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FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami
U.S. Corps of Engineers ERDC-CERL, 2902 Newmark Drive, Champaign, IL 61826, USA
U.S. Food and Drug Administration CDRH-OSEL-DB, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA
* Author to whom correspondence should be addressed.
Received: 14 April 2011; in revised form: 10 June 2011 / Accepted: 13 June 2011 / Published: 16 June 2011
Abstract: In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 103 spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 105 CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis.
Keywords: Fluorescence Resonance Energy Transfer (FRET); quantum dot (QD); displacement immunoassay; detection; biosensor; fluorescence; quenching; mold; fungi; spores
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Kattke, M.D.; Gao, E.J.; Sapsford, K.E.; Stephenson, L.D.; Kumar, A. FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami. Sensors 2011, 11, 6396-6410.
Kattke MD, Gao EJ, Sapsford KE, Stephenson LD, Kumar A. FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami. Sensors. 2011; 11(6):6396-6410.
Kattke, Michele D.; Gao, Elizabeth J.; Sapsford, Kim E.; Stephenson, Larry D.; Kumar, Ashok. 2011. "FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami." Sensors 11, no. 6: 6396-6410.