Electrochemical Detection of Viable Bacterial Cells Using a Tetrazolium Salt.

In this study, electrochemical detection of viable bacterial cells was performed using a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which was converted to an insoluble and redox active formazan compound in viable microbial cells. The insolubility of this formazan was effectively exploited as a surface-confined redox event. An indium-tin-oxide electrode was applied to a microbial suspension that had been incubated with MTT and was heated to dry for the extraction and adsorption of formazan. Drying led to the appearance of a distinctive voltammetric oxidation peak at +0.1 V vs Ag|AgCl, the magnitude of which was successfully correlated to the number of viable microbes in the suspension. Thus, the electrochemical detection of formazan was effectively coupled with the thermal lysis of microbes. It is also noteworthy that this lysis-adsorption technique was highly selective to the hydrophobic formazan molecule due to the removal of hydrophilic cell components during equilibration in a phosphate buffer before voltammetric measurement. This technique was capable of detecting microbes above 2.8 × 101 CFU mL-1 and required only a 1 h incubation. The results of this study indicate that the sensitivity of the present technique is up to 10 000-fold higher than that of MTT colorimetry. The higher sensitivity was mainly ascribed to the concentration of the microbially produced formazan on the electrode by thorough desiccation of the bacterial suspension.

Binding Constant of the Cell-shaped Cavity Formed on a Polymer for Escherichia coli O157.

The binding constant of receptors for small molecules, proteins, or antibodies is usually determined based on the concentrations of the ligand, receptor, and their complexes. The binding constant is used as a measure of the affinity between the ligand and the receptor. In the present study, we introduce a procedure to determine the binding constant of a cell-shaped cavity formed on a polymer by molecular imprinting for a whole cell. To determine the binding constant, we clarified the numbers of cavities and cells, based on the fluorescence of a single cell, and defined their concentrations. We successfully determined the binding constant of the complementary cavity for a whole cell (1.1 × 105 M-1). This is the first report to describe the binding constant of a complementary cavity for a whole cell.

Real-Time Evaluation of Bacterial Viability Using Gold Nanoparticles.

Real-time evaluation of bacterial viability is important for various purposes such as hygiene management, development of antibacterial agents, and effective utilization of bacterial resources. Here, we demonstrate a simple procedure for evaluating bacterial viability using gold nanoparticles (Au NPs). The color of bacterial suspensions containing Au NPs strongly depended on the bacterial viability. We found that the dispersion state of Au NPs affected the color of the suspension, based on the interaction of Au NPs with substances secreted by the bacteria. This color change was easily recognized with the naked eye, and viability was accurately determined by measuring the absorbance at a specific wavelength. This method was applicable to various bacterial species, regardless of whether they were Gram-positive or Gram-negative.

A rapid and specific bacterial detection method based on cell-imprinted microplates.

Bacterial detection has attracted substantial interest in recent years owing to its importance in biology, medical care, drug discovery, and public health. For such applications, bacterial cell-imprinting technologies are regarded as potential methods, as they can fabricate artificial tailor-made receptors for cellular recognition. In comparison to conventional methods, which generally require a few days for bacterial determination, cell-imprinted polymers can save a substantial amount of time. Here, we report a high-throughput bacterial detection method based on a cell-imprinted 96-well microplate. The fabrication of the bacterial cell-imprinted polypyrrole and nafion complex was accomplished on a gold nanoparticle-coated microplate. The cell-imprinted polymer complex on the microplate can spontaneously rebind and specifically detect target cells with high selectivity in a short time frame (within 30 min). Furthermore, the microplates could discriminate particular target Escherichia coli O157:H7 cells from bacterial mixtures. This simple method may be used for a variety of applications such as clinical testing, food safety, and continuous environmental monitoring.

Optical Elemental Analysis of Metals Using Shewanella oneidensis.

A simple method for the detection of metal ions in solution is proposed, using Shewanella oneidensis, which has the ability to reduce metal ions into metal nanoparticles on the cell surface. The method can be used to identify metal ions in solution using the light-scattering characteristics of the metal nanoparticles formed on the cells.

Specific single-molecule detection of glucose in a supramolecularly designed tunnel junction.

Scanning tunneling microscopy tips were functionalized with a boronic acid derivative. In combination with a similarly modified substrate, the molecular tip forms a supramolecular complex selectively with a glucose molecule. The conductance of the resulting single complex allows one to achieve the specific single-molecule detection of glucose.

