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.
Highlights
Recent terrorist attacks have reminded us of the immediate risk that biothreat agents pose, as well as the need for fast and accurate detection methods
quantum dot (QD) composition of the eluted fraction was characterized by UV-Vis spectroscopy, which indicated that QD-antibody conjugate concentration was 1.48 μM on average (Table 1)
QD-antibody conjugates contained ~4.2 antibodies for every QD; this antibody:QD ratio varied across multiple conjugation reactions, perhaps due to slight differences in the volume of activated QDs and reduced antibodies that was collected during each trial
Summary
Recent terrorist attacks have reminded us of the immediate risk that biothreat agents pose, as well as the need for fast and accurate detection methods. QDs can be linked to biomolecules, such as antibodies, to produce biosensors capable of fast, sensitive, and specific biological target detection [19] Such QD-antibody conjugates can be utilized as donors in FRET-based systems with organic quenchers as acceptors for simple and accurate target identification [20,21,22]. As with all FRET systems, several parameters determine the rate and efficiency of energy transfer, including: (1) quantum yield of the energy donor, (2) spectral overlap of the donor’s emission spectrum with the acceptor’s absorption spectrum, (3) orientation of the donor and acceptor transition dipoles, and (4) the distance between the donor and acceptor molecules [14,23] In addition to these classical parameters, it was necessary to address the issue of mold spore autofluorescence to obtain an optimal fluorescence signal. This study describes optimization and characterization of a FRET-based displacement immunoassay capable of sensitive and rapid biological target detection and identification
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