Biochemical Labels Research Articles

Multiplexed genomic encoding of non-canonical amino acids for labeling large complexes.

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

Advances and applications of isomotive dielectrophoresis for cell analysis.

Isomotive dielectrophoresis (isoDEP) is a unique electric field such that the gradient of the field-squared ([Formula: see text]) is constant, resulting a uniform dielectrophoretic force. The current status of isoDEP is presented in this review, and we will highlight the progress that has been achieved over the past 60 years in various avenues of isoDEP since H.A. Pohl initially described its premise. This article will discuss its applications and describe the various configurations of generating an isomotive force. Since H.A. Pohl introduced the theory of isoDEP, numerous authors have implemented isoDEP as a tool for the manipulation, sorting, separation, and characterization of polarizable particles without the need for biochemical labels or other bioengineered tagging. The growing field of microfluidics and electrokinetics has renewed interest in isoDEP, particularly for analytical characterization or separation of particles. Recent work has demonstrated that isoDEP can address some unmet needs for biomedical applications including single-cell analysis; moreover, advances in throughput as well as combining characterization and separation simultaneously will add significant value to isoDEP.

Recent advances and perspectives on capture and concentration of label-free rare cells for biomedical science and engineering research

Abstract Capture and concentration of the rare cells from a heterogeneous population plays an important role in the development of disease diagnostics, therapeutics, and biomedical research/applications. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer therapy and regenerative medicine. Large-scale supply of the rare cells at high purity and viability is essential, especially on stem cells, cancer stem cells (CSCs), and circulating tumor cells (CTCs). Conventional capture and concentration methods providing efficient and high throughput isolations are introduced; however, most of them require well-defined biochemical markers and immunolabeling procedures which are complicated, uneconomical, and often unreliable. In contrast, the emerging label-free cell capture methods based on biophysical and biomechanical properties of the desired cell population are growing. In this review, we aim to describe the status of label-free rare cell capture and concentration techniques and to highlight exciting new approaches in this field. Label-free rare cell isolation methods using intrinsic biophysical properties such as filtration, size, electrical polarizability, dielectrophoresis, optical dielectrophoresis, and hydrodynamic chromatography are introduced. Moreover, the microfluidic systems adopted to precisely handle cells and interface with other tools have been described earlier; thus, new approaches dealing with merging label-free selection methods and microfluidic systems are merely summarized. Furthermore, the capture and concentration methods using biochemical and materials properties such as cell-materials interaction, spheroid formation, and stem cells/CSCs niche mimicking are highlighted particularly. Advancing cell isolation technique provides the opportunity to avoid the shortcoming of using biochemical labels and presents better flexibility for subsequent characterization, such as on site screening, high throughput screening, and personalized medicine.

A novel lab-on-a-chip platform for spheroid metabolism monitoring.

Sensor-based cellular microphysiometry is a technique that allows non-invasive, label-free, real-time monitoring of living cells that can greatly improve the predictability of toxicology testing by removing the influence of biochemical labels. In this work, the Intelligent Mobile Lab for In Vitro Diagnostics (IMOLA-IVD) was utilized to perform cellular microphysiometry on 3D multicellular spheroids. Using a commercial 3D printer, 3×3 microwell arrays were fabricated to maintain nine previously cultured HepG2 spheroids on a single BioChip. Integrated layers above and under the spheroids allowed fluidic contact between spheroids in microwells and BioChip sensors while preventing wash out from medium perfusion. Spheroid culturing protocols were optimized to grow spheroids to a diameter of around 620μm prior to transfer onto BioChips. An ON/OFF pump cycling protocol was developed to optimize spheroid culture within the designed microwells, intermittently perfuse spheroids with fresh culture medium, and measure the extracellular acidification rate (EAR) and oxygen uptake rate (OUR) with the BioChips of the IMOLA-IVD platform. In a proof-of-concept experiment, spheroids were perfused for 36h with cell culture medium before being exposed to medium with 1% sodium dodecyl sulphate (SDS) to lyse cells as a positive control. These microphysiometry studies revealed a repeatable pattern of extracellular acidification throughout the experiment, indicating the ability to monitor real-time metabolic activity of spheroids embedded in the newly designed tissue encapsulation. After perfusion for 36h with medium, SDS exposure resulted in an instant decrease in EAR and OUR signals from 37mV/h (±5) to 8mV/h (±8) and from 308mV/h (±21) to -2mV/h (±13), respectively. The presented spheroid monitoring system holds great potential as a method to automate screening and analysis of pharmaceutical agents using 3D multicellular spheroid models.

