Fully automated chemiluminescence enzyme immunoassays showing high correlation with immunoprecipitation mass spectrometry assays for ß-amyloid (1-40) and (1-42) in plasma samples.

Blood based ß-amyloid (Aß) assays that can predict amyloid positivity in the brain are in high demand. Current studies that utilize immunoprecipitation mass spectrometry assay (IP-MS), which has high specificity for measuring analytes, have revealed that precise plasma Aß assays have the potential to detect amyloid positivity in the brain. In this study, we developed plasma Aß40 and Aß42 immunoassays using a fully automated immunoassay platform that is used in routine clinical practice. Our assays showed high sensitivity (limit of quantification: 2.46 pg/mL [Aß40] and 0.16 pg/mL [Aß42]) and high reproducibility within-run (coefficients of variation [CVs]: <3.7% [Aß40] and <2.0% [Aß42]) and within-laboratory (CVs: <4.6% [Aß40] and <5.3% [Aß42]). The interference from plasma components was less than 10%, and the cross-reactivity with various lengths of Aß peptides was less than 0.5%. In addition, we found a significant correlation between the IP-MS method and our immunoassay (correlation coefficients of Pearson's r: 0.91 [Aß40] and 0.82 [Aß42]). Our new method to quantify plasma Aß40 and Aß42 provides clinicians and patients with a way to continuously monitor disease progression.

Kinetics of Intracellular Electron Generation in Shewanella oneidensis MR-1.

Efficient utilization of bacterial bioresources requires quantitative evaluation of metabolic activity in living bacterial cells. Shewanella oneidensis MR-1 transfers electrons generated within the cell to the extracellular environment via the cytochrome complex in the inner/outer membranes and is one of the most useful bacteria for the recovery of metals, treatment of wastewater, and preparation of microbial fuel cells. Here, we performed a quantitative evaluation of electron generation based on individual enzyme reactions in S. oneidensis MR-1. By using potentiometric measurements, we have examined intracellular electron generation in bacterial suspensions of S. oneidensis supplemented with different carbon sources (formate, lactate, pyruvate, or acetyl coenzyme A) or ferricyanide, which was almost completely reduced to ferrocyanide during the incubation without affecting bacterial cell viability. The amount of electron generation strongly depended on the nature of the carbon source. Analysis of the obtained kinetic parameters of intracellular electron generation demonstrated that formate was the most effective carbon source, as it enabled 2.5-fold faster electron generation rate than other sources. We established that the respective contributions of lactate dehydrogenase, pyruvate dehydrogenase/pyruvate-formate-lyase, and tricarboxylic acid cycle to lactate metabolism were 62%, 31%, and 7.4%, correspondingly. Furthermore, we clarified that electrons may be generated at 1.6 × 10-12 A s-1 by ideal metabolism in a single living cell. These findings establish the basis for biological strategies of electron production and facilitate the utilization of S. oneidensis as a bioresource in practical applications, including energy production, environmental purification, and recovery of useful materials.

A Microbial Platform Based on Conducting Polymers for Evaluating Metabolic Activity.

Bacterial cells possessing a certain zeta potential are immobilized by electrochemical deposition within conducting polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) and polypyrrole (PPy). These conducting polymers serve as a biocompatible matrix for trapping bacteria on an indium-tin-oxide (ITO)-coated glass substrate. The biological functions of bacteria were not affected by the chemical structure and electrical conductivity of the matrix. The viability of the bacteria on the ITO glass was monitored by dark-field microscopy. The cell density of Escherichia coli increased logarithmically during incubation in nutrient broth medium, leading to definitive formation of a biofilm on PPy. The facultative E. coli anaerobe sustains metabolism under aerobic and anaerobic conditions, but proliferates more extensively in the presence of oxygen. The conducting PPy film also facilitates electrochemical evaluation of the respiratory activity of bacterial cells and establishes that facultative anaerobic and aerobic bacteria exhibit similar respiratory activities under aerobic conditions.

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.

Single cell immunodetection of Escherichia coli O157:H7 on an indium-tin-oxide electrode by using an electrochemical label with an organic-inorganic nanostructure.

A rapid and highly sensitive method is described for the detection of enterohemorrhagic Escherichia coli O157:H7. An organic-inorganic nanostructure in which numerous gold nanoparticles (AuNPs) are enclosed with polyaniline (PANI) was utilized as an electrochemical label. The nanostructure showed (a) strong light scattering intensity due to the coupling effect of the surface plasmon resonance based on the presence of AuNPs, and (b) high electrochemical response due to the redox activity of PANI. To achieve selectivity, antibody against E. coli O157:H7 was immobilized on the surface of the nanostructure. The method exploits the combination of strong adsorption of bacterial cells onto the indium-tin-oxide (ITO) glass electrode without any special processing and specific binding of the nanostructured label to E. coli O157:H7. This enables the electrochemical detection of a single cell on the ITO electrode. The electrochemical response to E. coli O157:H7 was 30-fold higher than that to other types of bacteria. This procedure can be applied to the determination of E. coli O157:H7 even in the presence of other bacteria. Graphical abstract Schematic of a voltammetric immunoassay for Escherichia coli O157:H7 by using a nanocomposite consisting of gold nanoparticles and polyaniline on an ITO electrode.

