Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.
20 Related JoVE Articles!
Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis
Institutions: Virginia Tech, Virginia Tech.
Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).
Biomedical Engineering, Issue 79, Medicine, Cellular Biology, Molecular Biology, Bioengineering, Anatomy, Physiology, Biophysics, Physics, Microfluidics, Cell Separation, Microfluidic Analytical Techniques, Electrophoresis, Microchip, cancer diagnosis, cell enrichment, cell sorting, microfluidics, dielectrophoresis, Lab on a chip, cells, imaging
Development of a 3D Graphene Electrode Dielectrophoretic Device
Institutions: Michigan Technological University, Michigan Technological University, XG Sciences, Inc..
The design and fabrication of a novel 3D electrode microdevice using 50 µm thick graphene paper and 100 µm double sided tape is described. The protocol details the procedures to construct a versatile, reusable, multiple layer, laminated dielectrophoresis chamber. Specifically, six layers of 50 µm x 0.7 cm x 2 cm graphene paper and five layers of double sided tape were alternately stacked together, then clamped to a glass slide. Then a 700 μm diameter micro-well was drilled through the laminated structure using a computer-controlled micro drilling machine. Insulating properties of the tape layer between adjacent graphene layers were assured by resistance tests. Silver conductive epoxy connected alternate layers of graphene paper and formed stable connections between the graphene paper and external copper wire electrodes. The finished device was then clamped and sealed to a glass slide. The electric field gradient was modeled within the multi-layer device. Dielectrophoretic behaviors of 6 μm polystyrene beads were demonstrated in the 1 mm deep micro-well, with medium conductivities ranging from 0.0001 S/m to 1.3 S/m, and applied signal frequencies from 100 Hz to 10 MHz. Negative dielectrophoretic responses were observed in three dimensions over most of the conductivity-frequency space and cross-over frequency values are consistent with previously reported literature values. The device did not prevent AC electroosmosis and electrothermal flows, which occurred in the low and high frequency regions, respectively. The graphene paper utilized in this device is versatile and could subsequently function as a biosensor after dielectrophoretic characterizations are complete.
Physics, Issue 88, graphene paper, dielectrophoresis, graphene electrodes, 3D laminated microdevice, polystyrene beads, cell diagnostics
Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow
Institutions: University of Illinois at Urbana-Champaign, University of Illinois at Urbana-Champaign, University of Illinois at Urbana-Champaign, University of Illinois at Urbana-Champaign.
Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.
Bioengineering, Issue 48, Dielectrophoresis, microfluidic, laminar flow, cell sorting, Human colon adenocarcinoma
Solid-state Graft Copolymer Electrolytes for Lithium Battery Applications
Institutions: Massachusetts Institute of Technology, Massachusetts Institute of Technology.
Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (<80 °C), flammable, and volatile organic electrolytes. These organic based electrolyte systems are viable at ambient temperatures, but require a cooling system to ensure that temperatures do not exceed 80 °C. These cooling systems tend to increase battery costs and can malfunction which can lead to battery malfunction and explosions, thus endangering human life. Increases in petroleum prices lead to a huge demand for safe, electric hybrid vehicles that are more economically viable to operate as oil prices continue to rise. Existing organic based electrolytes used in lithium ion batteries are not applicable to high temperature automotive applications. A safer alternative to organic electrolytes is solid polymer electrolytes. This work will highlight the synthesis for a graft copolymer electrolyte (GCE) poly(oxyethylene) methacrylate (POEM) to a block with a lower glass transition temperature (Tg
) poly(oxyethylene) acrylate (POEA). The conduction mechanism has been discussed and it has been demonstrated the relationship between polymer segmental motion and ionic conductivity indeed has a Vogel-Tammann-Fulcher (VTF) dependence. Batteries containing commercially available LP30 organic (LiPF6
in ethylene carbonate (EC):dimethyl carbonate (DMC) at a 1:1 ratio) and GCE were cycled at ambient temperature. It was found that at ambient temperature, the batteries containing GCE showed a greater overpotential when compared to LP30 electrolyte. However at temperatures greater than 60 °C, the GCE cell exhibited much lower overpotential due to fast polymer electrolyte conductivity and nearly the full theoretical specific capacity of 170 mAh/g was accessed.
