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Sensors 2011, 11(6), 6396-6410; doi:10.3390/s110606396
Article

FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami

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Received: 14 April 2011; in revised form: 10 June 2011 / Accepted: 13 June 2011 / Published: 16 June 2011
(This article belongs to the Special Issue Sensing with Quantum Dots)
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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 Fluorescence Resonance Energy Transfer (FRET); quantum dot (QD); displacement immunoassay; detection; biosensor; fluorescence; quenching; mold; fungi; spores
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

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.

AMA Style

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.

Chicago/Turabian Style

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.


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