Electroswitchable Emission and Coloration Based on Single-Molecular Fluoran Derivatives

Multifunctional molecular switches exhibiting changes in both emission and coloration in response to various external triggers such as thermal, photo, and electric stimuli have attracted much attention for potential application as chemical sensors, biochemical labels, molecular logic gates, molecular memories, and display devices. In this study, we demonstrated reversible electrochemically induced high-contrast switching of the emission and coloration of fluoran molecules associated with changes in the intramolecular structure. In order to investigate changes in the photophysical properties induced by electrical stimuli, we measured cyclic voltammogram, absorption spetra, emission spectra, and molecular orbital calculation. The mechanism for this electroswitching was attributed to the reversible electrochemically induced closing and opening of the lactone ring in the fluoran molecule. All neutral fluoran molecules were colorless and did not exhibit any fluorescence, while the oxidized (lactone ring-opened form) molecule was yellow and displayed a green fluorescence as a consequence of the extended, planar, conjugated system. Furthermore, this fluoran molecule achieved reversible electroswitchable emission and coloration with high on/off contrast[1]. In order to achieve a quick response time and high reversibility of the electroswitching optical device for modulating both emission and coloration, a 1,4-benzoquinone (BQ) molecule was introduced as a bifunctional material. The inclusion of a redox-active moiety on the auxiliary electrode maintained the charge balance and acted as an EC molecule (transparent/yellow in neutral/reduced state, respectively). BQ acted as a bifunctional molecule, leading to quick and stable switching of both emission and coloration[2]. Reference [1] K. Kanazawa, K. Nakamura and N. Kobayashi, J. Phys. Chem. A, 118, 6026–6033 (2014).[2] K. Kanazawa, K. Nakamura and N. Kobayashi, Sol. Ener. Mater. Solar Cell., 145, 42-53 (2016). Figure 1

Separation of Microvesicles from Serological Samples Using Deterministic Lateral Displacement Effect

Label-free isolation and detection of extracellular vesicles are essential in many diagnostic and therapeutic approaches. Recently, there has been an interest in methods that avoid the use of biochemical labels, intrinsic biomarkers, or electrical polarizability to identify microvesicles (also referred to as microparticles) from serological samples. Here, we report a microfluidic device to separate circulating extracellular vesicles from serological samples using the deterministic lateral displacement principle. The device continuously fractionates label-free extracellular vesicles and cells according to size and membrane flexibility by displacing them perpendicularly to the fluid flow direction in a micro-fabricated array of posts. Experimental data and computational fluid dynamic simulations are presented to create a compelling argument that microvesicles from serological samples could be separated by deterministic lateral displacement arrays. Direct separation of different-sized micro- and nanospheres were demonstrated using a multi-stage separation strategy thus offering a potential route for novel cancer diagnostic approaches where microvesicles can be targeted and intercepted during cell-to-cell communication.

Silicon nanograss based impedance biosensor for label free detection of rare metastatic cells among primary cancerous colon cells, suitable for more accurate cancer staging

Detection of rare metastatic cells within a benign tumor is a key challenge to diagnose the cancerous stage of the patients tested by clinical human biopsy or pap smear samples. We have fabricated and tested a nanograssed silicon based bioelectronic device with the ability of detecting a few human colon invasive cancer cells (SW48) in a mixed cell culture of primary cancerous colon cells (HT29) without any biochemical labels. A discernible impedance change was elicited after the presence of 5% metastatic cells in the whole benign sample. The electric field penetration as well as current flow to metastatic cells is different from benign ones due to their different membrane dielectric parameters. Beta dispersion as one of intrinsic bioelectrical properties of the cell membrane in blocking the stimulating current flow in the range of kHz is the specific parameter involved in our diagnosis approach. It can reflect in-depth information about the dielectric properties and the pathological condition of a cell before and after metastatic transformation. Electrically active doped silicon nanograss structures owing to their superior nanocontacts with cell membrane can detect any slight variations in current being originated from the presence of rare metastatic cells on the surface of the sensing electrode. The experimental results revealed that bare doped silicon microelectrodes are incapable of resolving different grades of attached cells.

