On-Demand Droplet Fusion: A Strategy for Stimulus-Responsive Biosensing in Solution

Abstract

A novel strategy is reported for biochemically controlled fusion of oil-in-water (O/W) droplets as an in-solution sensor for biological targets. Inspired by the SNARE complex in cells, the emulsions were stabilized by a combination of phospholipids, phospholipid–poly(ethylene glycol) conjugates, and cholesterol-anchored oligonucleotides. Prior to oligonucleotide binding, the droplets were stable in aqueous media, but hybridization of the oligonucleotides in a zipperlike fashion was shown to initiate droplet fusion. Using image analysis of content mixing of dye-loaded droplets, fusion specificity was studied and optimized as a function of interfacial chemistry. Changing the orientation of the anchored oligonucleotides, using long-chain phospholipids (C18 and C22), and binding a complementary oligonucleotide slowed or even halted fusion completely. Based on these studies, a sensor for the biomarker thrombin was designed using competitive binding of aptamer strands, with droplet fusion increasing as a function of thrombin addition in accordance with a simple binding model, with sensitivity down to 100 nM and with results in as little as 15 min. Future efforts will focus on utilizing this mechanism of content mixing to facilitate highly sensitive detection via modalities such as magnetoresistance or chemiluminescence.

Selective Vaporization of Superheated Nanodroplets for Rapid, Sensitive, Acoustic Biosensing

“…Formulation of Nanodroplets : Prior to droplet formulation, TBS was prepared to a final concentration of 1 × 10 −8 m Tris base (Fisher Scientific) and 1 × 10 −1 m NaCl (Fisher Scientific), adjusted to a pH of 8.0 with dilute HCl. A stock suspension of hydrated DPPC (Avanti Polar Lipids, Inc.) was prepared as described previously . The stock DPPC suspension was mixed with DSPE‐PEG2000 (Avanti Polar Lipids, Inc.) and DSPE‐PEG2000‐Biotin (Avanti Polar Lipids, Inc.) to make a final concentration of 1.3 × 10 −3 m /40 × 10 −6 m /15 × 10 −3 m , respectively, in TBS, and stirred at 75 °C for 30 min.…”

“…The acquired data also provided evidence as to the mechanism of droplet vaporization. First, the aggregation of droplets may lead to fusion and formation of nuclei for enhanced vaporization, but this was ruled out by the lack of any significant signal found in the highly aggregated 1 × 10 −6 m streptavidin samples prior to introduction of HIFU (Figure S1, Supporting Information) . Second, aggregation of droplets may lead to decreased Laplace pressure, but this is an unlikely explanation because the agglomerates appear to be intact by microscopy, so each of the droplets in the aggregate should still have the same surface area and thus the same Laplace pressure.…”

Lipid–oligonucleotide conjugates for bioapplications

“…(D) Stimulus-responsive droplet fusion for biosensing in an emulsion system. Reprinted with permission from [ 113 ]. Copyright (2014) American Chemical Society.…”

“…In Goodwin's work, a biosensor for thrombin was designed. Here, lipid–DNA-mediated fusion was triggered by competitive thrombin–aptamer binding [ 113 ]. Fluorescent-signal changes that resulted from component mixing, along with droplet fusion, could perform with high sensitivity down to 100 nM in only 15 minutes (Fig.…”

Mutually-Reactive, Fluorogenic Hydrocyanine/Quinone Reporter Pairs for In-Solution Biosensing via Nanodroplet Association

“…This paper presents an alternate strategy in which the aggregation of two oil droplets causes the mixing of two chemicals that react to produce an active fluorescent dye, thus creating fluorescence emission rather than shifting or modulating an existing signal. Previously, our lab developed oil droplets that could be induced to fuse together and mix their contents via DNA-mediated interactions inspired by the SNARE complex. , In the course of performing these studies, we found that simple aggregation could also allow contents to pass across the lipid monolayer, leading to content mixing even without fusion. Thus, droplets bearing reactive payloads could potentially be used as in-solution biosensors via analyte-induced aggregation.…”