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.

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.

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.

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.

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.

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.

Efficient collection and sensitive detection using conducting magnetic microbeads.

Conducting magnetic microbeads were successfully fabricated through a simple procedure that involves coating magnetic microbeads with gold nanoparticles. The formation of a gold layer on the bead enabled the simple introduction of a biotin probe onto the bead, resulting in the binding capacity with streptavidin being 10 times greater than that of commercially available biotin-binding magnetic beads. In addition to the high recovery via magnetic forces and high dispersibility in the sample solution, the accumulation of highly conductive beads on the electrode resulted in the amplification of the electrochemical response of the detection system. This paper reports the efficient collection and highly sensitive detection of target biomolecules using Au-coated magnetic microbeads.

Label-free and selective bacteria detection using a film with transferred bacterial configuration.

Specific identification of bacteria has been achieved through precisely transferred bacterial structure on the surface of overoxidized polypyrrole (OPPy) film. The recognition of target bacteria was successfully carried out in real time using OPPy film in combination with dielectrophoresis. The unique combination of both techniques made the specific detection of template bacilli possible at concentrations as low as 10(3) CFU/mL within 3 min, without any bacterial pretreatment. The observation of the movement of bacteria by using a fluorescent microscope revealed that living bacteria were being trapped vertically in the cavity created in the OPPy film. Further, the bacterial cavities had high selectivity and were able to discriminate particular target bacteria, Pseudomonas aeruginosa, out of bacterial mixtures containing Acinetobacter calcoaceticus, Escherichia coli, and Serratia marcescens, which are known to have a similar shape. This simple method can be used for a wide variety of applications in which rapid bacterial detection is required, such as food safety risk assessment, clinical point-of-care testing, and continuous environmental monitoring.

Chiral ligand exchange potentiometric aspartic acid sensors with polysiloxane films containing a chiral ligand N-carbobenzoxy-aspartic acid.

An enantioselective molecular sensor was fabricated by inserting a chiral ligand, N-carbobenzoxy-L-aspartic acid (N-CBZ-L-Asp) or N-CBZ-D-Asp, into an octadecylsiloxane (ODS) monolayer by polysiloxane film immobilization (PFI). The resulting system can recognize one enantiomer of aspartic acids (Asps) due to the chiral ligand exchange reaction at the N-CBZ-L-/D-Asp modified indium-tin oxide (ITO)-coated electrode. The enantioselective formation of diastereoisomeric complexes of Cu(II) with target enantiomers, in here L-/D-Asps, and N-CBZ-L-/D-Asp immobilized by PFI on the ITO electrode. Those diastereoisomeric complexes have different thermodynamic stabilities and Nernst factors and thus enable the sensors to convert the enantioselective recognition event into potential changes by detecting Asp enantiomers in a concentration range of (4.0 x 10(-8))-(8.9 x 10(-5)) M without any pre- or postseparation process. The enantiomeric selectivity coefficients of the sensors for the counterisomers were in the range of (4.0 x 10(-5))-(5.0 x 10(-5)).

Self-organization of an organic-inorganic hybrid nanomushroom by a simple synthetic route at the organic/water interface.

A new strategy for the synthesis of a mushroom-like aggregate, based on repeated sequences of a 3D gold nanoparticle-conducting polymer-gold nanoparticle arrangement, by a single-step process at the organic/water interface has been described.

Open bridge-structured gold nanoparticle array for label-free DNA detection.

We focused on changes in the electrical property of the open bridge-structured gold nanoparticles array consisting of 46-nm parent and 12-nm son gold nanoparticles by hybridization and applied it for a simple electrical DNA detection. Since a target DNA of a 24-mer oligonucleotide was added to the probe DNA modified 12-nm Au nanoparticles, which was arranged on the gap between the 46-nm Au particles, the response was read by an electrical readout system. Even in a simple measuring method, we obtained a rapid response to the cDNA with a high S/N ratio of 30 over a wide concentration range and a detection limit of 5.0 fmol. Moreover, the array discriminated 1-base mismatches, regardless of their location in the DNA sequence, which enabled us to detect single-nucleotide polymorphism, which is one of the important diagnoses, without any polymerase chain reaction amplification, sophisticated instrumentation, or fluorescent labeling through an easy-to-handle electrical readout system.

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.

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.

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.

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.

One-step preparation of positively-charged gold nanoraspberry.

Self-assembling particles were prepared by a new synthetic strategy for a raspberry-like aggregate, based on three-dimensional particle-aniline oligomer-particle repeated sequences; this one-step process, without the need for extra control, organic solvents, or ligand exchange, could further help in the realization of nanoscale electronics and molecular devices.

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.