Sequential Array Cytometry: Multi-Parameter Imaging with a Single Fluorescent Channel

:Heterogeneity within the human population and within diseased tissues necessitates a personalized medicine approach to diagnostics and the treatment of diseases. Functional assays at the single-cell level can contribute to uncovering heterogeneity and ultimately assist in improved treatment decisions based on the presence of outlier cells. We aim to develop a platform for high-throughput, single-cell-based assays using well-characterized hydrodynamic cell isolation arrays which allow for precise cell and fluid handling. Here, we demonstrate the ability to extract spatial and temporal information about several intracellular components using a single fluorescent channel, eliminating the problem of overlapping fluorescence emission spectra. Integrated with imaging technologies such as wide field-of-view lens-free fluorescent imaging, fiber-optic array scanning technology, and microlens arrays, use of a single fluorescent channel will reduce the cost of reagents and optical components. Specifically, we sequentially stain hydrodynamically trapped cells with three biochemical labels all sharing the same fluorescence excitation and emission spectrum. These markers allow us to analyze the amount of DNA, and compare nucleus-to-cytoplasm ratio, as well as glycosylation of surface proteins. By imaging cells in real-time we enable measurements of temporal localization of cellular components and intracellular reaction kinetics, the latter is used as a measurement of multi-drug resistance. Demonstrating the efficacy of this single-cell analysis platform is the first step in designing and implementing more complete assays, aimed toward improving diagnosis and personalized treatments to complex diseases.

Publisher’s Note: “Review Article—Dielectrophoresis: Status of the theory, technology, and applications” [Biomicrofluidics 4, 022811 (2010)

A review is presented of the present status of the theory, the developed technology and the current applications of dielectrophoresis (DEP). Over the past 10 years around 2000 publications have addressed these three aspects, and current trends suggest that the theory and technology have matured sufficiently for most effort to now be directed towards applying DEP to unmet needs in such areas as biosensors, cell therapeutics, drug discovery, medical diagnostics, microfluidics, nanoassembly, and particle filtration. The dipole approximation to describe the DEP force acting on a particle subjected to a nonuniform electric field has evolved to include multipole contributions, the perturbing effects arising from interactions with other cells and boundary surfaces, and the influence of electrical double-layer polarizations that must be considered for nanoparticles. Theoretical modelling of the electric field gradients generated by different electrode designs has also reached an advanced state. Advances in the technology include the development of sophisticated electrode designs, along with the introduction of new materials (e.g., silicone polymers, dry film resist) and methods for fabricating the electrodes and microfluidics of DEP devices (photo and electron beam lithography, laser ablation, thin film techniques, CMOS technology). Around three-quarters of the 300 or so scientific publications now being published each year on DEP are directed towards practical applications, and this is matched with an increasing number of patent applications. A summary of the US patents granted since January 2005 is given, along with an outline of the small number of perceived industrial applications (e.g., mineral separation, micropolishing, manipulation and dispensing of fluid droplets, manipulation and assembly of micro components). The technology has also advanced sufficiently for DEP to be used as a tool to manipulate nanoparticles (e.g., carbon nanotubes, nano wires, gold and metal oxide nanoparticles) for the fabrication of devices and sensors. Most efforts are now being directed towards biomedical applications, such as the spatial manipulation and selective separation/enrichment of target cells or bacteria, high-throughput molecular screening, biosensors, immunoassays, and the artificial engineering of three-dimensional cell constructs. DEP is able to manipulate and sort cells without the need for biochemical labels or other bioengineered tags, and without contact to any surfaces. This opens up potentially important applications of DEP as a tool to address an unmet need in stem cell research and therapy.

Review article-dielectrophoresis: status of the theory, technology, and applications.