Materials Science, Issue 78, Physics, Chemistry, Chemical Engineering, Chemistry and Materials, Engineering, Lithium Batteries, Polymer Electrolytes, Polyethylene oxide, Graft Copolymer, LiFePO4, synthesis, polymers
Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation
Institutions: University of Washington, Fred Hutchinson Cancer Research Center , University of Washington, Fred Hutchinson Cancer Research Center , Fred Hutchinson Cancer Research Center .
A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1
However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2
However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3
A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8
The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10
The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11
Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10
The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.
Bioengineering, Issue 55, Surface plasmon, optical trapping, optical tweezers, plasmonic trapping, cell manipulation, optical manipulation
On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids
Institutions: Stanford University .
Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2
and efficient separation and extraction based on ionic mobility.3
For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8
and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13
). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli
cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.
Bioengineering, Issue 61, Isotachophoresis, electrokinetics, microfluidics, sample preparation
Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures
Institutions: University of Houston.
The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3
, drug delivery4
, improved hydrocarbon recovery5-7
, and flowable electrodes for energy storage8
. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9
. Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10
. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37
. In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.
Chemistry, Issue 87, confocal microscopy, particle tracking, colloids, suspensions, confinement, gelation, microfluidics, image correlation, dynamics, suspension flow
Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids
Institutions: The University of Texas at Austin.
An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data.
The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.1
Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.
Physics, Issue 83, chemical engineering, settling velocity, Reynolds number, shear thinning, wall retardation
Reservoir Condition Pore-scale Imaging of Multiple Fluid Phases Using X-ray Microtomography
Institutions: Imperial College London.
X-ray microtomography was used to image, at a resolution of 6.6 µm, the pore-scale arrangement of residual carbon dioxide ganglia in the pore-space of a carbonate rock at pressures and temperatures representative of typical formations used for CO2
storage. Chemical equilibrium between the CO2
, brine and rock phases was maintained using a high pressure high temperature reactor, replicating conditions far away from the injection site. Fluid flow was controlled using high pressure high temperature syringe pumps. To maintain representative in-situ
conditions within the micro-CT scanner a carbon fiber high pressure micro-CT coreholder was used. Diffusive CO2
exchange across the confining sleeve from the pore-space of the rock to the confining fluid was prevented by surrounding the core with a triple wrap of aluminum foil. Reconstructed brine contrast was modeled using a polychromatic x-ray source, and brine composition was chosen to maximize the three phase contrast between the two fluids and the rock. Flexible flow lines were used to reduce forces on the sample during image acquisition, potentially causing unwanted sample motion, a major shortcoming in previous techniques. An internal thermocouple, placed directly adjacent to the rock core, coupled with an external flexible heating wrap and a PID controller was used to maintain a constant temperature within the flow cell. Substantial amounts of CO2
were trapped, with a residual saturation of 0.203 ± 0.013, and the sizes of larger volume ganglia obey power law distributions, consistent with percolation theory.
Medicine, Issue 96, Reservoir condition, micro-CT, Multi-phase, Carbon Storage, Capillary Trapping, Pore-scale
Transport of Surface-modified Carbon Nanotubes through a Soil Column
Institutions: Nalanda University, Uppsala University.
Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.
Chemistry, Issue 98, Carbon nanotubes, functionalization of carbon nanotubes, solution chemistry, flow rate, porous media
Picoinjection of Microfluidic Drops Without Metal Electrodes
Institutions: Unversity of California, San Francisco.
Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.
Bioengineering, Issue 86, Droplet microfluidics, picoinjection, lab on a chip, electrodes, microfabrication
Development of Whispering Gallery Mode Polymeric Micro-optical Electric Field Sensors
Institutions: Southern Methodist University.
Optical modes of dielectric micro-cavities have received significant attention in recent years for their potential in a broad range of applications. The optical modes are frequently referred to as "whispering gallery modes" (WGM) or "morphology dependent resonances" (MDR) and exhibit high optical quality factors. Some proposed applications of micro-cavity optical resonators are in spectroscopy1
, micro-cavity laser technology2
, optical communications3-6
as well as sensor technology. The WGM-based sensor applications include those in biology7
, trace gas detection8
, and impurity detection in liquids9
. Mechanical sensors based on microsphere resonators have also been proposed, including those for force10,11
and wall shear stress14
. In the present, we demonstrate a WGM-based electric field sensor, which builds on our previous studies15,16
. A candidate application of this sensor is in the detection of neuronal action potential.
