On the behavior of sub-micrometer polystyrene particles subjected to AC insulator-based dielectrophoresis.

Polymer beads, especially polystyrene particles, have been extensively used as model species in insulator-based dielectrophoresis (iDEP) studies. Their use in alternating current iDEP (AC-iDEP) is less explored; however, an assessment in the low-frequency regime (≤10 kHz) allows to link surface conduction effects with the surface properties of polymer particles. Here, we provide a case study for various experimental conditions assessing sub-micrometer polystyrene particles with AC-iDEP and link to accepted surface conduction theory to predict and experimentally verify the observed AC-iDEP trapping behavior based on apparent zeta potential and solution conductivity. We find excellent agreement with the theoretical predictions, but also the occurrence of concentration polarization electroosmotic flow under the studied conditions, which have the potential to confound acting dielectrophoresis conditions. Furthermore, we study a case relevant to the assessment of microplastics in human and animal body fluids by mimicking the protein adsorption of high abundant proteins in blood by coating polystyrene beads with bovine serum albumin, a highly abundant protein in blood. Theoretical predictions and experimental observations confirm a difference in observed AC-iDEP behavior between coated and non-coated particles, which might be exploited for future studies of microplastics in blood to assess their exposure to humans and animals.

Intracellular Amyloid-ß Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS.

Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-ß peptide (Aß) oligomers that appear as intermediates along the Aß aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aß from in vitro and animal models, there is little known about intracellular Aß in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aß species in specific brain cell subpopulations can provide insight into the role of Aß in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aß species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aß species from as few as 20 human brain cells.

Time-resolved serial femtosecond crystallography at the European XFEL.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

Numerical modeling reveals improved organelle separation for dielectrophoretic ratchet migration.

Organelle size varies with normal and abnormal cell function. Thus, size-based particle separation techniques are key to assessing the properties of organelle subpopulations differing in size. Recently, insulator-based dielectrophoresis (iDEP) has gained significant interest as a technique to manipulate sub-micrometer-sized particles enabling the assessment of organelle subpopulations. Based on iDEP, we recently reported a ratchet device that successfully demonstrated size-based particle fractionation in combination with continuous flow sample injection. Here, we used a numerical model to optimize the performance with flow rates a factor of three higher than previously and increased the channel volume to improve throughput. We evaluated the amplitude and duration of applied low-frequency DC-biased AC potentials improving separation efficiency. A separation efficiency of nearly 0.99 was achieved with the optimization of key parameters-improved from 0.80 in previous studies (Ortiz et al. Electrophoresis, 2022;43;1283-1296)-demonstrating that fine-tuning the periodical driving forces initiating the ratchet migration under continuous flow conditions can significantly improve the fractionation of organelles of different sizes.

Continuous organelle separation in an insulator-based dielectrophoretic device.

Heterogeneity in organelle size has been associated with devastating human maladies such as neurodegenerative diseases or cancer. Therefore, assessing the size-based subpopulation of organelles is imperative to understand the biomolecular foundations of these diseases. Here, we demonstrated a ratchet migration mechanism using insulator-based dielectrophoresis in conjunction with a continuous flow component that allows the size-based separation of submicrometer particles. The ratchet mechanism was realized in a microfluidic device exhibiting an array of insulating posts, tailoring electrokinetic and dielectrophoretic transport. A numerical model was developed to elucidate the particle migration and the size-based separation in various conditions. Experimentally, the size-based separation of a mixture of polystyrene beads (0.28 and 0.87 µ$\umu $ m) was accomplished demonstrating good agreement with the numerical model. Furthermore, the size-based separation of mitochondria was investigated using a mitochondria mixture isolated from HepG2 cells and HepG2 cells carrying the gene Mfn-1 knocked out, indicating distinct size-related migration behavior. With the presented continuous flow separation device, larger amounts of fractionated organelles can be collected in the future allowing access to the biomolecular signature of mitochondria subpopulations differing in size.

MIMAS: microfluidic platform in tandem with MALDI mass spectrometry for protein quantification from small cell ensembles.

