Single Cell Array Enclosed with a Photodegradable Hydrogel in Microwells for Image-Based Cell Classification and Selective Photorelease of Cells.

Single cell arrays provide an accurate classification of analyte cells through an image-based analysis of cellular phenotypes. Light-guided cell retrieval from a single cell array is a promising approach for the rapid and simple sorting of difficult to distinguish cells. In this study, we developed a single cell array enclosed with a photodegradable hydrogel in microwells to enable both comprehensive image-based single cell analysis and light-guided cell retrieval.

Large-Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria.

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

A Microfluidic Channel Method for Rapid Drug-Susceptibility Testing of Pseudomonas aeruginosa.

The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h.

Single-Cell Detection and Collection of Persister Bacteria in a Directly Accessible Femtoliter Droplet Array.

A directly accessible femtoliter droplet array as a platform for single-cell detection and collection of persister bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single cells, long-term culture and observation of single cells in droplets, and collection of identified persisters from single droplets are described in detail.

Label-free single-particle imaging of the influenza virus by objective-type total internal reflection dark-field microscopy.

Here we report label-free optical imaging of single particles of the influenza virus attached on a glass surface with a simple objective-type total internal reflection dark-field microscopy (TIRDFM). The capability of TIRDFM for the imaging of single viral particles was confirmed from fine correlation of the TIRDFM images with fluorescent immunostaining image and scanning electron microscopy image. The density of scattering spots in the TIRDFM images showed a good linearity against the virus concentration, giving the limit of detection as 1.

Large-scale femtoliter droplet array for digital counting of single biomolecules.

We present a novel device employing one million femtoliter droplets immobilized on a substrate for the quantitative detection of extremely low concentrations of biomolecules in a sample. Surface-modified polystyrene beads carrying either zero or a single biomolecule-reporter enzyme complex are efficiently isolated into femtoliter droplets formed on hydrophilic-in-hydrophobic surfaces. Using a conventional micropipette, this is achieved by sequential injection first with an aqueous solution containing beads, and then with fluorinated oil.

A single-cell drug efflux assay in bacteria by using a directly accessible femtoliter droplet array.

Active efflux of drugs, such as antibiotics, from a cell is one of the major mechanisms that cause multi-drug resistance in bacteria. Here we report a method to assess drug efflux activity in individual Escherichia coli cells enclosed and isolated in a directly accessible femtoliter droplet array with a fluorogenic compound. The inhibitory effect of a chemical compound on an exogenously expressed efflux pump system from pathogenic bacteria was easily detected at the single-cell level.

Mechanical modulation of catalytic power on F1-ATPase.

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F(1)-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle.

Evaluation of multidrug efflux pump inhibitors by a new method using microfluidic channels.

Fluorescein-di-β-D-galactopyranoside (FDG), a fluorogenic compound, is hydrolyzed by β-galactosidase in the cytoplasm of Escherichia coli to produce a fluorescent dye, fluorescein. We found that both FDG and fluorescein were substrates of efflux pumps, and have developed a new method to evaluate efflux-inhibitory activities in E. coli using FDG and a microfluidic channel device.

A single-molecule enzymatic assay in a directly accessible femtoliter droplet array.

The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult.

Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution.

Molecular motors such as kinesin, myosin, and F(1)-ATPase are responsible for many important cellular processes. These motor proteins exhibit nanometer-scale, stepwise movements on micro- to millisecond timescales. So far, methods developed to measure these small and fast movements with high spatial and temporal resolution require relatively complicated experimental systems.

Acceleration of the ATP-binding rate of F1-ATPase by forcible forward rotation.

F1-ATPase (F1) is a reversible ATP-driven rotary motor protein. When its rotary shaft is reversely rotated, F1 produces ATP against the chemical potential of ATP hydrolysis, suggesting that F1 modulates the rate constants and equilibriums of catalytic reaction steps depending on the rotary angle of the shaft. Although the chemomechanical coupling scheme of F1 has been determined, it is unclear how individual catalytic reaction steps depend on its rotary angle.

Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber.

Loop-mediated isothermal amplification (LAMP) is an original nucleic acid amplification method established by Notomi et al. LAMP is performed under isothermal condition, employing only a basic reaction protocol and minimal supporting electronics. These requirements prove to be viable for exploring the avenues to down-scale this biological reaction for Lab-on-a-chip application.

An integrated system for enzymatic cleavage and electrostretching of freely-suspended single DNA molecules.

A novel polyacrylamide gel-based femtolitre microchamber system for performing single-molecule restriction enzyme assay on freely-suspended DNA molecules and subsequent DNA electrostretching by applying an alternating electric field has been developed. We attempted the integration by firstly initiating restriction enzyme reaction on a fluorescent-stained lambdaDNA molecule, encapsulated in a microchamber, using magnesium as an external trigger. Upon complete digestion, the cleaved DNA fragments were electrostretched to analyze the DNA lengths optically.