Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250' and 280s Loops.

Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250' and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen-deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.

Proposal for a hybrid clock system consisting of passive and active optical clocks and a fully stabilized microcomb.

Optical atomic clocks produce highly stable frequency standards and frequency combs bridge clock frequencies with hundreds of terahertz difference. In this paper, we propose a hybrid clock scheme, where a light source pumps an active optical clock through a microresonator-based nonlinear third harmonic process, serves as a passive optical clock via indirectly locking its frequency to an atomic transition, and drives a chip-scale microcomb whose mode spacing is stabilized using the active optical clock. The operation of the whole hybrid system is investigated through simulation analysis. The numerical results show: (i) The short-term frequency stability of the passive optical clock follows an Allan deviation of σy(τ) = 9.3 × 10-14τ-1/2 with the averaging time τ, limited by the population fluctuations of interrogated atoms. (ii) The frequency stability of the active optical clock reaches σy(τ) = 6.2 × 10-15τ-1/2, which is close to the quantum noise limit. (iii) The mode spacing of the stabilized microcomb has a shot-noise-limited Allan deviation of σy(τ) = 1.9 × 10-11τ-1/2. Our hybrid scheme may be realized using recently developed technologies in (micro)photonics and atomic physics, paving the way towards on-chip optical frequency comparison, synthesis, and synchronization.

Optical sensors, 2022: introduction to the feature issue.

This feature issue of Optics Express highlights contributions from authors who presented their latest research at the OPTICA Optical Sensors and Sensing Congress, held in Vancouver, British Columbia, Canada from 11-15 July 2022. The feature issue comprises 9 contributed papers, which expand upon their respective conference proceedings. The published papers introduced here cover a range of timely research topics in optics and photonics for chip-based sensing, open-path and remote sensing and fiber devices.

Time-dependent Mandel Q parameter analysis for a hexagonal boron nitride single photon source.

The time-dependent Mandel Q parameter, Q(T), provides a measure of photon number variance for a light source as a function of integration time. Here, we use Q(T) to characterise single photon emission from a quantum emitter in hexagonal boron nitride (hBN). Under pulsed excitation a negative Q parameter was measured, indicating photon antibunching at an integration time of 100 ns. For larger integration times Q is positive and the photon statistics become super-Poissonian, and we show by comparison with a Monte Carlo simulation for a three-level emitter that this is consistent with the effect of a metastable shelving state. Looking towards technological applications for hBN single photon sources, we propose that Q(T) provides valuable information on the intensity stability of single photon emission. This is useful in addition to the commonly used g(2)(τ) function for the complete characterisation of a hBN emitter.

"Grafting-To" Covalent Binding of Plasmonic Nanoparticles onto Silica WGM Microresonators: Mechanically Robust Single-Molecule Sensors and Determination of Activation Energies from Single-Particle Events.

We hereby present a novel "grafting-to"-like approach for the covalent attachment of plasmonic nanoparticles (PNPs) onto whispering gallery mode (WGM) silica microresonators. Mechanically stable optoplasmonic microresonators were employed for sensing single-particle and single-molecule interactions in real time, allowing for the differentiation between binding and non-binding events. An approximated value of the activation energy for the silanization reaction occurring during the "grafting-to" approach was obtained using the Arrhenius equation; the results agree with available values from both bulk experiments and ab initio calculations. The "grafting-to" method combined with the functionalization of the plasmonic nanoparticle with appropriate receptors, such as single-stranded DNA, provides a robust platform for probing specific single-molecule interactions under biologically relevant conditions.

Single Virus Detection on Silicon Photonic Crystal Random Cavities.

On-chip silicon microcavity sensors are advantageous for the detection of virus and biomolecules due to their compactness and the enhanced light-matter interaction with the analyte. While their theoretical sensitivity is at the single-molecule level, the fabrication of high quality (Q) factor silicon cavities and their integration with optical couplers remain as major hurdles in applications such as single virus detection. Here, label-free single virus detection using silicon photonic crystal random cavities is proposed and demonstrated. The sensor chips consist of free-standing silicon photonic crystal waveguides and do not require pre-fabricated defect cavities or optical couplers. Residual fabrication disorder results in Anderson-localized cavity modes which are excited by a free space beam. The Q ≈105 is sufficient for observing discrete step-changes in resonance wavelength for the binding of single adenoviruses (≈50 nm radius). The authors' findings point to future applications of CMOS-compatible silicon sensor chips supporting Anderson-localized modes that have detection capabilities at the level of single nanoparticles and molecules.

Nonlinear Sensing with Whispering-Gallery Mode Microcavities: From Label-Free Detection to Spectral Fingerprinting.

Optical microresonators have attracted intense interests in highly sensitive molecular detection and optical precision measurement in the past decades. In particular, the combination of a high quality factor with a small mode volume significantly enhances the nonlinear light-matter interaction in whispering-gallery mode (WGM) microresonators, which greatly boost nonlinear optical sensing applications. Nonlinear WGM microsensors not only allow for label-free detection of molecules with an ultrahigh sensitivity but also support new functionalities in sensing such as the specific spectral fingerprinting of molecules with frequency conversion involved. Here, we review the mechanisms, sensing modalities, and recent progresses of nonlinear optical sensors along with a brief outlook on the possible future research directions of this rapidly advancing field.

