Rapid Buffer Exchange Research Articles

Supercoiling DNA optically

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

A Microfluidic-Based Microscopy Platform for Continuous Interrogation of Trypanosoma brucei during Environmental Perturbation.

The African trypanosome, Trypanosoma brucei, is the causative agent of human African trypanosomiasis (HAT). African trypanosomes are extracellular parasites that possess a single flagellum that imparts a high degree of motility to the microorganisms. In addition, African trypanosomes show significant metabolic and structural adaptation to environmental conditions. Analysis of the ways that environmental cues affect these organisms generally requires rapid perfusion experiments in combination with single-cell imaging, which are difficult to apply under conditions of rapid motion. Microfluidic devices have been used previously as a strategy for trapping small motile cells in a variety of organisms, including trypanosomes; however, in the past, such devices required individual fabrication in a cleanroom, limiting their application. Here we demonstrate that a commercial microfluidic device, typically used for bacterial trapping, can trap bloodstream and procyclic form trypanosomes, allowing for rapid buffer exchange via perfusion. As a result, time-lapse single-cell microscopy images of these highly motile parasites were acquired during environmental variations. Using these devices, we have been able to perform and analyze perfusion-based single-cell tracking experiments of the responses of the parasite to changes in glucose availability, which is a major step in resolving the mechanisms of adaptation of kinetoplasts to their individual biological niches; we demonstrate utility of this tool for making measurements of procyclic form trypanosome intracellular glucose levels as a function of changes in extracellular glucose concentrations. These experiments demonstrate that cytosolic glucose equilibrates with external conditions as fast as, or faster than, the rate of solution exchange in the instrument.

Automated Capillary Electrophoresis System for Fast Single-Cell Analysis

Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. Eighty-eight percent of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.

1P221 急速緩衝液交換法による時間分解全反射赤外分光法の開発(13C. 生体膜・人工膜:興奮・チャンネル,ポスター,日本生物物理学会年会第51回(2013年度))

1P221 急速緩衝液交換法による時間分解全反射赤外分光法の開発(13C. 生体膜・人工膜:興奮・チャンネル,ポスター,日本生物物理学会年会第51回(2013年度))

Microfluidic-Enabled Real-Time Imaging of Lipid Domains in Bilayer Membranes

A microfluidic bilayer lipid membrane system supporting correlated opto-electrical membrane characterization has been developed and applied to the real time imaging of lipid domains by confocal microscopy. Annulus supported bilayers are formed by a novel “kiss and retreat” method and the device supports rapid buffer exchange in both chambers. Additionally, the system has been extended for the creation of solvent-free asymmetric bilayers by a microfluidic adaptation of the monolayer folding method. Because the dynamic folding process itself may be imaged, the very early stages of phase separation and domain growth may be observed. By enabling the simultaneous measurement of transmembrane current and optical observation of lipid microdomains in real-time, the microfluidic platforms open the door to new multi-domain measurements of dynamic lipid-lipid and lipid-ligand interactions that are not feasible using established tools. (left) Confocal image sequences of liquid ordered domains in a bilayer formed by kiss and retreat method. Under ∼5 Pa transmembrane pressure, lo domains are observed to emerge, grow and merge. (right) Confocal image sequence of bilayer formation by monolayer folding, sequentially revealing a single monolayer, the folding process, and the initial emergence of liquid ordered domains.

Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry.

A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel, where cellular constituents were separated, and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the alpha and beta subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute, demonstrating the potential of this method for high-throughput single cell analysis.

Microfluidic Device for Single-Molecule Experiments with Enhanced Photostability

A microfluidic device made of polydimethylsiloxane (PDMS) addresses key limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Photobleaching is dramatically reduced by deoxygenation via gas diffusion through porous channel walls. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation minimizes surface-sample contacts and allows the in situ addition and combination of other reagents.

Single-biomolecule observation with micro one-way valves for rapid buffer exchange

This paper describes a method for the rapid exchange of buffer solution during single-molecule observation. We use a simple, transparent, all-plastic one-way valve integrated on a coverslip on a flow cell. The valve is formed using a membrane made of parylene covering a microhole. It opens when a buffer solution is introduced from the microhole (the flow pushes the cover membrane) and closes when suctioning the buffer solution (the membrane is pulled back and seals the microhole to prevent the diffusion of the solution). To check valve performance, we observed the response of a rotary biomotor, F1-ATPase, for several buffer solutions in the fabricated chamber.

Electromechanical Properties of Pressure-Actuated Poly(dimethylsiloxane) Microfluidic Push-Down Valves

Pressure-actuated poly(dimethylsiloxane) (PDMS) valves have been characterized with respect to their electromechanical properties. Measurements of the valve opening and closing times, threshold pressures, and impedance spectra for closed valves can be used to assess the quality of the devices in general, determine their suitability for specialized applications, such as providing electrical isolated fluidic compartments for planar patch clamping, and specify ideal operating conditions. For our particular valve designs, we report valve opening times of the order of 10-100 micros, making them suitable for rapid buffer exchange applications. They can effectively provide reversible electrical isolation between adjacent fluidic compartments with typical resistances of 5 Gohms in the closed state, which meets the gigaohm requirement for patch clamping applications.