Understanding cell-to-cell variation at the molecular level provides relevant information about biological phenomena and is critical for clinical and biological research. Proteins carry important information not available from single-cell genomics and transcriptomics studies; however, due to the minute amount of proteins in single cells and the complexity of the proteome, quantitative protein analysis at the single-cell level remains challenging. Here, we report an integrated microfluidic platform in tandem with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the detection and quantification of targeted proteins from small cell ensembles (> 10 cells). All necessary steps for the assay are integrated on-chip including cell lysis, protein immunocapture, tryptic digestion, and co-crystallization with the matrix solution for MALDI-MS analysis. We demonstrate that our approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 released from MCF-7 breast cancer cells, ranging from 26 to 223 cells lysed on-chip (8.75 nL wells). A limit of detection (LOD) of 11.22 nM was determined, equivalent to 5.91 × 107 protein molecules per well. Additionally, the microfluidic platform design was further improved, establishing the successful quantification of Bcl-2 protein from MCF-7 cell ensembles ranging from 8 to 19 cells in 4 nL wells. The LOD in the smaller well designs for Bcl-2 resulted in 14.85 nM, equivalent to 3.57 × 107 protein molecules per well. This work shows the capability of our approach to quantitatively assess proteins from cell lysate on the MIMAS platform for the first time. These results demonstrate our approach constitutes a promising tool for quantitative targeted protein analysis from small cell ensembles down to single cells, with the capability for multiplexing through parallelization and automation.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Quantitative Approach for Protein Analysis in Small Cell Ensembles by an Integrated Microfluidic Chip with MALDI Mass Spectrometry.

Increasing evidence has demonstrated that cells are individually heterogeneous. Advancing the technologies for single-cell analysis will improve our ability to characterize cells, study cell biology, design and screen drugs, and aid cancer diagnosis and treatment. Most current single-cell protein analysis approaches are based on fluorescent antibody-binding technology. However, this technology is limited by high background and cross-talk of multiple tags introduced by fluorescent labels. Stable isotope labels used in mass cytometry can overcome the spectral overlap of fluorophores. Nevertheless, the specificity of each antibody and heavy-metal-tagged antibody combination must be carefully validated to ensure detection of the intended target. Thus, novel single-cell protein analysis methods without using labels are urgently needed. Moreover, the labeling approach targets already known motifs, hampering the discovery of new biomarkers relevant to single-cell population variation. Here, we report a combined microfluidic and matrix-assisted laser desorption and ionization (MALDI) mass spectrometric approach for the analysis of protein biomarkers suitable for small cell ensembles. All necessary steps for cell analysis including cell lysis, protein capture, and digestion as well as MALDI matrix deposition are integrated on a microfluidic chip prior to the downstream MALDI-time-of-flight (TOF) detection. For proof of principle, this combined method is used to assess the amount of Bcl-2, an apoptosis regulator, in metastatic breast cancer cells (MCF-7) by using an isotope-labeled peptide as an internal standard. The proposed approach will eventually provide a new means for proteome studies in small cell ensembles with the potential for single-cell analysis and improve our ability in disease diagnosis, drug discovery, and personalized therapy.

Length-Selective Dielectrophoretic Manipulation of Single-Walled Carbon Nanotubes.

Single-walled carbon nanotubes (SWNTs) possess unique physical, optical, and electrical properties with great potential for future nanoscale device applications. Common synthesis procedures yield SWNTs with large length polydispersity and varying chirality. Electrical and optical applications of SWNTs often require specific lengths, but the preparation of SWNTs with the desired length is still challenging. Insulator-based dielectrophoresis (iDEP) integrated into a microfluidic device has the potential to separate SWNTs by length. Semiconducting SWNTs of varying length suspended with sodium deoxycholate (NaDOC) show unique dielectrophoretic properties at low frequencies (<1 kHz) that were exploited here using an iDEP-based microfluidic constriction sorter device for length-based sorting. Specific migration directions in the constriction sorter were induced for long SWNTs (≥1000 nm) with negative dielectrophoretic properties compared to short (≤300 nm) SWNTs with positive dielectrophoretic properties. We report continuous fractionation conditions for length-based iDEP migration of SWNTs, and we characterize the dynamics of migration of SWNTs in the microdevice using a finite element model. Based on the length and dielectrophoretic characteristics, sorting efficiencies for long and short SWNTs recovered from separate channels of the constriction sorter amounted to >90% and were in excellent agreement with a numerical model for the sorting process.

