Photonic-Plasmonic Coupling Enhanced Fluorescence Enabling Digital-Resolution Ultrasensitive Protein Detection.

Assays utilizing fluorophores are common throughout life science research and diagnostics, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve higher signal-to-noise. We describe how the synergistic coupling of plasmonic and photonic modes can significantly boost the emission from fluorophores. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) with the absorption and emission spectrum of the fluorescent dye, a 52-fold improvement in signal intensity is observed, enabling individual PFs to be observed and digitally counted, where one PF tag represents one detected target molecule. The amplification can be attributed to the strong near-field enhancement due to the cavity-induced activation of the PF, PC band structure-mediated improvement in collection efficiency, and increased rate of spontaneous emission. The applicability of the method by dose-response characterization of a sandwich immunoassay for human interleukin-6, a biomarker used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease is demonstrated. A limit of detection of 10 fg mL-1 and 100 fg mL-1 in buffer and human plasma respectively, is achieved, representing a capability nearly three orders of magnitude lower than standard immunoassays.

Photonic Crystal Enhanced Fluorescence: A Review on Design Strategies and Applications.

Nanoscale fluorescence emitters are efficient for measuring biomolecular interactions, but their utility for applications requiring single-unit observations is constrained by the need for large numerical aperture objectives, fluorescence intermittency, and poor photon collection efficiency resulting from omnidirectional emission. Photonic crystal (PC) structures hold promise to address the aforementioned challenges in fluorescence enhancement. In this review, we provide a broad overview of PCs by explaining their structures, design strategies, fabrication techniques, and sensing principles. Furthermore, we discuss recent applications of PC-enhanced fluorescence-based biosensors incorporated with emerging technologies, including nucleic acids sensing, protein detection, and steroid monitoring. Finally, we discuss current challenges associated with PC-enhanced fluorescence and provide an outlook for fluorescence enhancement with photonic-plasmonics coupling and their promise for point-of-care biosensing as well monitoring analytes of biological and environmental relevance. The review presents the transdisciplinary applications of PCs in the broad arena of fluorescence spectroscopy with broad applications in photo-plasmonics, life science research, materials chemistry, cancer diagnostics, and internet of things.

A photonic resonator interferometric scattering microscope for label-free detection of nanometer-scale objects with digital precision in point-of-use environments.

Label-free detection and digital counting of nanometer-scaled objects such as nanoparticles, viruses, extracellular vesicles, and protein molecules enable a wide range of applications in cancer diagnostics, pathogen detection, and life science research. Here, we report the design, implementation, and characterization of a compact Photonic Resonator Interferometric Scattering Microscope (PRISM) designed for point-of-use environments and applications. The contrast of interferometric scattering microscopy is amplified through a photonic crystal surface, upon which scattered light from an object combines with illumination from a monochromatic source. The use of a photonic crystal substrate for interferemetric scattering microscopy results in reduced requirements for high-intensity lasers or oil-immersion objectives, thus opening a pathway toward instruments that are more suitable for environments outside the optics laboratory. The instrument incorporates two innovative elements that facilitate operation on a desktop in ordinary laboratory environments by users that do not have optics expertise. First, because scattering microscopes are extremely sensitive to vibration, we incorporated an inexpensive but effective solution of suspending the instrument's main components from a rigid metal framework using elastic bands, resulting in an average of 28.7 dBV reduction in vibration amplitude compared to an office desk. Second, an automated focusing module based on the principle of total internal reflection maintains the stability of image contrast over time and spatial position. In this work, we characterize the system's performance by measuring the contrast from gold nanoparticles with diameters in the 10-40 nm range and by observing various biological analytes, including HIV virus, SARS-CoV-2 virus, exosome, and ferritin protein.

A Photonic Resonator Interferometric Scattering Microscope for Label-free Detection of Nanometer-Scale Objects with Digital Precision in Point-of-Use Environments.

Label-free detection and digital counting of nanometer-scaled objects such as nanoparticles, viruses, extracellular vesicles, and protein molecules enable a wide range of applications in cancer diagnostics, pathogen detection, and life science research. The contrast of interferometric scattering microscopy is amplified through a photonic crystal surface, upon which scattered light from an object combines with illumination from a monochromatic plane wave source. The use of a photonic crystal substrate for interference scattering microscopy results in reduced requirements for high-intensity lasers or oil-immersion objectives, thus opening a pathway toward instruments that are more suitable for environments outside the optics laboratory. Here, we report the design, implementation, and characterization of a compact Photonic Resonator Interferometric Scattering Microscope (PRISM) designed for point-of-use environments and applications. The instrument incorporates two innovative elements that facilitate operation on a desktop in ordinary laboratory environments by users that do not have optics expertise. First, because scattering microscopes are extremely sensitive to vibration, we incorporated an inexpensive but effective solution of suspending the instrument's main components from a rigid metal framework using elastic bands, resulting in an average of 28.7 dBV reduction in vibration amplitude compared to an office desk. Second, an automated focusing module based on the principle of total internal reflection maintains the stability of image contrast over time and spatial position, facilitating automated data collection. In this work, we characterize the system's performance by measuring the contrast from gold nanoparticles with diameters in the 10-40 nm range and by observing various biological analytes, including HIV virus, SARS-CoV-2 virus, exosomes, and ferritin protein.

Label-Free Digital Detection of Intact Virions by Enhanced Scattering Microscopy.

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.

Detailed Balance Broken by Catch Bond Kinetics Enables Mechanical-Adaptation in Active Materials.

Unlike nearly all engineered materials which contain bonds that weaken under load, biological materials contain "catch" bonds which are reinforced under load. Consequently, materials, such as the cell cytoskeleton, can adapt their mechanical properties in response to their state of internal, non-equilibrium (active) stress. However, how large-scale material properties vary with the distance from equilibrium is unknown, as are the relative roles of active stress and binding kinetics in establishing this distance. Through course-grained molecular dynamics simulations, the effect of breaking of detailed balance by catch bonds on the accumulation and dissipation of energy within a model of the actomyosin cytoskeleton is explored. It is found that the extent to which detailed balance is broken uniquely determines a large-scale fluid-solid transition with characteristic time-reversal symmetries. The transition depends critically on the strength of the catch bond, suggesting that active stress is necessary but insufficient to mount an adaptive mechanical response.

KERA: analysis tool for multi-process, multi-state single-molecule data.

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA's utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.

Effects of Intercalating Molecules on the Polymer Properties of DNA.

Atomic force microscopy (AFM) enables determination of physical properties from single DNA molecules. Insertion of aromatic molecules into the structure of DNA results in morphological changes. However, the accompanying changes to elastic properties due to this insertion are not fully understood. AFM was used to examine the morphological effects of intercalator binding and report changes in the elastic properties of intrinsically straight DNA molecules. The persistence length and polymer extension were characterized in the presence of three intercalating molecules: ethidium bromide and the less well studied chloroquine and acridine. It was found that all three intercalators significantly increased the bending persistence length. In addition, an analysis of the normal bending modes of the static molecules corroborated these results. This approach of measuring binding effects of intercalators on DNA physical properties using a model system of intrinsically straight DNA is applicable to other DNA binding ligands and other modes of DNA interaction.

Dynamics and selective remodeling of the DNA-binding domains of RPA.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-stranded DNA (ssDNA) intermediates, activating the DNA-damage response and handing off ssDNA to the appropriate downstream players. Six DNA-binding domains (DBDs) in RPA promote high-affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA-interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.