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

Sensors

Michele D. Kattke²  Elizabeth J. Gao²  Kim E. Sapsford¹  Larry D. Stephenson² 

[1] U.S. Food and Drug Administration CDRH-OSEL-DB, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA; E-Mail:;U.S. Corps of Engineers ERDC-CERL, 2902 Newmark Drive, Champaign, IL 61826, USA; E-Mails:

关键词: Fluorescence Resonance Energy Transfer (FRET);    quantum dot (QD);    displacement immunoassay;    detection;    biosensor;    fluorescence;    quenching;    mold;    fungi;    spores;   

DOI  :  10.3390/s110606396

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2011 by the authors; licensee MDPI, Basel, Switzerland.