Staining-Enhanced Peroxidase-Mimicking Gold Nanoparticles in Nano-ELISA for Highly Sensitive Detection of Klebsiella pneumoniae.

Klebsiella pneumoniae, a member of the family Enterobacteriaceae, is a rod-shaped, Gram-negative bacterium, mainly found in the hospital environment and medical tools. It is the leading cause of nosocomial infection, characterized by bloodstream infection, wound site infection, urinary tract infection, and sepsis, mostly in older adults, newborn infants, and immunocompromised patients. This present study demonstrated a novel diagnostic method for K. pneumoniae detection based on the gold nanozyme activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The nanozyme activity of AuNPs with staining enhancement was statistically three times higher than that of the bare AuNPs in solid absorption at 650 nm. Nano-ELISA with staining enhancement could detect as low as 102 CFUs/mL of K. pneumoniae concentration, as the cutoff value was determined to be 0.158, which boosted the sensitivity of the immunoreactions by up to 100-fold. The detection limit of our assays was 26.023 CFUs/mL, and the limit of quantification was 78.857 CFUs/mL. There was no cross-reaction against other bacteria, which proved the immunoassays' remarkable specificity for recognizing K. pneumoniae. Taken together, we successfully developed and optimized the highly sensitive and decently specific nano-ELISA strategy that might be applicable for detecting various other bacterial pathogens.

Disease spectrum and management of children admitted with acute respiratory infection in Viet Nam.

OBJECTIVE: To assess the acute respiratory infection (ARI) disease spectrum, duration of hospitalisation and outcome in children hospitalised with an ARI in Viet Nam. METHODS: We conducted a retrospective descriptive study of ARI admissions to primary (Hoa Vang District Hospital), secondary (Da Nang Hospital for Women and Children) and tertiary (National Hospital of Paediatrics in Ha Noi) level hospitals in Viet Nam over 12 months (01/09/2015 to 31/08/2016). RESULTS: Acute respiratory infections accounted for 27.9% (37 436/134 061) of all paediatric admissions; nearly half (47.6%) of all children admitted to Hoa Vang District Hospital. Most (64.6%) of children hospitalised with an ARI were <2 years of age. Influenza/pneumonia accounted for 69.4% of admissions; tuberculosis for only 0.3%. Overall 284 (0.8%) children died; most deaths (269/284; 94.7%) occurred at the tertiary referral hospital. The average duration of hospitalisation was 7.6 days (median 7 days). The average direct hospitalisation cost per ARI admission was 157.5 USD in Da Nang Provincial Hospital. In total, 62.6% of admissions were covered by health insurance. CONCLUSION: Acute respiratory infection is a major cause of paediatric hospitalisation in Viet Nam, characterised by prolonged hospitalisation for relatively mild disease. There is huge potential to reduce unnecessary hospital admission and cost.

Modified screen printed electrode for development of a highly sensitive label-free impedimetric immunosensor to detect amyloid beta peptides.

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting approximately 26 million people world-wide, and the number is increasing as life expectancy increases. Since the only reliable diagnosis for the pathology is histochemical post-mortem examination, there is a rather urgent need for reliable, sensitive and quick detection techniques. Amyloid beta, being one of the established and widely accepted biomarkers of AD is a target biomolecule. Herein, we present fabrication of a labelless impedimetric amyloid beta immunosensor on carbon DEP (disposable electrochemical printed) chip. Three types of amyloid ß impedimetric immunosensors were fabricated in a systematic step-wise manner in order to understand the effects that each surface modification chemistry had on detection sensitivity. We found that compared to a bare electrode, surface modification through formation of SAM of AuNPs increased sensitivity by approximately three orders of magnitude (LoD from 2.04 µM to 2.65 nM). A further modification using protein G, which helps orientate antibodies to an optimum position for interaction with antigen, lowered the LoD further to 0.57 nM. We have demonstrated that the presence of one of the most abundance proteins in biological fluids, bovine serum albumin (BSA), did not interfere with the sensitivity of the sensor. Since the DEP chips are disposable and the detection platform label-free, the developed sensor is relatively fast and cheap. These methods could easily be applied for detection of other antigens, with selection of the detection platform based on the desired for sensitivity.

Quantitative analysis of GAP-43 expression by neurons in microcultures using cell-ELISA.

A cell-ELISA technique is described which allows the quantification of GAP-43 protein in a large number of microcultures of adult dorsal root ganglion neurons. GAP-43 is measured in the 1-10 ng range, corresponding to the amount of GAP-43 present in fewer than 500 DRG neurons. Specificity of the assay is confirmed using Western blotting and immunocytochemistry. The GAP-43 content of adult DRG microcultures rises during 2 weeks in culture, although the number of surviving neurons decreases. The GAP-43 content of cultured adult DRG neurons is not increased by chronic exposure to added nerve growth factor after 7 days in vitro. However, GAP-43 is increased in DRG taken from animals with prior peripheral nerve injury, and is decreased by chronic exposure to dibutyryl cyclic AMP after 7 days in vitro. The method affords the sensitivity and statistical power to document modest changes in GAP-43 protein abundance in complex cultures.

Acetylcholinesterase: electrostatic steering increases the rate of ligand binding.

Brownian dynamics simulations have been used to calculate the diffusion-controlled rate constants for the binding of a positively charged ligand to several models of acetylcholinesterase (AChE). The crystal structure was used to define the detailed topography and the active sites of the dimeric enzyme. The electric field around AChE was then computed by solving the Poisson equation for different charge distributions in the enzyme at zero ionic strength. These fields were used in turn to calculate the forces on the diffusing ligand. Significant increases in the rate constant resulted in going from a model with no charges to one with the net charges concentrated at the centers of the monomers and then to a model with a realistic distribution of charges throughout the enzyme. The results show that electrostatic steering of ligands contributes to the high rate constants that are observed experimentally for AChE.