Microfluidic device coupled with a microfabricated oxygen electrode for the measurement of bactericidal activity of neutrophil-like cells.

A microfluidic device coupled with a microfabricated Clark-type oxygen electrode was used to measure the bactericidal activity of neutrophil-like cells differentiated from HL-60 cells. The neutrophil-like cells and Escherichia coli (E. coli) cells were cultured in the same medium, which was introduced into the flow channel of the device. Changes in the respiratory activity of E. coli were measured as changes in the consumption of dissolved oxygen. As the activity of the neutrophil-like cells increased, the rate of elimination of E. coli increased. The accompanying decrease in the number of E. coli reduced the consumption of dissolved oxygen. The changes were actually observed as changes in generated current. A distinct difference in changes in dissolved oxygen concentrations was observed between E. coli cells co-incubated with IFN-γ-activated or non-activated neutrophil-like cells. The required sample volume was less than 10 µL, and results could be obtained within 1-2 h. The device may be useful for the assessment of psychological stresses that affect the activity of neutrophils.

A Versatile and Rapidly Deployable Device to Enable Spatiotemporal Observations of the Sessile Microbes and Environmental Surfaces.

Although microbes typically associate with surfaces, detailed observations of surface-associated microbes on natural substrata are technically challenging. We herein introduce a flow channel device named the Stickable Flow Device, which is easily configurable and deployable on various surfaces for the microscopic imaging of environmental microbes. We demonstrated the utility of this device by creating a flow channel on different types of surfaces including live leaves. This device enables the real-time imaging of bacterial biofilms and their substrata. The Stickable Flow Device expands the limits of conventional real-time imaging systems, thereby contributing to a deeper understanding of microbe-surface interactions on various surfaces.

Microfluidic Separation of Redox Reactions for Coulometry Based on Metallization at the Mixed Potential.

Coulometric detection of an analyte in a solution at nanoliter scale was conducted by having redox reactions proceed simultaneously on a platinum electrode. The analyte was oxidized on a part of the electrode in one flow channel and silver was deposited on an array of circular microelectrodes formed in another flow channel at a mixed potential. Coulometric determination of the deposited silver showed a steep change in the generated charge as a result of the complete oxidation of silver. The short measurement time after the start of the coulometry suppressed the increase in background charge, resulting in significant lowering of the detection limit. The lower detection limit for H2O2 was 30 nM (3σ). To improve selectivity and minimize the influence of coexisting interferents, the shifting of the mixed potential, application of a permselective membrane, and electrochemical elimination of the interferents were effective modifications.

Programmed Transport and Release of Cells by Self-Propelled Micromotors.

Autonomous transport and release of bacterial cells by self-propelled micromotors were achieved. The motors consisted of zinc and platinum hemispheres formed on polystyrene beads and moved as a result of simultaneous redox reactions occurring on both metal ends. The highly negative redox potential of zinc enabled the selection of a wide variety of organic redox compounds as fuels, such as methanol and p-benzoquinone. The movement of motors was observed in solutions of fuels. To realize autonomous capture, transport, and release of cargo, a self-assembled monolayer (SAM) was formed on the platinum part of the motor. This SAM could be desorbed by coupling the reaction with the dissolution of zinc, which could also be controlled by adjusting the concentration of Zn(2+) ions. Escherichia coli (E. coli) cells were captured by the motor (due to hydrophobic interactions), transported, and released following SAM desorption at the mixed potential.

Switchable Hydrophobic Valve for Controlled Microfluidic Processing.

A simple microfluidic valve, without any moving parts, is presented that can control solution flow on demand in microchannels of many different materials using a low-power electric signal. Many independently operating valves can easily be integrated into complex microfluidic systems. The valve consists of a self-assembled monolayer (SAM) formed on a platinum electrode that is incorporated directly in the microchannel. The normally-on valve stops the solution flow due to a hydrophobic SAM on the electrode surface. The solution is allowed to pass the valve by applying a potential to the electrode, which removes the SAM due to reductive desorption. The valve operation is highly stable and has switching times of the order of 1 s. The valve is ideal for controlled solution manipulation in integrated micro-analytical systems and autonomous microfluidic systems.

Taking it one step at a time in homologous recombination repair.

The individual steps in the process of homologous recombination are particularly amenable to analysis by single-molecule imaging and manipulation experiments. Over the past 20 years these have provided a wealth of new information on the DNA transactions that make up this vital process. Exciting progress in developing new tools and techniques to analyze more complex components, dynamic reaction steps and molecular coordination continues at a rapid pace. Here we highlight recent results and indicate some emerging techniques likely to produce the next stage of advanced insight into homologous recombination. In this and related fields the future is bright.

Microdevice for on-site fish freshness checking based on K-value measurement.

An electrochemical microfluidic device with two sensing sites in the upper and lower streams of a flow channel was fabricated to measure the K-value as a means of evaluating the freshness of fish. In this device, plugs of solutions were processed using mechanisms to place a plug at the sensing site and to merge and mix two plugs in a single flow channel. The sums of ATP-related compound concentrations used for the calculation of the K-value were measured at the first and second sensing sites. The ratio of the output currents agreed well with the K-value calculated from predetermined concentrations in standard solutions. The K-values of jack mackerel, yellow tail, and sea bream extracts were then obtained using the device and were found to agree well with those obtained by high-performance liquid chromatography (HPLC). In addition, the changes in the K-value with time were observed to depend strongly on the type of fish for these three fish extracts.

