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...

Aptamers have been utilized as biosensors because they can be readily adapted to sensor platforms and signal transduction schemes through both rational design and selection. One highly generalizable scheme for the generation of the so-called aptamer beacons involves denaturing the aptamer with antisense oligonucleotides. For example, rational design methods have been utilized to adapt anti-thrombin aptamers to function as biosensors by hybridizing an antisense oligonucleotide containing a quencher to the aptamer containing a fluorescent label. In the presence of thrombin, the binding equilibrium is shifted, the antisense oligonucleotide dissociates, and the beacon lights up. By changing the affinity of the antisense oligonucleotide for the aptamer beacon, it has proven possible to change the extent of activation of the beacon. More importantly, modulating interactions between the antisense oligonucleotide and the aptamer strongly influences the kinetics of activation. Comparisons across multiple, designed aptamer beacons indicate that there is a strong inverse correlation between the thermodynamics of hybridization and the speed of activation, a finding that should prove to be generally useful in the design of future biosensors. By pre-organizing the thrombin-binding quadruplex within the aptamer the speed of response can be greatly increased. By integrating these various interactions, we were ultimately able to design aptamer beacons that were activated by threefold within 1 min of the addition of thrombin.

We have developed a coaxial flow focusing geometry that can be fabricated using soft lithography in poly(dimethylsiloxane) (PDMS). Like coaxial flow focusing in glass capillary microfluidics, our geometry can form double emulsions in channels with uniform wettability and of a size much smaller than the channel dimensions. However, In contrast to glass capillary coaxial flow focusing, our geometry can be fabricated using lithographic techniques, allowing it to be integrated as the drop making unit in parallel drop maker arrays. Our geometry enables scalable formation of emulsions down 7 μm in diameter, in large channels that are robust against fouling and clogging.

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