Identification and Quantitation of Bacillus globigii Using Metal Enhanced Electrochemical Detection and Capillary Biosensor

Abstract

Presented herein are two detection strategies for the identification and quantification of Bacillus globigii, a spore forming nonpathogenic simulant of Bacillus anthracis. The first strategy involves a label-free, metal-enhanced electrochemical immunosensor for the quantitative detection of Bacillus globigii (atrophaeus). The immunosensor comprises of antibacillus globigii (BG) antibody self-assembled onto a gold quartz crystal electrode via cystamine bond. A solid-phase monolayer of silver underpotentially deposited onto the cystamine modified-Au-electrode surface is used as the redox probe. The monolayer was also generated by adsorbing silver nanoparticles on the gold electrode. When the antibody-modified electrode is exposed to BG spores, the antibody-antigen (Ab-Ag) complex formed insulated the electrode surface toward the silver redox probe. The variation of redox current was found to be proportional to the concentration of the BG spores between 1 x 10(2)-3.5 x 10(4) spores/mL. A detection limit of 602 spores/mL was obtained, which is well-below the infectious dose of anthrax spores at 2.5 x 10(5) spores/mL. The second approach involves the use of ultrasensitive portable capillary biosensor (UPAC) to detect the spores. The capillary is an enclosed system that acts as the flow cell, the waveguide, and the solid support for immobilized bimolecular probes. An evanescent excitation generates a signal from an antigen-antibody-fluorophore complex, which propagates along the capillary and is guided to the detector. A limit of detection of 112 spores/mL was reported using the UPAC sensor. Both methods showed lower detection limits compared to the conventional ELISA. The effect of potential interferants tested using Bacillus pumilus confirmed the selectivity for the analyte. This work should allow the first responders to rapidly detect and quantify Bacillus globigii spores at concentrations that are well-below the infectious dose.

Development of a high-performance immunolatex based on “soft landing” antibody immobilization mechanism

“…With the development of biotechnology and nanotechnology, immobilized proteins, especially antibodies and enzymes, have been widely used as biosensors [1], microarrays [2][3][4][5], and immunoparticles [6][7][8]. These applications require immobilized proteins to maintain their conformation and proper active site orientation toward the bulk solution.…”

Effects of inactivation methods on the analysis of Bacillus atrophaeus endospores using real‐time PCR and MALDI‐TOF‐MS

“…Further examination determined in a rather straightforward way, how each treatment affected the characteristic MALDI-TOF-MS spectra of the endospores. There are in fact several studies that suggest B. atrophaeus spores as a substitute for B. anthracis [16,[37][38][39] and although there may be other species that might be even better suited as simulants for B. anthracis such as B. cereus and B. thuringiensis, we focused on B. atrophaeus as a well-established BWA simulant. They indicate that each bacterial species has its own optimal inactivation parameters (incubation time, temperature, reagent concentration, etc.).…”

“…Some studies that have investigated the effects of heat and chemicals on virulent B. anthracis show that these methods are suited for endospore inactivation [35,36] but some major species attributes such as endospore cortex and stress resistance may still be different [28]. There are in fact several studies that suggest B. atrophaeus spores as a substitute for B. anthracis [16,[37][38][39] and although there may be other species that might be even better suited as simulants for B. anthracis such as B. cereus and B. thuringiensis, we focused on B. atrophaeus as a well-established BWA simulant. The main reason why B. atrophaeus was used is that all the inactivation methods presented here completely inactivate B. atrophaeus spores.…”

A new substrate for alkaline phosphatase based on quercetin pentaphosphate

“…para-nitrophenyl phosphate is converted to a para-nitrophenol in enzymatic assay. Our previous work has utilized immunoassays that employ enzyme-labeled antibodies to provide detection signals by hydrolysis of a phosphate-derived substrate [15]. Since QPP has five phosphate moieties we believe the new emerging peak at 324 nm is due to hydrolysis of the phosphate ester to phenolic form (quercetin) by ALP enzyme.…”