Colocalization of quantum dots by reactive molecules carried by motor proteins on polarized microtubule arrays.

The field of microfluidics has drastically contributed to downscale the size of benchtop experiments to the dimensions of a chip without compromising results. However, further miniaturization and the ability to directly manipulate individual molecules require a platform that permits organized molecular transport. The motor proteins and microtubules that carry out orderly intracellular transport are ideal for driving in vitro nanotransport. Here, we demonstrate that a reconstruction of the cellular kinesin/dynein-microtubule system in nanotracks provides a molecular total analysis system (MTAS) to control massively parallel chemical reactions. The mobility of kinesin and a microtubule dissociation method enable orientation of a microtubule in an array for directed transport of reactive molecules carried by kinesin or dynein. The binding of glutathione S-transferase (GST) to glutathione (GSH) and the binding of streptavidin to biotin are visualized as colocalizations of quantum dots (Q-dots) when motor motilities bring them into contact. The organized nanotransport demonstrated here suggests the feasibility of using our platform to perform parallel biochemical reactions focused at the molecular level.

Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54.

High-speed AFM (atomic force microscopy also called scanning force microscopy) provides nanometre spatial resolution and sub-second temporal resolution images of individual molecules. We exploit these features to study diffusion and motor activity of the RAD54 DNA repair factor. Human RAD54 functions at critical steps in recombinational-DNA repair. It is a member of the Swi2/Snf2 family of chromatin remodelers that translocate on DNA using ATP hydrolysis. A detailed single molecular description of DNA-protein interactions shows intermediate states and distribution of variable states, usually hidden by ensemble averaging. We measured the motion of individual proteins using single-particle tracking and observed that random walks were affected by imaging-buffer composition. Non-Brownian diffusion events were characterized in the presence and in the absence of nucleotide cofactors. Double-stranded DNA immobilized on the surface functioned as a trap reducing Brownian motion. Distinct short range slides and hops on DNA were visualized by high-speed AFM. These short-range interactions were usually inaccessible by other methods based on optical resolution. RAD54 monomers displayed a diffusive behavior unrelated to the motor activity.

Motion of the Ca2+-pump captured.

Studies of ion pumps, such as ATP synthetase and Ca(2+)-ATPase, have a long history. The crystal structures of several kinds of ion pump have been resolved, and provide static pictures of mechanisms of ion transport. In this study, using fast-scanning atomic force microscopy, we have visualized conformational changes in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) in real time at the single-molecule level. The analyses of individual SERCA molecules in the presence of both ATP and free Ca(2+) revealed up-down structural changes corresponding to the Albers-Post scheme. This fluctuation was strongly affected by the ATP and Ca(2+) concentrations, and was prevented by an inhibitor, thapsigargin. Interestingly, at a physiological ATP concentrations, the up-down motion disappeared completely. These results indicate that SERCA does not transit through the shortest structure, and has a catalytic pathway different from the ordinary Albers-Post scheme under physiological conditions.

Acid-sensing ion channel (ASIC) 1a undergoes a height transition in response to acidification.

The acid-sensing ion channel (ASIC) 1a is known to assemble as a homotrimer. Here, we used atomic force microscopy to image ASIC1a, integrated into lipid bilayers, at pH 7.0 and pH 6.0. The triangular appearance of the channel was clearly visible. A height distribution for the channels at pH 7.0 had two peaks, at 2 and 4 nm, likely representing the intracellular and extracellular domains, respectively. At pH 6.0 the 2-nm peak remained, but the higher peak shifted to 6 nm. Hence, the extracellular domain of the channel becomes 'taller' after acidification.

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy.

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within approximately 50nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein-DNA interaction in sub-seconds time scale.

Human transcriptional coactivator PC4 stimulates DNA end joining and activates DSB repair activity.

Human transcriptional coactivator PC4 is a highly abundant nuclear protein that is involved in diverse cellular processes ranging from transcription to chromatin organization. Earlier, we have shown that PC4, a positive activator of p53, overexpresses upon genotoxic insult in a p53-dependent manner. In the present study, we show that PC4 stimulates ligase-mediated DNA end joining irrespective of the source of DNA ligase. Pull-down assays reveal that PC4 helps in the association of DNA ends through its C-terminal domain. In vitro nonhomologous end-joining assays with cell-free extracts show that PC4 enhances the joining of noncomplementary DNA ends. Interestingly, we found that PC4 activates double-strand break (DSB) repair activity through stimulation of DSB rejoining in vivo. Together, these findings demonstrate PC4 as an activator of nonhomologous end joining and DSB repair activity.

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping.

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1-3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 +/- 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL.

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.

Fabrication of an atomic resolution low cost STM-SNOM hybrid probe.

We derived a simple method to fabricate STM-SNOM hybrid probes obtained from commercial cheap communication optical fibers. The tips are fabricated by a methodology that combines two well-known techniques: the selective attack by a buffered solution and the protected layer chemical etching, in a single new one-step technique. The tailored probes are then sputtered by metal and mounted on a STM setup. The usual difficulties of integrating the optical fiber in the STM head are solved originally with a particular home made mount described in details. We will show that the resulting probes reach atomic resolution on both vertical and horizontal scale, and that the optical imaging is free of artifacts and satisfactory with a lateral resolution in the order of lamda/20, as far as we know the finest resolution obtained with a system based on a hybrid fiber probe. We believe that our methodology is very interesting for its simplicity of realization and for the good resolving power in both SNOM and STM modes.