A review is presented of the present status of the theory, the developed technology and the current applications of dielectrophoresis (DEP). Over the past 10 years around 2000 publications have addressed these three aspects, and current trends suggest that the theory and technology have matured sufficiently for most effort to now be directed towards applying DEP to unmet needs in such areas as biosensors, cell therapeutics, drug discovery, medical diagnostics, microfluidics, nanoassembly, and particle filtration. The dipole approximation to describe the DEP force acting on a particle subjected to a nonuniform electric field has evolved to include multipole contributions, the perturbing effects arising from interactions with other cells and boundary surfaces, and the influence of electrical double-layer polarizations that must be considered for nanoparticles. Theoretical modelling of the electric field gradients generated by different electrode designs has also reached an advanced state. Advances in the technology include the development of sophisticated electrode designs, along with the introduction of new materials (e.g., silicone polymers, dry film resist) and methods for fabricating the electrodes and microfluidics of DEP devices (photo and electron beam lithography, laser ablation, thin film techniques, CMOS technology). Around three-quarters of the 300 or so scientific publications now being published each year on DEP are directed towards practical applications, and this is matched with an increasing number of patent applications. A summary of the US patents granted since January 2005 is given, along with an outline of the small number of perceived industrial applications (e.g., mineral separation, micropolishing, manipulation and dispensing of fluid droplets, manipulation and assembly of micro components). The technology has also advanced sufficiently for DEP to be used as a tool to manipulate nanoparticles (e.g., carbon nanotubes, nano wires, gold and metal oxide nanoparticles) for the fabrication of devices and sensors. Most efforts are now being directed towards biomedical applications, such as the spatial manipulation and selective separationenrichment of target cells or bacteria, high-throughput molecular screening, biosensors, immunoassays, and the artificial engineering of three-dimensional cell constructs. DEP is able to manipulate and sort cells without the need for biochemical labels or other bioengineered tags, and without contact to any surfaces. This opens up potentially important applications of DEP as a tool to address an unmet need in stem cell research and therapy.

Label-free cell separation and sorting in microfluidic systems

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. FigureA wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties

Dielectrophoresis: A Review of Applications for Stem Cell Research

Dielectrophoresis can discriminate distinct cellular identities in heterogeneous populations, and monitor cell state changes associated with activation and clonal expansion, apoptosis, and necrosis, without the need for biochemical labels. Demonstrated capabilities include the enrichment of haematopoetic stem cells from bone marrow and peripheral blood, and adult stem cells from adipose tissue. Recent research suggests that this technique can predict the ultimate fate of neural stem cells after differentiation before the appearance of specific cell-surface proteins. This review summarises the properties of cells that contribute to their dielectrophoretic behaviour, and their relevance to stem cell research and translational applications.

Cell Physiometry Tools Based on Dielectrophoresis

A cell physiometry system is described for characterizing and separating cells, and performing cell-based assays, using dielectrophoresis (DEP). Cells, or mixtures of cells, are suspended in a chamber containing an array of microelectrodes located on the chamber's bottom surface. A sequence of radio frequency signals is automatically applied to the microelectrodes, and images of the DEP-induced motions of the cells are captured and analyzed to determine a characteristic parameter known as the DEP cross-over frequency. Once a cell population has been characterized in this way, the same apparatus can be used to selectively isolate cells for additional biochemical, physical, or genetic analysis. Biochemical labels or bioengineered tags such as fluorescent markers or antibody-coated beads are not required. Cell separations are achieved by “electronically tuning” into the different cell types by superimposing different signal frequencies onto the microelectrodes, using both stationary and traveling wave DEP signals. Examples of separating different types of blood cell are given, together with descriptions of cell-based bioassays that monitor physiological changes that accompany trans-membrane signaling events, apoptosis, and the aging of cell cultures.

The neostriatal mosaic: compartmental distribution of calcium-binding protein and parvalbumin in the basal ganglia of the rat and monkey.

Calcium-binding protein (CaBP) and parvalbumin are two proteins that are expressed in brain and bind calcium in the micromolar range. The immunohistochemical distribution of these two proteins was examined in the basal ganglia of rats and rhesus monkeys. In the striatum, CaBP immunoreactivity is localized to a subset of striatonigral projection neurons; CaBP-positive neurons are distributed in areas containing somatostatin-immunoreactive fibers and not in the complementary areas containing dense mu opiate-receptor binding. These biochemical labels mark, respectively, the matrix and patch compartments of the striatum. Previous studies have shown that striatal matrix neurons project to the substantia nigra pars reticulata, whereas striatal patch neurons project to the substantia nigra pars compacta. Consistent with the restricted localization of CaBP in the matrix projection neurons is the confinement of CaBP-immunoreactive afferent fibers to the pars reticulata. CaBP is also localized to a portion of dopaminergic and a few nondopaminergic neurons in the substantia nigra pars compacta and in most dopaminergic neurons in the ventral tegmental area. Parvalbumin immunoreactivity is localized to a subset of substantia nigra pars reticulata neurons and their axons. In the lateral striatum, some medium-sized aspiny interneurons are also parvalbumin immunoreactive. The distinct distributions of CaBP and parvalbumin in the basal ganglia are discussed in terms of their possible roles as intracellular calcium buffer systems related to the physiologic response properties of the neurons in which they are contained.