Shape Memory Characteristics of O157-Antigenic Cavities Generated on Nanocomposites Consisting of Copolymer-Encapsulated Gold Nanoparticles.

Nanometer-sized composite particles, which consisted of gold nanoparticles encapsulated by an N-isopropylacrylamide copolymer, were successfully synthesized using a one-step process. Shape complementary cavities of the O157-antigen were formed on the composite utilizing temperature-dependent affinity changes of the copolymer. The composite bound to enterohemorrhagic Escherichia coli (E. coli) O157 at 298 K and enhanced light-scattering intensity of the cell due to the optical properties of the gold nanoparticles. Moreover, the composite showed excellent selectivity (>15) against other types of E. coli such as O26 and O Rough. Recognition of the O157-antigen ceased upon heating to 313 K but was restored upon cooling to 298 K. During repeated temperature cycling around the phase transition temperature of the copolymer (305 K), the composite reproducibly showed recognition behavior at 298 K. The binding ability of the composite could be switched reversibly. Therefore, it was concluded that the molecular structure of the O157-antigen was memorized by the composite, rather than being molded into it. This technique is applicable not only for the detection of a target bacterium but also for an identification of new bacterial threats by the simple formation of the specific antigen-imprinted composite.

Spontaneous and specific binding of enterohemorrhagic Escherichia coli to overoxidized polypyrrole-coated microspheres.

Specific identification of enterohemorrhagic Escherichia coli was achieved using microspheres coated with overoxidized polypyrrole. The microspheres are well dispersed in aqueous media, and they specifically, spontaneously, and efficiently bind E. coli O157:H7 through surface area effects. In addition, we found that light-scattering by a single microsphere depended linearly on the number of bound cells.

Investigation Concerning the Formation Process of Gold Nanoparticles by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a facultative anaerobic bacterium that is known to transfer electrons generated during metabolism to various metal ions and produce nanoparticles on the bacterial surface. In this study, we tracked the formation of gold nanoparticles (Au NPs) on the S. oneidensis cell surfaces and investigated the roles of membrane proteins and extracellular polysaccharides in this process by spectrometry, zeta potential analysis, and electron microscopy.

Optical Characterization of Gold Nanoparticle Layers Formed on Plastic Microbeads.

Generally, the characterization of a metal layer formed on a planar substrate has been achieved using scanning electron microscopy and transmission electron microscopy. These techniques provide details of the surface and/or the cross-section of a planar structure with high resolution. However, the evaluation of sphere-like structures is troublesome owing to the necessity to observe a sample from various angles and/or to calculate the yield from many values obtained for many samples, since the conventional methods can observe a sample only from one direction. We have developed a simple evaluation method for a thin metal layer on plastic microbeads based on its light-scattering properties using dark-field microscopy coupled with a spectrometer. The light-scattering intensity of gold-nanoparticle-coated microbeads depends significantly on the gold coverage. We believe that our study is significant because it describes the development and evaluation of the surface coverage of a thin metal layer on a sphere-like microstructure.

Light-scattering Characteristics of Metal Nanoparticles on a Single Bacterial Cell.

Metal nanoparticles express unique light-scattering characteristics based on the localized surface plasmon resonance, which depends on the metal species, particle size, and aggregation state of the nanoparticles. Therefore, we focused on the light-scattering characteristics of metal nanoparticles, such as silver, gold, and copper oxide, adsorbed on a bacterium. Monodisperse silver nanoparticles expressed the strongest scattered light among them, and showed various colors of scattered light. Although a monodisperse gold nanoparticle produced monochromatic light (green color), the color of the scattered light strongly depended on the aggregation state of the nanoparticles on a bacterium. On the other hand, copper oxide nanoparticles expressed monochromatic light (blue color), regardless of their aggregation states on a bacterium. We examined details concerning the light-scattering characteristics of metal nanoparticles, and discussed the possibility of their applications to bacterial cell imaging.

Optical Evaluation of the Surface Coverage of Silver Nanoparticle-coated Plastic Microbeads.

A simple evaluation method has been developed for a metal thin layer on a sphere-like plastic microstructure, based on its light-scattering property since, according to our results, the light-scattering intensity of silver-coated microbeads depends significantly on the silver coverage. Our attempt was carried out by using a dark-field microscopy coupled with a spectrometer.

Voltammetric detection and profiling of isoprenoid quinones hydrophobically transferred from bacterial cells.