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.

The appropriate sample-handling procedure for measuring the plasma ß-amyloid level using a fully automated immunoassay.

Plasma ß-amyloid (Aß) assays are a promising tool for Alzheimer's disease diagnosis in clinical practice. To obtain reliable results, establishing an appropriate sample-handling procedure for each analytical platform is warranted. This study proposes an appropriate sample-handling procedure using HISCL analyzer by elucidating the individual/combined effects of pre-analytical parameters on plasma Aß42/Aß40 levels. We investigated the effects of various pre-analytical parameters, including storage times for whole blood, plasma, and freezing conditions, on plasma Aß42/Aß40 levels, and confirmed if these values met the acceptable criteria. Plasma Aß42/Aß40 levels were acceptable in all conditions. We determined our protocol by confirming that plasma Aß42/Aß40 levels remained acceptable when combining pre-analytical parameters. We established an appropriate sample-handling protocol that ensures reliable measurement of plasma Aß42/Aß40 levels using HISCL analyzer. We believe the Aß assay, with our protocol, shows promise for aiding AD diagnosis in clinical settings.

Mirror-image streptavidin with specific binding to L-biotin, the unnatural enantiomer.

The streptavidin-biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method is affected by endogenous D-biotin in serum sample measurements (biotin interference). While several efforts using alternative high-affinity binding systems (e.g., genetically modified streptavidin and biotin derivatives) have been attempted, these efforts have all led to reduction in affinity. To solve this interference issue, the enantiomer of streptavidin was synthesized, which enabled specific binding to L-biotin. We successfully obtained a functional streptavidin molecule by peptide synthesis using D-amino acids and an in vitro folding technique. Several characterizations, including size exclusion chromatography (SEC), circular dichroism spectra (CD), and heat denaturation experiments collectively confirmed the higher-order enantiomer of natural streptavidin had been formed with comparable stability to the natural protein. L-biotin specific binding of this novel molecule enabled us to avoid biotin interference in affinity measurements using the Biacore system and enzyme-linked immunosorbent assay (ELISA). We propose the enantiomer of streptavidin as a potential candidate to replace the natural streptavidin-biotin system, even for in vivo use.

Fully automated and highly specific plasma ß-amyloid immunoassays predict ß-amyloid status defined by amyloid positron emission tomography with high accuracy.

BACKGROUND: Clinicians, researchers, and patients alike would greatly benefit from more accessible and inexpensive biomarkers for neural ß-amyloid (Aß). We aimed to assess the performance of fully automated plasma Aß immunoassays, which correlate significantly with immunoprecipitation mass spectrometry assays, in predicting brain Aß status as determined by visual read assessment of amyloid positron emission tomography (PET). METHODS: The plasma Aß42/Aß40 ratio was measured using a fully automated immunoassay platform (HISCL series) in two clinical studies (discovery and validation studies). The discovery and validation sample sets were retrospectively and randomly selected from participants with early Alzheimer's disease (AD) identified during screening for the elenbecestat Phase 3 program. RESULTS: We included 197 participants in the discovery study (mean [SD] age 71.1 [8.5] years; 112 females) and 200 in the validation study (age 70.8 [7.9] years; 99 females). The plasma Aß42/Aß40 ratio predicted amyloid PET visual read status with areas under the receiver operating characteristic curves of 0.941 (95% confidence interval [CI] 0.910-0.973) and 0.868 (95% CI 0.816-0.920) in the discovery and validation studies, respectively. In the discovery study, a cutoff value of 0.102 was determined based on maximizing the Youden Index, and the sensitivity and specificity were calculated to be 96.0% (95% CI 90.1-98.9%) and 83.5% (95% CI 74.6-90.3%), respectively. Using the same cutoff value, the sensitivity and specificity in the validation study were calculated to be 88.0% (95% CI 80.0-93.6%) and 72.0% (95% CI 62.1-80.5%), respectively. CONCLUSIONS: The plasma Aß42/Aß40 ratio measured using the HISCL series achieved high accuracy in predicting amyloid PET status. Since our blood-based immunoassay system is less invasive and more accessible than amyloid PET and cerebrospinal fluid testing, it may contribute to the diagnosis of AD in routine clinical practice.

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.

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.