstrating at least 2 orders of magnitude higher migration speeds as compared to previous ANM systems. We also induce dANM for mouse liver mitochondria and envision that the separation mechanisms described here provide size selectivity required in future separations of organelles, nanoparticles, and protein nanocrystals.

Protein identification and quantification in individual cells is essential to understand biological processes such as those involved in cell apoptosis, cancer, biomarker discovery, disease diagnostics, pathology, or therapy. Compared with present single cell genome analysis, probing the protein content of single cells has been hampered by the lack of a protein amplification technique. Here, we report the development of a quantitative mass spectrometric approach combined with microfluidic technology reaching the detection sensitivity of high abundant proteins in single cells. A microfluidic platform with a series of chambers and valves, ensuring a set of defined wells for absolute quantification of targeted proteins, was developed and combined with isotopic labeling strategies employing isobaric tags for relative and absolute quantitation (iTRAQ)-labels. To this aim, we adapted iTRAQ labeling to an on-chip protocol. Simultaneous protein digestion and labeling performed on the microfluidic platform rendered the labeling strategy compatible with all necessary manipulation steps on-chip, including the matrix delivery for MALDI-TOF analysis. We demonstrate this approach with the apoptosis related protein Bcl-2 and quantitatively assess the number of Bcl-2 molecules detected. We anticipate that this approach will eventually allow quantification of protein expression on the single cell level.

Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies.

Protein crystallography is transitioning into a new generation with the introduction of the X-ray free electron laser, which can be used to solve the structures of complex proteins via serial femtosecond crystallography. Sample characteristics play a critical role in successful implementation of this new technology, whereby a small, narrow protein crystal size distribution is desired to provide high quality diffraction data. To provide such a sample, we developed a microfluidic device that facilitates dielectrophoretic sorting of heterogeneous particle mixtures into various size fractions. The first generation device demonstrated great potential and success toward this endeavor; thus, in this work, we present a comprehensive optimization study to improve throughput and control over sorting outcomes. First, device geometry was designed considering a variety of criteria, and applied potentials were modeled to determine the scheme achieving the largest sorting efficiency for isolating nanoparticles from microparticles. Further, to investigate sorting efficiency within the nanoparticle regime, critical geometrical dimensions and input parameters were optimized to achieve high sorting efficiencies. Experiments revealed fractionation of nanobeads from microbeads in the optimized device with high sorting efficiencies, and protein crystals were sorted into submicrometer size fractions as desired for future serial femtosecond crystallography experiments.

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 Kramer’s 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.

Insulator-based dielectrophoresis is a relatively new analytical technique with a large potential for a number of applications, such as sorting, separation, purification, fractionation, and preconcentration. The application of insulator-based dielectrophoresis (iDEP) for biological samples, however, requires the precise control of the microenvironment with temporal and spatial resolution. Temperature variations during an iDEP experiment are a critical aspect in iDEP since Joule heating could lead to various detrimental effects hampering reproducibility. Additionally, Joule heating can potentially induce thermal flow and more importantly can degrade biomolecules and other biological species. Here, we investigate temperature variations in iDEP devices experimentally employing the thermosensitive dye Rhodamin B (RhB) and compare the measured results with numerical simulations. We performed the temperature measurement experiments at a relevant buffer conductivity range commonly used for iDEP applications under applied electric potentials. To this aim, we employed an in-channel measurement method and an alternative method employing a thin film located slightly below the iDEP channel. We found that the temperature does not deviate significantly from room temperature at 100 μS/cm up to 3000 V applied such as in protein iDEP experiments. At a conductivity of 300 μS/cm, such as previously used for mitochondria iDEP experiments at 3000 V, the temperature never exceeds 34 °C. This observation suggests that temperature effects for iDEP of proteins and mitochondria under these conditions are marginal. However, at larger conductivities (1 mS/cm) and only at 3000 V applied, temperature increases were significant, reaching a regime in which degradation is likely to occur. Moreover, the thin layer method resulted in lower temperature enhancement which was also confirmed with numerical simulations. We thus conclude that the thin film method is preferable providing closer agreement with numerical simulations and further since it does not depend on the iDEP channel material. Overall, our study provides a thorough comparison of two experimental techniques for direct temperature measurement, which can be adapted to a variety of iDEP applications in the future. The good agreement between simulation and experiment will also allow one to assess temperature variations for iDEP devices prior to experiments.

The trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment.

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.

Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these “difficult to crystallize” proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.Keywords: concentration gradients; membrane protein; numerical simulation; SONICC

Structure elucidation of large membrane protein complexes is still a considerable challenge, yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected submicrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ∼100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography.Keywords: dielectrophoresis; membrane protein; microfluidic; nanocrystallography; sorting

A microfluidics-based laboratory experiment for the analysis of DNA fragments in an analytical undergraduate course is presented. The experiment is set within the context of food species identification via amplified DNA fragments. The students are provided with berry samples from which they extract DNA and perform polymerase chain reaction (PCR) with strawberry-specific primers. The resulting PCR products are analyzed using the Agilent Bioanalyzer. Using the raw data, the students are tasked to identify the strawberry sample. This course serves as a practical introduction into microfluidic-based capillary gel electrophoresis as well as a primer for biomolecular DNA analysis.Keywords: Analytical Chemistry; Bioanalytical Chemistry; Electrophoresis; Food Science; Hands-On Learning/Manipulatives; Laboratory Instruction; Microscale Lab; Nucleic Acids/DNA/RNA; Separation Science; Upper-Division Undergraduate;

The ability to detect and quantify proteins of individual cells in high throughput is of enormous biological and clinical relevance. Most methods currently in use either require the measurement of large cell populations or are limited to the investigation of few cells at a time. In this report, we present the combination of a polydimethylsiloxane-based microfluidic device to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) that allows the detection of as few as 300 molecules at the peptide level and ∼106 to 107 molecules at the protein level. Moreover, we performed an immunoassay with subsequent MALDI-TOF-MS to capture and detect insulin immobilized on a surface (∼0.05 mm2) in this device with a detection limit of 106 insulin molecules. This microfluidic-based approach therefore begins to approach the sample handling and sensitivity requirements for MS-based single-cell analysis of proteins and peptides and holds the potential for easy parallelization of immunoassays and other highly sensitive protein analyses.