Stand-off biodetection with free-space coupled asymmetric microsphere cavities.

Asymmetric microsphere resonant cavities (ARCs) allow for free-space coupling to high quality (Q) whispering gallery modes (WGMs) while exhibiting highly directional light emission, enabling WGM resonance measurements in the far-field. These remarkable characteristics make "stand-off" biodetection in which no coupling device is required in near-field contact with the resonator possible. Here we show asymmetric microsphere resonators fabricated from optical fibers which support dynamical tunneling to excite high-Q WGMs, and demonstrate free-space coupling to modes in an aqueous environment. We characterize the directional emission by fluorescence imaging, demonstrate coupled mode effects due to free space coupling by dynamical tunneling, and detect adsorption kinetics of a protein in aqueous solution. Based on our approach, new, more robust WGM biodetection schemes involving microfluidics and in-vivo measurements can be designed.

Nanosecond time scale transient optoplasmonic detection of single proteins.

Optical detection of individual proteins with high bandwidth holds great promise for understanding important biological processes on the nanoscale and for high-throughput fingerprinting applications. As fluorescent labels impose restrictions on detection bandwidth and require time-intensive and invasive processes, label-free optical techniques are highly desirable. Here, we read out changes in the resonantly scattered field of individual gold nanorods interferometrically and use photothermal spectroscopy to optimize the experiment's parameters. This interferometric plasmonic scattering enables the observation of single proteins as they traverse plasmonic near fields of gold nanorods with unprecedented temporal resolution in the nanosecond-to-microsecond range.

Photothermal Spectro-Microscopy as Benchmark for Optoplasmonic Bio-Detection Assays.

Optoplasmonic bio-detection assays commonly probe the response of plasmonic nanostructures to changes in their dielectric environment. The accurate detection of nanoscale entities such as virus particles, micelles and proteins requires optimization of multiple experimental parameters. Performing such optimization directly via analyte recognition is often not desirable or feasible, especially if the nanostructures exhibit limited numbers of analyte binding sites and if binding is irreversible. Here we introduce photothermal spectro-microscopy as a benchmarking tool for the characterization and optimization of optoplasmonic detection assays.

Towards next-generation label-free biosensors: recent advances in whispering gallery mode sensors.

Whispering gallery mode biosensors have been widely exploited over the past decade to study molecular interactions by virtue of their high sensitivity and applicability in real-time kinetic analysis without the requirement to label. There have been immense research efforts made for advancing the instrumentation as well as the design of detection assays, with the common goal of progressing towards real-world sensing applications. We therefore review a set of recent developments made in this field and discuss the requirements that whispering gallery mode label-free sensors need to fulfill for making a real world impact outside of the laboratory. These requirements are directly related to the challenges that these sensors face, and the methods proposed to overcome them are discussed. Moving forward, we provide the future prospects and the potential impact of this technology.

Label-free optical detection of single enzyme-reactant reactions and associated conformational changes.

Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.

In Situ Observation of Single-Molecule Surface Reactions from Low to High Affinities.

In situ observation of single-molecule surface reactions from low to high affinities is achieved by resonant coupling between optical whispering-gallery modes and the localized surface plasmon of nanorods. Transient and permanent interactions between ligands (thiol, amine) and the gold surface are monitored without labels, allowing direct determination of the associated kinetic constants and rapid development of new functionalization protocols.

Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform.

Biosensing relies on the detection of molecules and their specific interactions. It is therefore highly desirable to develop transducers exhibiting ultimate detection limits. Microcavities are an exemplary candidate technology for demonstrating such a capability in the optical domain and in a label-free fashion. Additional sensitivity gains, achievable by exploiting plasmon resonances, promise biosensing down to the single-molecule level. Here, we introduce a biosensing platform using optical microcavity-based sensors that exhibits single-molecule sensitivity and is selective to specific single binding events. Whispering gallery modes in glass microspheres are used to leverage plasmonic enhancements in gold nanorods for the specific detection of nucleic acid hybridization, down to single 8-mer oligonucleotides. Detection of single intercalating small molecules confirms the observation of single-molecule hybridization. Matched and mismatched strands are discriminated by their interaction kinetics. Our platform allows us to monitor specific molecular interactions transiently, hence mitigating the need for high binding affinity and avoiding permanent binding of target molecules to the receptors. Sensor lifetime is therefore increased, allowing interaction kinetics to be statistically analysed.