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Biosensors (Basel). 2016 Dec 20;6(4). pii: E61. doi: 10.3390/bios6040061.

Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry.

Author information

1
Black Ivory Biotech, Inc., P.O. Box 893128, Mililani, HI 96789, USA. dfw808@yahoo.com.
2
Black Ivory Biotech, Inc., P.O. Box 893128, Mililani, HI 96789, USA. hewbrian@gmail.com.
3
Holatron Systems, LLC., 833 Ilaniwai Street, Suite 2, Honolulu, HI 96813, USA. charlie@holatron.com.
4
Mike Jen Software Services, 1035 Aster Ave #2103, Sunnyvale, CA 94086, USA. mike.jen@gmail.com.
5
Black Ivory Biotech, Inc., P.O. Box 893128, Mililani, HI 96789, USA. biogabe@gmail.com.

Abstract

Portable detection and quantitation methods for Bacillus anthracis (anthrax) spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min) result in short sample-to-signal times (less than an hour). With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field.

KEYWORDS:

amperometry; anthrax spores; biodetection; bioterrorism; immunoassay; portable assay

PMID:
27999382
PMCID:
PMC5192381
DOI:
10.3390/bios6040061
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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