Review of optical sensing and manipulation of chiral molecules and nanostructures with the focus on plasmonic enhancements [Invited].

Chiral molecules are ubiquitous in nature; many important synthetic chemicals and drugs are chiral. Detecting chiral molecules and separating the enantiomers is difficult because their physiochemical properties can be very similar. Here we review the optical approaches that are emerging for detecting and manipulating chiral molecules and chiral nanostructures. Our review focuses on the methods that have used plasmonics to enhance the chiroptical response. We also review the fabrication and assembly of (dynamic) chiral plasmonic nanosystems in this context.

Combining Whispering-Gallery Mode Optical Biosensors with Microfluidics for Real-Time Detection of Protein Secretion from Living Cells in Complex Media.

The noninvasive monitoring of protein secretion of cells responding to drug treatment is an effective and essential tool in latest drug development and for cytotoxicity assays. In this work, a surface functionalization method is demonstrated for specific detection of protein released from cells and a platform that integrates highly sensitive optical devices, called whispering-gallery mode biosensors, with precise microfluidics control to achieve label-free and real-time detection. Cell biomarker release is measured in real time and with nanomolar sensitivity. The surface functionalization method allows for antibodies to be immobilized on the surface for specific detection, while the microfluidics system enables detection in a continuous flow with a negligible compromise between sensitivity and flow control over stabilization and mixing. Cytochrome c detection is used to illustrate the merits of the system. Jurkat cells are treated with the toxin staurosporine to trigger cell apoptosis and cytochrome c released into the cell culture medium is monitored via the newly invented optical microfluidic platform.

Tracking micro-optical resonances for identifying and sensing novel procaspase-3 protein marker released from cell cultures in response to toxins.

The response of cells to toxins is commonly investigated by detecting intracellular markers for cell death, such as caspase proteins. This requires the introduction of labels by the permeabilization or complete lysis of cells. Here we introduce a non-invasive tool for monitoring a caspase protein in the extracellular medium. The tool is based on highly sensitive optical micro-devices, referred to as whispering-gallery mode biosensors (WGMBs). WGMBs are functionalized with antibodies for the specific and label-free detection of procaspase-3 released from human embryonic kidney HEK293 and neuroglioma H4 cells after introducing staurosporine and rotenone toxins, respectively. Additional tests show that the extracellular accumulation of procaspase-3 is concomitant with a decrease in cell viability. The hitherto unknown release of procaspase-3 from cells in response to toxins and its accumulation in the medium is further investigated by Western blot, showing that the extracellular detection of procaspase-3 is interrelated with cytotoxicity of alpha-synuclein protein (aSyn) overexpressed in H4 cells. These studies provide evidence for procaspase-3 as a novel extracellular biomarker for cell death, with applications in cytotoxicity tests. Such WGMBs could be applied to further identify as-yet unknown extracellular biomarkers using established antibodies against intracellular antigens.

Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

Optical tracking of anomalous diffusion kinetics in polymer microspheres.

In this Letter we propose the use of whispering gallery mode resonance tracking as a label-free optical means to monitor diffusion kinetics in glassy polymer microspheres. Approximate solutions to the governing diffusion equations are derived for the case of slow relaxation and small Stefan number. Transduction of physical changes in the polymer, including formation of a rubbery layer, swelling, and dissolution, into detectable resonance shifts are described using a perturbative approach. Concrete examples of poly(methyl methacrylate) and polystyrene spheres in water are considered.

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.

Ultraspecific and highly sensitive nucleic acid detection by integrating a DNA catalytic network with a label-free microcavity.

Nucleic acid detection with label-free biosensors circumvents costly fluorophore functionalization steps associated with conventional assays by utilizing transducers of impressive ultimate detection limits. Despite this technological prowess, molecular recognition at a surface limits the biosensors' sensitivity, specificity, and reusability. It is therefore imperative to integrate novel molecular approaches with existing label-free transducers to overcome those limitations. Here, we demonstrate this concept by integrating a DNA strand displacement circuit with a micron-scale whispering gallery mode (WGM) microsphere biosensor. The integrated biosensor exhibits at least 25-fold improved nucleic acid sensitivity, and sets a new record for label-free microcavity biosensors by detecting 80 pM (32 fmol) of a 22nt oligomer; this improvement results from the catalytic behavior of the circuit. Furthermore, the integrated sensor exhibits extremely high specificity; single nucleotide variants yield 40- to 100-fold lower signal. Finally, the same physical sensor was demonstrated to alternatingly detect 2 different nucleic acid sequences through 5 cycles of detection, showcasing both its reusability and its versatility.

Optimizing detection limits in whispering gallery mode biosensing.