Fabrication of simple micro one-way valves of rapid buffer exchange in a chamber for single bio-molecules observation

Fabrication of simple micro one-way valves of rapid buffer exchange in a chamber for single bio-molecules observation

Light-Activated Desorption of Photoactive Polyelectrolytes from Supported Lipid Bilayers

Phospholipid vesicles and supported bilayers have emerged as a promising platform for the development of biorecognition devices. To expand the capabilities of such biochips, it becomes desirable to direct and control the assembly of lipid structures into more sophisticated architectures. As one step toward this goal, we demonstrate the photoregulated desorption of a new class of polymer from lipid bilayers. The neutral, hydrophobic polymer resides within the bilayer under mild pH and ambient conditions. However, it contains side groups that can undergo excited state proton transfer (ESPT). The polymer therefore behaves as a polyelectrolyte when exposed to IN light. With the ensuing increase in hydrophilicity, the molecule is spontaneously ejected from the bilayer. Quartz crystal microbalance measurements with dissipation monitoring (QCM-D) have recorded this process and have shown that a rapid buffer exchange during light exposure results in efficient removal of the polymer from the system. Three polymers were tested in all: a polyanion, a polyeation, and a polyzwitterion. A one-step approach to the synthesis of the monomer, performed under relatively mild reaction conditions, made it possible to synthesize each polymer in one step. (Less)

Integration of polymeric membranes with microfluidic networks for bioanalytical applications.

The concept of microfluidics has significantly influenced the design and the implementation of modern bioanalytical systems due to the fact that these miniaturized devices can handle and manipulate samples in a much more efficient way than conventional instruments. In an analogy to the development of microelectronics, increasingly sophisticated devices with greater functionalities have become one of the major goals being pursued in the area of micrototal analysis systems. The incorporation of polymeric membranes into microfluidic networks has therefore been employed in an effort to enhance the functionalities of these microfabricated devices. These commercially available membranes are porous, flexible, mechanically robust and compatible with plastic microfluidic networks. The large surface area-to-volume ratio of porous membrane media is particularly important for achieving rapid buffer exchange during microdialysis and obtaining ultrahigh concentration of adsorbed enzymes for various biochemical reactions. Furthermore, the membrane pore diameter in the sub-microm range eliminates the constraints of diffusional mass-transfer resistance for performing chiral separation using adsorbed protein as the chiral stationary phase. A review on the recent advancement in the integration of polymeric membranes with microfluidic networks is presented for their widespread applications in bioanalytical chemistry.

Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5.

Seven-transmembrane segment, G protein-coupled receptors play central roles in a wide range of biological processes, but their characterization has been hindered by the difficulty of obtaining homogeneous preparations of native protein. We have created paramagnetic proteoliposomes containing pure and oriented CCR5, a seven-transmembrane segment protein that serves as the principal coreceptor for human immunodeficiency virus (HIV-1). The CCR5 proteoliposomes bind the HIV-1 gp120 envelope glycoprotein and conformation-dependent antibodies against CCR5. The binding of gp120 was enhanced by a soluble form of the other HIV-1 receptor, CD4, but did not require additional cellular proteins. Paramagnetic proteoliposomes are uniform in size, stable in a broad range of salt concentrations and pH, and can be used in FACS and competition assays typically applied to cells. Integral membrane proteins can be inserted in either orientation into the liposomal membrane. The magnetic properties of these proteoliposomes facilitate rapid buffer exchange useful in multiple applications. As an example, the CCR5-proteoliposomes were used to select CCR5-specific antibodies from a recombinant phage display library. Thus, paramagnetic proteoliposomes should be useful tools in the analysis of membrane protein interactions with extracellular and intracellular ligands, particularly in establishing screens for inhibitors.

Analysis of transcription complexes and effects of ligands by microelectrospray ionization mass spectrometry.

The human vitamin D receptor (VDR) and retinoid X receptor-alpha (RXRalpha) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known endogenous ligands 1alpha,25-dihydroxyvitamin D3 (1, 25-[OH]2D3) and 9-cis-retinoic acid (9-c-RA). We used rapid buffer exchange gel filtration in conjunction with microelectrospray ionization mass spectrometry (microESI-MS) to study the binding of these receptors to the osteopontin vitamin D response element (OP VDRE). In the absence of DNA, both VDR and RXRalpha existed primarily as monomers, but in the presence of OP VDRE, homodimeric RXRalpha and heterodimeric RXRalpha-VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA increased RXRalpha homodimer-OP VDRE complexes, and addition of 1,25-(OH) 2D3 resulted in formation of 1, 25-(OH)2D 3-VDR-RXRalpha-OP VDRE complexes. Addition of low-affinity binding ligands had no detectable effect on the VDR-RXRalpha-OP VDRE transcription complex. These results demonstrate the utility of microESI-MS in analyzing multimeric, high-molecular-weight protein-protein and protein-DNA complexes, and the effects of ligands on these transcriptional complexes.

Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor.

A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 degrees C, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.

A microcell for small-volume dialysis

We have designed a microcell for use in the dialysis of microliter sample volumes. The microcell was easily constructed from inexpensive laboratory materials and allowed for a rapid buffer exchange with total protein recovery.