Quantum‐Dot Aptamer Beacons for the Detection of Proteins

Abstract

Quantum dots (QDs) offer a number of advantages over standard fluorescent dyes for monitoring biological systems in real time, including greater photostability, larger effective Stokes shifts, longer fluorescent lifetimes, and sharper emission bands than traditional organic fluorophores. In addition, QDs all respond to the same excitation wavelength, but emit at different wavelengths; this should allow for the multiplex detection of different analytes in parallel.Recent work has also demonstrated that QDs can be used in the construction of biosensors that signal by fluorescence resonance energy transfer (FRET). For example, a quantum-dotbased molecular beacon has been described in which the nonfluorescent dye DABCYL was used to quench the fluorescence of the quantum dot. In the presence of a target DNA, the opening of the molecular beacon resulted in an approximately fivefold increase in fluorescence of the quantum dot.[1] Similarly, a QD-based protein biosensor that can detect the small molecule maltose has been designed. In one configuration of this system, a maltose-binding protein was complexed to the ZnS shell of a CdSe QD through a 5-histidine tail appended to the protein. Binding of a dye-labeled cyclodextrin molecule to the protein resulted in a loss of photoluminescence, which could be restored by the displacement of the bound cyclodextrin by the addition of maltose.[2]We have now designed a quantum-dot-based aptamer biosensor. Like molecular beacons, aptamer beacons rely on analyte-dependent conformational changes that can alter the proximity of optical reporters to one another, and thus can potentially alter the fluorescence resonance energy transfer (FRET) between these reporters. Aptamer beacons have previously been designed for a variety of targets including small molecules, such as ATP and cocaine, and protein targets, such as PDGF, thrombin, and the HIV-1 Tat. [3][4][5][6][7][8][9] We utilized an aptamer that is known to bind the blood-clotting protein thrombin as a model system.[10] While thrombin aptamer beacons that utilize organic fluorophores have previously been developed, [5][6][7] the adaptation of these strategies to the development of a quantum-dot aptamer beacon (QDB) was not straightforward, primarily because the inorganic quantum dot is much larger and brighter than its organic partner. The development of quantum-dot beacons therefore relied upon two important design features.First, in order to ensure that a substantive conformational change would occur upon analyte binding, we synthesized a quantum-dot beacon based on a two-piece aptamer beacon originally developed by Li et al. in 2002. The final construct consisted of the anti-thrombin aptamer conjugated to a quantum dot (Figure 1 A; underlined), and an oligonucleotide quencher conjugate that hybridized to and disrupted the aptamer structure (Figure 1 A; gray). In the presence of the target protein, thrombin, the quadruplex conformation of the aptamer should be preferentially stabilized, resulting in the displacement of the antisens...

Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles

“…Alternatively, nanoparticle derivatization can be carried out in organic solution (Pathak et al 2001) prior to the coupling to amino-modified DNA oligomers, or the 5 end phosphate group of DNA can be reacted with EDC and imidazole to primary amino groups (Hermanson 2008) present on the particle surface (Skaff & Emrick 2003). Apart from covalent conjugation chemistry, avidin-biotin has also been used as the noncovalent receptor-ligand system for the binding of DNA to nanoparticles, both with biotin-modified DNA (Levy et al 2005) and biotin-modified nanoparticles (Niemeyer et al 1998). …”

Protein detection via direct enzymatic amplification of short DNA aptamers

“…(9)(10)(11)(12) A wealth of sensitive aptamer-based biodetection approaches have been reported in which aptamers were labeled with molecules such as redox probes, fluorescent dyes, or nanocrystals to be an integral part of signal transduction. (13)(14)(15)(16)(17)(18)(19)(20)(21) In addition, researchers have recently taken advantage of PCR to amplify DNA aptamers for sensitive detection of protein targets. Le and coworkers describe the isolation of a protein-aptamer complex via capillary electrophoresis, followed by PCR amplification of the bound ssDNA and subsequent visualization and quantification by gel electrophoresis.…”

Energy Transfer-Based Multiplex Analysis Using Quantum Dots

“…Specific uptake of QD-Apt(Dox) conjugates into target cancer cell through PSMA mediate endocytosis evokes the release of Dox from the QD-Apt(Dox) conjugates, resulting in the recovery of fluorescence from both QD and Dox and the intracellular delivery of Dox inside cancer cells. Levy et al demonstrated an aptamer beacon-based approach for detecting the protein thrombin with QD-based fluorescence readout, in which the detection limit was determined to be 1 μM (Levy et al, 2005). Most recently, Cheng et al Once MUC1 peptide binds to the aptamer strand on the surface of QD, fluorescence intensity successively decreases through FRET effect, showing low detection limit for MUC1 (nanomolar level) and a linear response found in blood serum.…”