title: FRET-based quantum dot immunoassay for rapid and sensitive detection of Aspergillus amstelodami.
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 10(3) spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 10(5) 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.
authors.affiliation: US Corps of Engineers ERDC-CERL, Champaign, IL 61826, USA. Michele.D.Kattke@usace.army.mil
authors.forename: Michele D
authors.initials: MD
authors.lastname: Kattke
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Gao
authors.forename: Kim E
authors.initials: KE
authors.lastname: Sapsford
authors.forename: Larry D
authors.initials: LD
authors.lastname: Stephenson
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001230:Aspergillus / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D045663:Quantum Dots; D015203:Reproducibility of Results; D059629:Signal-To-Noise Ratio; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013170:Spores; D013234:Stem Cells
chemical_list: D000906:Antibodies