Carbon nanotube dielectrophoresis: Theory and applications.

Carbon nanotubes (CNTs) are one of the most extensively studied nanomaterials in the 21st century. Since their discovery in 1991, many studies have been reported advancing our knowledge in terms of their structure, properties, synthesis, and applications. CNTs exhibit unique electrothermal and conductive properties which, combined with their mechanical strength, have led to tremendous attention of CNTs as a nanoscale material in the past two decades. To introduce the various types of CNTs, we first provide basic information on their structure followed by some intriguing properties and a brief overview of synthesis methods. Although impressive advances have been demonstrated with CNTs, critical applications require purification, positioning, and separation to yield desired properties and functional elements. Here, we review a versatile technique to manipulate CNTs based on their dielectric properties, namely dielectrophoresis (DEP). A detailed discussion on the DEP aspects of CNTs including the theory and various technical microfluidic realizations is provided. Various advancements in DEP-based manipulations of single-walled and multiwalled CNTs are also discussed with special emphasis on applications involving separation, purification, sensing, and nanofabrication.

Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser.

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Electric Triggering for Enhanced Control of Droplet Generation.

Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures. This increases the time and resources required for sample preparation and experimentation. The intrinsic pulsed nature of all current X-ray sources is a major reason why such large amounts of sample are required. Any crystal sample that is delivered in the path of the X-ray beam during its "off-time" is wasted. To address this large sample consumption issue, we developed a 3D printed microfluidic system with integrated metal electrodes for water-in-oil droplet generation to dynamically create and manipulate aqueous droplets. We demonstrate on-demand droplet generation using DC potentials and the ability to tune the frequency of droplet generation through the application of AC potentials. More importantly, to assist with the synchronization of droplets and XFEL pulses, we show that the device can induce a phase shift in the base droplet generation frequency. This novel approach to droplet generation has the potential to reduce sample waste by more than 95% for SFX experiments with XFELs performed with liquid jets and can operate under low- and high-pressure liquid injection systems.

Microfluidic sample delivery for serial crystallography using XFELs.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) is an emerging field for structural biology. One of its major impacts lies in the ability to reveal the structure of complex proteins previously inaccessible with synchrotron-based crystallography techniques and allowing time-resolved studies from femtoseconds to seconds. The nature of this serial technique requires new approaches for crystallization, data analysis, and sample delivery. With continued advancements in microfabrication techniques, various developments have been reported in the past decade for innovative and efficient microfluidic sample delivery for crystallography experiments using XFELs. This article summarizes the recent developments in microfluidic sample delivery with liquid injection and fixed-target approaches, which allow exciting new research with XFELs. Graphical abstract.

Deterministic Ratchet for Sub-micrometer (Bio)particle Separation.

Resolving the heterogeneity of particle populations by size is important when the particle size is a signature of abnormal biological properties leading to disease. Accessing size heterogeneity in the sub-micrometer regime is particularly important to resolve populations of subcellular species or diagnostically relevant bioparticles. Here, we demonstrate a ratchet migration mechanism capable of separating sub-micrometer sized species by size and apply it to biological particles. The phenomenon is based on a deterministic ratchet effect, is realized in a microfluidic device, and exhibits fast migration allowing separation in tens of seconds. We characterize this phenomenon extensively with the aid of a numerical model allowing one to predict the speed and resolution of this method. We further demonstrate the deterministic ratchet migration with two sub-micrometer sized beads as model system experimentally as well as size-heterogeneous mouse liver mitochondria and liposomes as model system for other organelles. We demonstrate excellent agreement between experimentally observed migration and the numerical model.

Continuous Separation of DNA Molecules by Size Using Insulator-Based Dielectrophoresis.

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 Probed with Insulator-Based Dielectrophoresis.

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.

Toward Analysis of Proteins in Single Cells: A Quantitative Approach Employing Isobaric Tags with MALDI Mass Spectrometry Realized with a Microfluidic Platform.

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.

Deterministic Absolute Negative Mobility for Micro- and Submicrometer Particles Induced in a Microfluidic Device.

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.

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 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.

High throughput protein nanocrystal fractionation in a microfluidic sorter.

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.