The electric field sensor is based on polymeric multi-layered dielectric microspheres. The external electric field induces surface and body forces on the spheres (electrostriction effect) leading to elastic deformation. This change in the morphology of the spheres, leads to shifts in the WGM. The electric field-induced WGM shifts are interrogated by exciting the optical modes of the spheres by laser light. Light from a distributed feedback (DFB) laser (nominal wavelength of ~ 1.3 μm) is side-coupled into the microspheres using a tapered section of a single mode optical fiber. The base material of the spheres is polydimethylsiloxane (PDMS). Three microsphere geometries are used: (1) PDMS sphere with a 60:1 volumetric ratio of base-to-curing agent mixture, (2) multi layer sphere with 60:1 PDMS core, in order to increase the dielectric constant of the sphere, a middle layer of 60:1 PDMS that is mixed with varying amounts (2% to 10% by volume) of barium titanate and an outer layer of 60:1 PDMS and (3) solid silica sphere coated with a thin layer of uncured PDMS base. In each type of sensor, laser light from the tapered fiber is coupled into the outermost layer that provides high optical quality factor WGM (Q ~ 106
). The microspheres are poled for several hours at electric fields of ~ 1 MV/m to increase their sensitivity to electric field.
Mechanical Engineering, Issue 71, Physics, Optics, Materials Science, Chemical Engineering, electrostatics, optical fibers, optical materials, optical waveguides, optics, optoelectronics, photonics, geometrical optics, sensors, electric field, dielectric resonators, micro-spheres, whispering gallery mode, morphology dependent resonance, PDMS
AC Electrokinetic Phenomena Generated by Microelectrode Structures
Institutions: Drexel University, Drexel University.
The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2
. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5
, environmental monitoring6
and homeland security7
In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior.
It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.
Bioengineering, Issue 17, AC Electrokinetics, AC Electroosmosis, Dielectrophoresis, Electrothermal Effect, Microelectrode, Microfluidics, Simulation, Microsphere, Microfabrication
Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy
Institutions: University of Guelph.
Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus
USA100 cells on stainless steel and gold using KPFM.
Bioengineering, Issue 93, Kelvin probe force microscopy, atomic force microscopy, surface potential, stainless steel, microbial attachment, bacterial biofilms, methicillin-resistant Staphylococcus aureus
Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions
Institutions: University of Michigan - Ann Arbor.
The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4
make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5
in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11
. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12
. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13
, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14
onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11
Bioengineering, Issue 77, Chemical Engineering, Biochemistry, Biophysics, Electrical Engineering, Nanotechnology, Biosensing Techniques, carbon nanotubes (synthesis and properties), bioelectronic instruments (theory and techniques), Carbon nanotube, biosensor, frequency mixing, biotin, streptavidin, poly-dimethylsiloxane
Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2
. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5
. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6
. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7
. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9
. Bevacizumab however failed to prolong overall survival in a recent phase III trial26
. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12
, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores
Institutions: University of Ottawa, University of Ottawa.
Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1
. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3
and size control is nontrivial4,5
. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ
in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.
Physics, Issue 80, Nanopore, Solid-State, Size Control, Noise Reduction, Translocation, DNA, High Electric Fields, Nanopore Conditioning
The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry
Institutions: Washington University in St. Louis.
The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV
1.5), expressed in Xenopus
oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV
1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1
. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.
Developmental Biology, Issue 85, Voltage clamp, Cut-open, Oocyte, Voltage Clamp Fluorometry, Sodium Channels, Ionic Currents, Xenopus laevis
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
Institutions: Freie Universität Berlin.
Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102
repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4
, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.
Biophysics, Issue 88, bacteriorhodopsin, channelrhodopsin, attenuated total reflection, proton transfer, protein dynamics, infrared spectroscopy, time-resolved spectroscopy, step-scan, membrane proteins, singular value decomposition