We have developed a novel bacterial detection technique by desiccating a bacterial suspension deposited on an electrode. It was also found that the use of an indium-tin-oxide (ITO) electrode dramatically improved the resolution of the voltammogram, allowing us to observe two pairs of redox peaks, each assigned to the adsorption of isoprenoid ubiquinone (UQn) and menaquinone (MKn), which were present in the bacterial cell envelopes, giving midpeak potentials of -0.015 and -0.25 V versus Ag|AgCl|saturated KCl| at pH 7.0, respectively. Most of the microorganisms classified in both the Gram-negative and -positive bacteria gave well-defined redox peaks, demonstrating that this procedure made the detection of the quinones possible without solvent extraction. It has been demonstrated that the present technique can be used not only for the detection of bacteria, but also for profiling of the isoprenoid quinones, which play important roles in electron and proton transfer in microorganisms. In this respect, the present technique provides a much more straightforward way than the solvent extraction in that one sample can be prepared in 1 min by heat evaporation of a suspension containing the targeted bacteria, which has been applied on the ITO electrode.

Fluorescence Enhancement of Nanoraspberry Hot-spot Source Composed of Gold Nanoparticles and Aniline Oligomers.

In this study, we examined raspberry-shaped organic/inorganic hybrid structure for potential development of a nanoantenna system capable of detecting and labeling biomolecules. The structure is characterized by a high density of gold nanoparticles (AuNPs) separated by closely packed aniline oligomers that serve as a linkage between adjacent particles. In particular, the structure was based on repeated sequences of AuNP-aniline oligomer-AuNP in a three-dimensional arrangement, which enabled the creation of optical hot spots that can hold multiple molecules. We examine the expression of such features by focusing on the structure and characteristics of the hybrid. We demonstrate that these optical hot spots enhance the dye fluorescence without quenching. As a result, we were able to create a nanoantenna structure enabling the efficient use of light.

Electrochemical evaluation of poly(3,4-ethylenedioxythiophene) films doped with bacteria based on viability analysis.

To immobilize viable bacteria on an electrode, we present a novel and straightforward technique that relies on the negative ζ-potentials of bacteria for insertion into conducting polymers as dopants. In the present study, we conducted an electrochemical polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) doped with various gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Shewanella oneidensis. The PEDOT film doped with bacteria indicated a typical redox response, high conductivity, and electrochemical stability. Fluorescence microscopy confirmed that approximately 90% of the bacteria incorporated into the PEDOT film at >0.5 µm in thickness were viable.

Nanoantennas as biomarkers for bacterial detection.

Understanding the biology of bacteria is critical for exploiting their beneficial properties and for preventing and treating bacterial diseases. Nanobioscience is an area that has recently seen major scientific progress. Here, we demonstrate that a raspberry-shaped nanostructure with a high density of gold nanoparticles acts like an excellent antenna due to its optical properties, which permit sensitive detection and analysis of bacterial cells. By using antibodies, these nanoantennas can be engineered to recognize only specific bacterial species. This system provides a new technique that will allow for more sensitive detection of specific bacteria.

Development of an observation platform for bacterial activity using polypyrrole films doped with bacteria.

In our study, various bacteria, including Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Acinetobacter calcoaceticus, Serratia marcescens, Shewanella oneidensis) and Gram-positive (Bacillus subtilis) bacteria, were straightforwardly immobilized into the conducting polymers (CPs) by electrochemical deposition. The doping state of bacteria in the polymer films (polypyrrole and poly(3,4-ethylenedioxythiophene)) varied according to the polymerization conditions. The viability of bacteria in the polymers and of those adsorbed on various substrates was evaluated. The activity of bacteria doped on the polymer film was evaluated by cyclic voltammetry in a thin-layer cell.

One-by-one single-molecule detection of mutated nucleobases by monitoring tunneling current using a DNA tip.

A DNA molecule was utilized as a probe tip to achieve single-molecule genetic diagnoses. Hybridization of the probe and target DNAs resulted in electron tunneling along the emergent double-stranded DNA. Simple stationary monitoring of the tunneling current leads to single-molecule DNA detection and discovery of base mismatches and methylation.

Construction of nanoantennas on the bacterial outer membrane.

We demonstrate a simple manipulation of gold nanoparticles that creates a structure-dependent nanometer-scale antenna on the surface of bacteria. Our studies illuminate the concept of the "effective use of light" based on the absorption and emission of light by antennas formed on bacteria.