A theoretical analysis of detection limits in swept-frequency whispering gallery mode biosensing modalities is presented based on application of the Cramér-Rao lower bound. Measurement acuity factors are derived assuming the presence of uncoloured and 1/ f Gaussian technical noise. Frequency fluctuations, for example arising from laser jitter or thermorefractive noise, are also considered. Determination of acuity factors for arbitrary coloured noise by means of the asymptotic Fisher information matrix is highlighted. Quantification and comparison of detection sensitivity for both resonance shift and broadening sensing modalities are subsequently given. Optimal cavity and coupling geometries are furthermore identified, whereby it is found that slightly under-coupled cavities outperform critically and over coupled ones.

Hybrid quantum-classical polarizability model for single molecule biosensing.

Optical whispering gallery mode biosensors are able to detect single molecules through effects of their polarizability. We address the factors that affect the polarizability of amino acids, which are the building blocks of life, via electronic structure theory. Amino acids are detected in aqueous environments, where their polarizability is different compared to the gasphase due to solvent effects. Solvent effects include structural changes, protonation and the local field enhancement through the solvent (water). We analyse the impact of these effects and find that all contribute to an increased effective polarizability in the solvent. We also address the excess polarizability relative to the displaced water cavity and develop a hybrid quantum-classical model that is in good agreement with self-consistent calculations. We apply our model to calculate the excess polarizability of 20 proteinogenic amino acids and determine the minimum resolution required to distinguish the different molecules and their ionised conformers based on their polarizability.

Thermo-Optoplasmonic Single-Molecule Sensing on Optical Microcavities.

Whispering-gallery-mode (WGM) resonators are powerful instruments for single-molecule sensing in biological and biochemical investigations. WGM sensors leveraged by plasmonic nanostructures, known as optoplasmonic sensors, provide sensitivity down to single atomic ions. In this article, we describe that the response of optoplasmonic sensors upon the attachment of single protein molecules strongly depends on the intensity of WGM. At low intensity, protein binding causes red shifts of WGM resonance wavelengths, known as the reactive sensing mechanism. By contrast, blue shifts are obtained at high intensities, which we explain as thermo-optoplasmonic (TOP) sensing, where molecules transform absorbed WGM radiation into heat. To support our conclusions, we experimentally investigated seven molecules and complexes; we observed blue shifts for dye molecules, amino acids, and anomalous absorption of enzymes in the near-infrared spectral region. As an example of an application, we propose a physical model of TOP sensing that can be used for the development of single-molecule absorption spectrometers.

Whispering-Gallery Mode Optoplasmonic Microcavities: From Advanced Single-Molecule Sensors and Microlasers to Applications in Synthetic Biology.

Optical microcavities, specifically, whispering-gallery mode (WGM) microcavities, with their remarkable sensitivity to environmental changes, have been extensively employed as biosensors, enabling the detection of a wide range of biomolecules and nanoparticles. To push the limits of detection down to the most sensitive single-molecule level, plasmonic nanorods are strategically introduced to enhance the evanescent fields of WGM microcavities. This advancement of optoplasmonic WGM sensors allows for the detection of single molecules of a protein, conformational changes, and even atomic ions, marking significant contributions in single-molecule sensing. This Perspective discusses the exciting research prospects in optoplasmonic WGM sensing of single molecules, including the study of enzyme thermodynamics and kinetics, the emergence of thermo-optoplasmonic sensing, the ultrasensitive single-molecule sensing on WGM microlasers, and applications in synthetic biology.

Roadmap on optical sensors.

Optical sensors and sensing technologies are playing a more and more important role in our modern world. From micro-probes to large devices used in such diverse areas like medical diagnosis, defence, monitoring of industrial and environmental conditions, optics can be used in a variety of ways to achieve compact, low cost, stand-off sensing with extreme sensitivity and selectivity. Actually, the challenges to the design and functioning of an optical sensor for a particular application requires intimate knowledge of the optical, material, and environmental properties that can affect its performance. This roadmap on optical sensors addresses different technologies and application areas. It is constituted by twelve contributions authored by world-leading experts, providing insight into the current state-of-the-art and the challenges their respective fields face. Two articles address the area of optical fibre sensors, encompassing both conventional and specialty optical fibres. Several other articles are dedicated to laser-based sensors, micro- and nano-engineered sensors, whispering-gallery mode and plasmonic sensors. The use of optical sensors in chemical, biological and biomedical areas is discussed in some other papers. Different approaches required to satisfy applications at visible, infrared and THz spectral regions are also discussed.

Interference of conically scattered light in surface plasmon resonance.

Surface plasmon polaritons on thin metal films are a well studied phenomena when excited using prism coupled geometries such as the Kretschmann attenuated total reflection configuration. Here we describe a novel interference pattern in the conically scattered light emanating from such a configuration when illuminated by a focused beam. We observe conditions indicating only self-interference of scattered surface plasmon polaritions without any contributions from specular reflection. The spatial evolution of this field is described in the context of Fourier optics and has applications in highly sensitive surface plasmon based biosensing.